Josh was raised in the town of Wimberley, only 30 minutes southwest of Austin. His love for Austin and its surrounding hill country is what brought Josh into Real Estate. Josh began his career in the homebuilding industry in 1998, then ventured into sales after receiving his degree in Computer Science, and becoming a licensed Real Estate Agent. His vast knowledge and understanding of home construction and the Austin Real Estate market, makes him an excellent resource for his clients, whether buying or selling. In his spare time, Josh enjoys playing soccer, swimming in one of the many rivers and lakes around Austin, kayaking, and spending time with family.


text:
New sports pitch for Weston College's university campus
Henry Woodsford
An artist's impression of the propsed health and active living skills centre.
Weston College has applied for planning permission to build a new artificial sports pitch.
The all-weather pitch will be 61 metres wide and 97.4 metres in length and will be located directly behind the college's university campus, in Loxton Road.
It will have a total area of 7132sqm and form part of the college's proposed new £13.3million Health and Active Living Skills Centre, also in Loxton Road.
MORE: Weston College to spend more than £13million on new healthcare centre.
The floodlit pitch will include a single-storey sports equipment store which will have a ridge height of 3.6 metres.
In the design and access statement, the college said it wishes 'to minimise the impact on the houses to the north of the college campus'.
A new footpath will be located to the east of the pitch to connect it to the equipment store and to footpaths within the campus, while it stresses no trees will be removed 'to provide ease of access to the proposed health centre'.

redpajama_set_name: RedPajamaCommonCrawl


I was shopping in Nordstrom recently and found the cutest necessities for this summer! This black dress by LEITH is so simple and timeless, you can wear it anywhere for any occasion. I've worn this dress to a wedding, out to brunch with cute flats, or just walking around enjoying NYC. The best part of this dress, is that it's only $69!


text:
Test Code Page

Current location is <current-location></current-location>

<code-tabs>

No linenums at code-tabs level

  class {
    foo(param: string) {}
  }

  <h1>Heading</h1>
  { "key": "value" }

linenums=true at code-tabs level

  class {
    foo(param: string) {}
  }

  <h1>Heading</h1>
  { "key": "value" }

No linenums at code-tabs level; linenums=true for HTML pane

  class {
    foo(param: string) {}
  }

  <h1>Heading</h1>
  { "key": "value" }

<code-example>

One line.

const foo = 'bar'

Multi-line, linenums=false.

<hero-details nghost-pmm-5>
  <h2 ngcontent-pmm-5>Bah Dah Bing</h2>
  <hero-team ngcontent-pmm-5 nghost-pmm-6>
    <h3 _ngcontent-pmm-6>Headless Team</h3>
  </hero-team>
</hero-details>

Default linenums.

<hero-details nghost-pmm-5>
  <h2 ngcontent-pmm-5>Mister Fantastic</h2>
  <hero-team ngcontent-pmm-5 nghost-pmm-6>
    <h3 _ngcontent-pmm-6>Headless Team</h3>
  </hero-team>
</hero-details>

Header on this one.

  <hero-details nghost-pmm-5>
    <h2 ngcontent-pmm-5>Mister Fantastic</h2>
    <hero-team ngcontent-pmm-5 nghost-pmm-6>
      <h3 _ngcontent-pmm-6>Winning Team</h3>
    </hero-team>
  </hero-details>

An "avoid" header on this one.

  <hero-details nghost-pmm-5>
    <h2 ngcontent-pmm-5>Mister Fantastic</h2>
    <hero-team ngcontent-pmm-5 nghost-pmm-6>
      <h3 _ngcontent-pmm-6>Losing Team</h3>
    </hero-team>
  </hero-details>

## Backticked code blocks

```html
  <hero-details>
    <h2>Mister Fantastic</h2>
    <hero-team>
      <h3>Losing Team</h3>
    </hero-team>
  </hero-details>
```

<live-example>

Plain live-example

Try this <live-example></live-example>.

live-example with title atty

<live-example header="Good Example"></live-example>

live-example with title body

<live-example header="Good Example">Try this great example</live-example>

live-example with name

<live-example name="testy" header="Better Example"></live-example>

live-example with spacey name and stackblitz

<live-example name=" testy " stackblitz="super-stackblitz"></live-example>

live-example with name and stackblitz but no download

<live-example name="testy" stackblitz="super-stackblitz" noDownload></live-example>

live-example embedded with name and stackblitz

<live-example embedded name="testy" stackblitz="super-stackblitz"></live-example>

More text follows ...

redpajama_set_name: RedPajamaGithub


text:
(845) 353-1313 10 Park Street • Nyack NY • 10960
Sundays at 7
Cold Readings
Tech Classes
Box Office Policies
Program Advertising
Home » Shows » Calendar Girls
2019-20 SEASON:
Next Fall
Born Yesterday
written by Tim Firth, based on Miramax motion picture by Juliette Towhidi and Tim Firth
directed by Margaret Young
Friday July 10th 2020 — Saturday August 1st 2020
When Annie's husband John dies of leukemia, she and best friend Chris resolve to raise money for a new settee in the local hospital waiting room. They manage to persuade four fellow Women's Institute (WI) members to pose nude with them for an "alternative" calendar with a little help from hospital porter and amateur photographer Lawrence, and much to the horror of their WI chairman Marie. The news of the women's charitable venture spreads like wildfire, and hordes of press soon descend on the small village of Knapeley in Yorkshire, England. The calendar is a success, but Chris and Annie's friendship is put to the test under the strain of their new-found fame, and Chris is forced to question her real motivation behind doing the calendar. Tim Firth's hilarious play is based on the true story of eleven British WI members who famously posed nude for a calendar to raise money for the Leukemia Research Fund in 1999, and was made into a hit movie in 2003 with Helen Mirren and Julie Walters. Calendar Girls is a funny, moving show about friendship, courage, passion and risk-taking. Based on the true story and hit movie.
Presented by special arrangement with Samuel French, Inc.
Production Staff / Crew
Director: Margaret Young
Elmwood Playhouse
Tue - Fri: 9AM – 3PM
"The Little Foxes" Opens at Elmwood This Weekend
Elmwood Playhouse Announces Cast and Crew for "NEXT FALL"
Elmwood represented at AACT Community Theatre Management Conference
@BadCaseOfSerpes You are correct sir!	3 weeks ago
Click to submit news article »
Copyright Elmwood Playhouse. All Rights Reserved ©2019


text:
Baden-Baden 2014: Puccini: Manon Lescaut
Philharmonia Choir Vienna, Eva-Maria Westbroek, Lester Lynch, Massimo Giordano, Liang Li, Bogdan Mihai, Reinhard Dorn, Magdalena Kožená, Kresimir Spicer, Arthur Espiritu, Johannes Kammler
Richard Eyre, direction; Robert Howell stage; Fotini Dimou, costumes; Peter Mumford, light design
Prog. No.
János Darvas
EuroArts Music in co-production with SWR/arte & NHK
A rococo beauty who ultimately drags everything into the abyss, Puccini's Manon Lescaut is never fully satisfied. She becomes addicted to the admiring glances she receives from others, and the luxury and diamonds in which she bathes don't warm her for long. In this, his first successful opera, the composer paints a portrait of a woman who has everything, only to lose it because she is incapable of holding on to anything. Likewise Puccini, who identified closely with his heroine, helping himself to many things. He took the orchestra from Verdi, expanding it with phrases and themes he found in Wagner and the French people. To all of this he added something from within himself – a melody more breathless than that of Verdi, but in many ways, sweeter. Add to that an ingenious fire in which ideas flow and the themes are inspired. The fact that the Berliner Philharmoniker turn to Puccini – this beloved yet ostracized composer – is symbolic. Here is someone who was thought to be outmoded for a long time, but there is really so much more to be discovered about him and his work. The tale of one who becomes a model but remains a girl, is very contemporary.
Opera in four acts by Giacomo Puccini after Histoire du Chevalier Des Grieux et de Manon Lescaut des Abbé Prévost


Despite facing many of the same barriers that wheelchair-using doctors encounter, a growing army of nurses on wheels is fighting the system and advocating for changes that will benefit everyone, with and without disabilities.
Disabled Docs - Healing the Medical Model?
Cory Lee Woodard has become the face of accessible travel, and now he has another honor to add to his growing list: New Mobility's 2018 Person of the Year.
Living a fearless life after a spinal cord injury doesn't mean living with a lack of fear. In fact, it's the opposite. It's having fears and choosing to go forward despite them.
Finding a silver lining amidst pain, unwanted health issues and society's cruel eye is a lot for anyone to handle.


text:
search filter All ContentAll JournalsJournal of General Physiology
Milestones in Physiology
Article| December 28 2009
Inactivation of the KcsA potassium channel explored with heterotetramers
Dvir Rotem,
Dvir Rotem
Department of Chemistry, University of Oxford, Oxford OX1 3TA, England, UK
Amy Mason,
Amy Mason
Hagan Bayley
Correspondence to Hagan Bayley: hagan.bayley@chem.ox.ac.uk
Abbreviations used in this paper:
DPhPC
1,2-diphytanoyl-sn-glycero-3-phosphocholine
IVTT
in vitro transcription and translation
1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine
POPG
1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)]
tobacco etch virus
wild-type
Received: July 29 2009
Accepted: December 08 2009
© 2009 Rotem et al.
This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.jgp.org/misc/terms.shtml). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).
J Gen Physiol (2010) 135 (1): 29–42.
https://doi.org/10.1085/jgp.200910305
Dvir Rotem, Amy Mason, Hagan Bayley; Inactivation of the KcsA potassium channel explored with heterotetramers. J Gen Physiol 1 January 2010; 135 (1): 29–42. doi: https://doi.org/10.1085/jgp.200910305
The tetrameric prokaryotic potassium channel KcsA is activated by protons acting on the intracellular aspect of the protein and inactivated through conformational changes in the selectivity filter. Inactivation is modulated by a network of interactions within each protomer between the pore helix and residues at the external entrance of the channel. Inactivation is suppressed by the E71A mutation, which perturbs the stability of this network. Here, cell-free protein synthesis followed by protein purification by sodium dodecyl sulfate–polyacrylamide gel electrophoresis was used to produce heterotetramers of KcsA that contain different combinations of wild-type and E71A subunits. Single-channel recordings from these heterotetramers reveal how the network of interactions in individual protomers affects ionic conduction and channel inactivation, suggesting that the latter is a cooperative process.
The potassium channel KcsA from Streptomyces lividans has been used as a model for understanding the structure–function relationships of ion channels (Doyle et al., 1998; MacKinnon, 2004; Gouaux and Mackinnon, 2005). It is a small tetrameric protein with a pore region that resembles that of larger potassium channels (Doyle et al., 1998; MacKinnon, 2004; Gouaux and Mackinnon, 2005). The channel is activated by protons acting on the intracellular aspect of the protein (Cuello et al., 1998; Heginbotham et al., 1999; Thompson et al., 2008) and inactivated through conformational changes in the selectivity filter (Blunck et al., 2006; Cordero-Morales et al., 2006a,b, 2007). Macroscopic current measurements of KcsA channels have shown that after exposure to low pH, the channels are rapidly activated and subsequently enter an inactivated state from which recovery is slow (Gao et al., 2005; Cordero-Morales et al., 2006b; Chakrapani et al., 2007a). As a result, under steady-state conditions, the channel has a low open probability (Cordero-Morales et al., 2006b; Chakrapani et al., 2007b). In single-channel recordings, this activity is characterized by short bursts of activity that are separated by long periods of inactivity (Cordero-Morales et al., 2006b; Chakrapani et al., 2007b). In addition, the steady-state gating of KcsA at low pH is affected by the applied potential (Cuello et al., 1998; Heginbotham et al., 1999; Cordero-Morales et al., 2006a), which influences the rate of entry and exit into the inactivated state as reflected in macroscopic currents (Chakrapani et al., 2007a).
The inactivation of KcsA is modulated by intraprotomeric interactions between Glu71 and Trp67 in the pore helix and Asp80 in the external entrance of the channel (Fig. 1). Therefore, the inactivation of KcsA channels can be described as an allosteric process, where the conformation of the regulatory site (the network of interactions between the pore helix and the external entrance) affects ion permeability through the selectivity filter. The mutation E71A, which disrupts this network, greatly reduces the rate of inactivation as observed in macroscopic currents (Cordero-Morales et al., 2006a,b, 2007 ,Chakrapani et al., 2007a,b) and produces a remarkably high steady-state open probability in single-channel recordings at low pH (Cordero-Morales et al., 2006a,b, 2007; Chakrapani et al., 2007b). Further, it has been proposed that crystal structures of wild-type (WT) KcsA in the presence of high K+ concentrations, where the selectivity filter contains two ions distributed over the four binding sites (Zhou et al., 2001), represent the permeable (active) configuration of the KcsA selectivity filter (Fig. 1). On the other hand, the structures of WT KcsA at low K+ concentrations (Zhou et al., 2001) and the crystal structure of the M96V mutant (Lockless et al., 2007), where the selectivity filter contains one ion distributed over the two remaining binding sites, are thought to represent the configuration of the selectivity filter during inactivation.
C-type inactivation of eukaryotic voltage-dependent K+ (Kv) channels, which occurs in response to prolonged depolarization, has also been associated with conformational changes in the selectivity filter (Yellen, 1998). C-type inactivation of Kv channels is a cooperative process that involves concerted conformational changes of all four subunits (Ogielska et al., 1995; Panyi et al., 1995). The KcsA inactivation process shares features with C-type inactivation: (a) mutation of Glu71 and Tyr82 in KcsA (Cordero-Morales et al., 2006a,b), and the corresponding residues in Shaker Kv channels, Val438 (Yifrach and MacKinnon, 2002) and Thr449 (López-Barneo et al., 1993; Ogielska et al., 1995), respectively, all affect the inactivation of the channels; (b) the rate of inactivation of KcsA and Shaker Kv channels has a similar dependence on the extracellular potassium ion concentration (López-Barneo et al., 1993; Cordero-Morales et al., 2006b; Chakrapani et al., 2007a). These results, together with the sequence and structural similarities of KcsA and eukaryotic K+ channels in the region of the selectivity filter (Long et al., 2005), suggest that the inactivation processes of the channels are mechanistically similar.
Single-channel recordings are widely used to clarify the mechanisms of action of ion channels. The majority of single-channel activity measurements on ion channels that have been performed in planar lipid bilayers, including those on KcsA channels, have been of proteins overexpressed and purified from bacteria. The ion channels are typically reconstituted into lipid vesicles, which are subsequently fused with bilayers (e.g., Schrempf et al., 1995; Heginbotham et al., 1999). Although this approach has produced valuable data and expanded our understanding of the functional properties of ion channels, time-consuming overexpression and purification steps are involved. It has previously been shown that some β barrel pores, such as staphylococcal α-hemolysin, and the small viral K+ channel Kcv can be expressed in a cell-free system and purified by SDS-PAGE before direct insertion into planar lipid bilayers (Braha et al., 1997; Shim et al., 2007; Mason, 2008). Cell-free protein synthesis has advantages over conventional protein expression techniques, including its rapidity and the capability to synthesize proteins with unnatural or labeled amino acids (Spirin, 2002; Wang et al., 2006). Cell-free synthesis has been used to produce many membrane proteins, including KcsA channels (van Dalen et al., 2000). In addition, it has been shown that KcsA, like the α-hemolysin pore and the Kcv channel, maintains its oligomeric structure during SDS-PAGE (Heginbotham et al., 1997). These results suggested that KcsA tetramers might also be purified directly from polyacrylamide gels in a functional form, which is the approach taken here.
Cell-free protein synthesis followed by direct protein purification by SDS-PAGE was previously exploited to form heteromers of WT and chemically modified mutant subunits of α-hemolysin (Braha et al., 1997). More recently, when this technique was used, the mutant α-hemolysin subunits contained oligoaspartate extensions at their C termini (Howorka et al., 2001). Accordingly, it was possible to separate heteromers with different combinations of WT and mutated subunits based on their differing electrophoretic mobilities. Electrophoresis of KcsA heteromers formed by the coexpression of subunits with different peptide extensions at their C termini was used previously to demonstrate that KcsA forms tetramers (Heginbotham et al., 1997). However, these channels were not purified from the gels and used for functional studies. In the present work, heteromers that contained WT and E71A KcsA protomers with different peptide extensions at their N termini were produced and separated by SDS-PAGE. The extensions were then cleaved by proteolysis, and single-channel recordings were made with heteromers in which the protomers differed from each other only at the desired site of mutation, position 71.
By this approach, we reveal how regulatory sites in the KcsA channel affect inactivation. These regulatory sites are the networks of interaction within individual protomers between the pore helix and the external entrance of the channel. Based on our results, we propose an inactivation mechanism that features cooperativity between the four subunits. In addition, this study shows that the E71A mutation causes the formation of a ring of charges at the extracellular entrance of the channel that increases inward ionic conduction.
Unless otherwise stated, chemicals and oligonucleotides were purchased from Sigma-Aldrich. Enzymes and prestained gel markers were purchased from New England Biolabs, Inc. and lipids from Avanti Polar Lipids, Inc.
Genetic constructs
Constructs were assembled in the pT7-SC1 expression vector (Cheley et al., 1997) and verified by sequencing of the entire genes. The KcsA gene encoding the point mutant E71A was generated by PCR mutagenesis and in vivo recombination as described previously (Jones, 1995; Howorka and Bayley, 1998). The same technique was used to generate the genes for KcsA with N-terminal polypeptide extensions by using overlapping primers to form the coding sequences for the extensions. Two extensions were generated: "long," with the amino acid sequence MHHHHHHNNNNNNNNRLEQKLISEEDLL…, and "short," with the sequence MHHHHHHL.... In both cases, the sequences were followed by a tobacco etch virus (TEV) protease recognition site (ENLYFQG) and eight asparagine residues.
Cell-free coupled transcription and translation
Polypeptides were generated by coupled in vitro transcription and translation (IVTT) by using an Escherichia coli T7-S30 extract system for circular DNA (Promega). The complete 1-mM amino acid mixture minus cysteine and the complete 1-mM amino acid mixture minus methionine were mixed in equal volumes to obtain the working amino acid solution required to generate high concentrations of the proteins. The amino acids (10 µl) were mixed with premix solution (40 µl), 4 µl [35S]methionine (1,175 Ci mmol−1, 10 mCi ml−1; MP Biomedicals) and 30 µl T7 S30 extract supplemented with 20 µg ml−1 (final concentration) rifampicin (Cheley et al., 1997). To form heteromers, different ratios of the plasmid DNA encoding WT KcsA with the long polypeptide extension and E71A-KcsA with the short polypeptide extension were mixed (total volume 16 µl, from 400–ng µl−1 stock solutions of each DNA). Synthesis was performed for 60 min at 37°C to generate 100 µl IVTT polypeptide solution. Laemmli sample buffer (2×, 100 µl; Bio-Rad Laboratories) was then added and the sample was loaded, without heating, into two 5-mm wells of a 15-cm-long, 1.5-mm-thick 10% SDS polyacrylamide gel. Both the stacking and the separation gels were supplemented with 10 mM KCl. The gel was run overnight at 45 V, and then for a few more hours at 100 V to achieve the maximal separation between the different heteromers. The unfixed gel was vacuum dried without heating onto 3MM chromatography paper (GE Healthcare) and visualized by exposure to film (BioMax MR-1; Kodak). Each of the heteromer-containing bands was cut from the gel by using the autoradiogram as a guide. The excised pieces were rehydrated with buffer (200–300 µl) containing 10 mM HEPES, pH 7.0, and 10 mM KCl for 1 h. After removing the paper, each gel strip was thoroughly crushed in the solution, and the protein was separated from the gel by centrifugation through a 0.2-µm cellulose acetate filter (Rainin). The protein was stored frozen at −80°C.
Proteolysis with TEV protease
The polypeptide extensions of the heteromers were removed by proteolysis with TEV protease. Before proteolysis, the storage buffer of the protein was exchanged for 50 mM Tris HCl, pH 8.0, 0.5 mM EDTA, 0.02% (wt/vol) n-dodecyl-β-d-maltoside, and 10 mM KCl by dilution and concentration by centrifugation, three times at 14,000 g for 18 min, with a filter (YM-10 Microcon; Millipore). 1 mM (final concentration) dithiothreitol and AcTEV protease (1 µl, 10 U µl−1; Invitrogen) were added to a protein sample (50 µl) (Parks et al., 1994; Suh-Lailam and Hevel, 2009). The sample was incubated overnight at 25°C and stored at −80°C. This material was used directly for electrical recordings.
Single-channel electrical recording and analysis
Single-channel recordings were performed by using the folded planar lipid bilayer method (Montal and Mueller, 1972). The apparatus consisted of a 0.025-mm-thick Teflon septum glued between two Delrin chambers. The septum contained an aperture of 100-µm diameter, which was treated with 10% (vol/vol) hexadecane in pentane. Each chamber was filled with 1 ml of filter-sterilized buffered salt solution. Unless otherwise stated, the salt solutions were 200 mM KCl and 10 mM HEPES, pH 7.0, in the cis chamber and 200 mM KCl and 10 mM succinic acid, pH 4.0, in the trans chamber (solutions were titrated with KOH or HCl).
1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) and 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (POPG) (8 µl; 7.5 and 2.5 mg ml−1, respectively, in pentane) or 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC; 8 µl; 10 mg ml−1 in pentane) were added to the surface of each chamber, and the pentane was allowed to evaporate to leave behind a lipid monolayer. A lipid bilayer was formed by lowering and raising the liquid levels in the chambers below and above the aperture.
The electrical current was detected with two Ag/AgCl electrodes, amplified with a patch clamp amplifier (Axopatch 200B; MDS Analytical Technologies), filtered with a low-pass Bessel filter (80 dB per decade) with a corner frequency of 5 kHz, and digitized with an A/D converter (DigiData 1320; MDS Analytical Technologies) at a sampling frequency of 50 kHz. In all experiments, the cis chamber was at ground. A positive current represents the movement of cations from trans to cis. The bilayer recording platform, chambers, amplifying headstage, and micromanipulator were enclosed in a Faraday cage to shield against ambient electrical noise. KcsA channels were directly inserted into the bilayer from the cis (ground) chamber either by adding the protein to the bath solution and stirring or by using an agarose probe, as described previously (Holden and Bayley, 2005).
Traces of 2-min duration (beginning in the open state) were further filtered with a digital Gaussian filter at 1 kHz in Clampfit 10 (MDS Analytical Technologies). Amplitude histograms were fitted to Gaussian functions, and the quoted unitary conductance values were obtained from the peak (mean) values of the fits. Open probabilities were determined with the Single-Channel Search module from Clampfit. When the activity of more than one channel was observed in a trace, the single-channel open probability was determined by dividing the total open probability (NPo) by the total number of active channels (N). NPo is calculated by Clampfit:
NPo=∑1NntnT,
where n is the number of channels in level n (1, 2, … N), tn is the total amount of time spent in level n during the entire trace, and T is the total recording time. A value for N was obtained from the highest current level observed during a recording.
In vitro expression of functional KcsA channels
KcsA was expressed and labeled with [35S]methionine in a cell-free IVTT system. Analysis by SDS-PAGE revealed two major bands with Mr values of ∼15 and ∼45 kD (Fig. 2 A). The molecular mass of KcsA, predicted from the polypeptide sequence, is 18 kD for the monomer and hence 72 kD for the tetramer. Tetrameric KcsA has been shown previously to migrate in SDS polyacrylamide gels with Mr < 72 kD (Heginbotham et al., 1997), and we surmised that the ∼45-kD band is the tetramer.
For single-channel recording, the KcsA tetramers formed by IVTT were isolated from an SDS polyacrylamide gel by elution into 10 mM HEPES, pH 7.0. The protein was added to the cis chamber (ground) of a bilayer apparatus, and the solution was stirred until single-channel activity was seen. Alternatively, the protein was introduced directly into the bilayer by using an agarose probe (Holden and Bayley, 2005). During this study, folded lipid bilayers were used (Montal and Mueller, 1972), composed initially of either DPhPC or a 3:1 ratio of POPE and POPG.
Previous studies have suggested a requirement for a negatively charged lipid for channel activity (Heginbotham et al., 1998; Valiyaveetil et al., 2002). Typically then, for single-channel recordings, KcsA is reconstituted into vesicles and fused with bilayers of 3:1 POPE/POPG (Heginbotham et al., 1999; LeMasurier et al., 2001; Marius et al., 2008) or soybean asolectin lipids (Cortes et al., 2001). However, we found that KcsA functions in a lipid bilayer composed solely of DPhPC with similar activity, in terms of channel gating and ion conductance, compared with that observed in POPE/POPG bilayers (Fig. 2, B and C). As the S30 cell extract contains a high level of E. coli lipids (van Dalen et al., 2002; Conlan, 2003), it is possible that the tetramer formed by IVTT contains tightly bound PG, which might be retained throughout the purification process and thereby allow the channel to function in a DPhPC bilayer. All subsequent experiments were performed with DPhPC bilayers.
To be active in reconstituted systems, the intracellular domain of KcsA should be at pH <5 (Cuello et al., 1998). Therefore, the chamber solutions were buffered with 10 mM HEPES, pH 7.0, in the cis chamber, and with 10 mM succinic acid, pH 4.0, in the trans chamber. This ensured that the active channels were of known orientation with respect to the bilayer (Heginbotham et al., 1999). The cis chamber therefore represented the extracellular side of a cell and the trans chamber the intracellular side. The channels displayed the well-documented activity of KcsA: burst-like behavior, with a higher open probability at positive potentials compared with negative potentials (Fig. 2 C) (Cordero-Morales et al., 2006b; Chakrapani et al., 2007b). The openings at negative potentials were significantly noisier than at positive potentials (not depicted) and the I-V curve displayed partial outward rectification (Fig. 3). WT KcsA had a conductance of 108 ± 8 pS at +100 mV and 50 ± 10 pS at −100 mV in 200 mM KCl. These values are in reasonable agreement with previously reported values of 56 pS at +100 mV and 31 pS at −100 mV recorded in 100 mM KCl (Heginbotham et al., 1999). This anticipated single-channel behavior demonstrates that it is possible to examine the functional properties of a K+ channel expressed in vitro and purified directly from a gel, thereby eliminating the time-consuming labor of traditional protocols.
Formation of heteromeric channels
It had been shown that KcsA heteromers with different combinations of subunits, containing short and long peptide extensions, can be separated by SDS-PAGE (Heginbotham et al., 1997). Therefore, we attempted to form heteromeric KcsA channels by co-translation of two different constructs. We added DNAs encoding WT and E71A KcsA polypeptides, which contained, respectively, long and short peptide extensions at the N terminus (see Materials and methods), to an IVTT mix. When the products were examined by SDS-PAGE, the E71A tetramer was not observed, and it was supposed that it either did not form or dissociated into monomers in SDS. Cations known to permeate or strongly block K+ channels (such as K+, Rb+, and Cs+) confer stability to the WT tetramer (Krishnan et al., 2005). Indeed, the addition of 10 mM KCl to the SDS polyacrylamide gels increased the stability of the E71A tetramer sufficiently for purification for single-channel recording. Therefore, 10 mM KCl was routinely added to gels and to the storage solutions of the purified protein.
When the WT/E71A tetramers were analyzed by SDS-PAGE, a ladder of five evenly spaced bands was evident (Fig. 4). The different heteromeric combinations had been separated according to their molecular masses, conferred by the N-terminal extensions. However, by using this method it was not possible to distinguish between the two potential WT2E71A2 tetramers, where the mutant protomers are in either symmetric or asymmetric configurations (Fig. 4). The tetramers with different ratios of WT and E71A subunits were isolated from the gel, and the protein was eluted into 10 mM KCl and 10 mM HEPES, pH 7.0. Most of the purified protein retained its tetrameric structure, as shown by the rerunning of samples in SDS polyacrylamide gels (Fig. 5). The ratios of the two subunits in the dissociated tetramers were as anticipated based on the expected "parent" tetramer structures (Fig. 5).
For further work, the peptide extensions at the N termini of the KcsA subunits in the tetramers were removed by proteolysis with TEV protease to form subunits that differed from each other only at the mutated position 71 (Fig. 5). After the proteolysis step, all the subunits in a tetramer (WT or E71A) retain a short extension at the N terminus comprising one Gly residue followed by eight Asn residues (G8N). Interestingly, in the absence of the G8N linker between the N terminus of the protomers and the TEV recognition site, protease cleavage was very inefficient (not depicted). It has been determined by site-directed spin labeling (Cortes et al., 2001) and nuclear magnetic resonance spectroscopy (Baker et al., 2007) that the N terminus of the KcsA channel forms an α helix. It is possible that this well-ordered secondary structure prevents the binding of the protease or inhibits protease activity when it is adjacent to the recognition site. After the proteolysis step, the KcsA tetramers with different ratios of WT and E71A subunits all formed active channels in planar lipid bilayers (Fig. 6). Comparisons of the properties of WT and E71A KcsA channels with or without the G8N extension demonstrated that the presence of the extension did not affect the activity of channels in terms of their unitary conductance values (Fig. 3) and gating activities (compare Figs. 2 and 6).
The effect of the E71A mutation on the KcsA inward conductance
The I-V curve of WT KcsA in 200 mM KCl reflects significant outward rectification (Fig. 3). By comparison, under the same conditions, E71A homotetramers show mild inward rectification. Although the mutation has little effect on the magnitude of the current in the outward direction, it has a major effect on the inward current, e.g., at −100 mV the conductance of WT channels is 50 ± 10 pS (n = 4), and the conductance of E71A channels is about three times higher, 148 ± 5 pS (n = 4). Similar results were previously published (Thompson et al., 2008). The E71V homotetramer in planar lipid bilayers showed a similar phenomenon, with an even larger inward conductance (Choi and Heginbotham, 2004). The E71A homotetramers exhibited significant noise in single-channel recordings (Fig. 2). The origin of the noise is unclear, but it is of a similar magnitude in currents recorded from WT tetramers.
Comparison of the crystal structures of WT (Zhou et al., 2001) and E71A (Cordero-Morales et al., 2006b) KcsA channels suggests a possible explanation for the differences in the conductance values. In the WT KcsA crystal structure, the side chain of Asp80 faces into the protein core (Zhou et al., 2001) (Fig. 1). In one of the two structures of E71A (called "flipped"), the Asp80 side chain moves 8 Å and is situated in the extracellular entrance of the channel, exposed to the bulk solvent (Fig. 1) (Cordero-Morales et al., 2006b). The authors suggest that eliminating the carboxyl–carboxylate interaction between Asp80 and Glu71 increases the flexibility of Asp80 side chain and allows the observed movement (Cordero-Morales et al., 2006b). Based on the conformational change observed in the flipped structure, the exposed carboxyl side chains of Asp80 creates an external ring of negative charge (Fig. 1). This ring would increase the inward conductance of the channel by decreasing the height of the energy barrier for ion entry through the external entrance into the selectivity filter.
The effect of charged rings on conduction has been studied in various ion channels, including BK potassium channels (Qian et al., 2002; Brelidze et al., 2003; Carvacho et al., 2008), acetylcholine receptors (Imoto et al., 1988), and mutated KcsA (Nimigean et al., 2003). For all of these channels, it was demonstrated that the presence of negatively charged amino acids in one of the entrances into the channel increases the rate of transport of cations from this entrance to the other side of the membrane.
To further explore the effect of a negatively charged ring at the extracellular entrance of E71A KcsA, we determined the effect of pH on the conductance of the channel. If the source of the increase of inward conductance in E71A KcsA channels is a ring of charge at the extracellular entrance formed by the exposed carboxyl side chains of Asp80, it is predicted that a reduction in the pH on the extracellular side of the channel would cause a decrease in the inward current as a result of protonation of the Asp side chains. When the pH in the cis chamber (the extracellular side of the channel) is decreased from pH 7.0 to 3.0, only the inward conductance of E71A KcsA is reduced, whereas for WT KcsA, both the inward and the outward conductances are unaffected (Fig. 7). Therefore, the results support the idea that the rise in the inward conductance of the mutated channel is caused, at least in part, by the exposure of the Asp80 carboxyl side chains at the extracellular entrance of the channel, as shown in the flipped E71A KcsA crystal structure. On the other hand, the WT KcsA inward conductance was not changed at low extracellular pH because the carboxyl side chain of Asp80 is buried inside the protein, as shown in the WT crystal structure (Fig. 1), and does not form a ring of charge at the extracellular entrance.
Measurements on the WT/E71A heteromers gave a further opportunity to determine the effect of Ala substitution at position 71 on the channel conductance (Fig. 8 A). The I-V curves of the WT/E71A heteromers show that as the number of E71A subunits in the tetramers is increased, the inward current increases (Fig. 8 B). These results suggest that each of the E71A subunits in the tetramer makes a cumulative contribution to the elevation of the inward current.
Inactivation of KcsA
Proton-dependent activation of KcsA is followed by an inactivation process similar to C-type inactivation. As a result, at low pH, homotetrameric WT KcsA channels are characterized by short bursts of activity that are separated by long periods of inactivity (Cordero-Morales et al., 2006b; Chakrapani et al., 2007b). Under our conditions, WT4 KcsA channels behave as described in the earlier reports (Fig. 6). WT4 channels indeed have a low open probability, Po < 0.1, and as the bilayer is depolarized, the open probability of the channel increases (Fig. 9 A and Table I) (Cordero-Morales et al., 2006a; Chakrapani et al., 2007b). The inactivation gate is located in the selectivity filter of the channel, and inactivation is suppressed by the E71A mutation (Cordero-Morales et al., 2006b). In contrast with WT4, previous work has shown that the open probability of E71A4 at low pH is close to one, Po = 1 (Cordero-Morales et al., 2006b; Chakrapani et al., 2007b; Thompson et al., 2008). In the experiments presented here, E71A4 channels exhibited variable and lower open probabilities compared with those reported previously (e.g., Po = 0.58 ± 0.18 at +50 mV; n = 12) (Fig. 9 A and Table I). The basis of the differences between this study and previous work is not clear. Nevertheless, we do find that the open probability of E71A4 is much higher than that of WT4 (Fig. 9 A and Table I) and is practically voltage independent (Cordero-Morales et al., 2007).
Measurements of the single-channel activity of the WT/E71A heteromers gave a closer view of the mechanism behind the transition of the selectivity filter between active and inactive states. The open probability of WT3E71A1 channels, where one WT subunit is substituted with one E71A subunit, was higher than that of WT4 channels at all the potentials examined (Fig. 9 A and Table I). However, like that of WT4 channels, the open probability of WT3E71A1 channels was voltage dependent, and it increased as the membrane was depolarized. At high depolarizing potentials (above +50 mV), the open probability of WT3E71A1 channels approached the same level as that of the E71A4 channels (Fig. 9 A and Table I). Because of the high variability in the open probability values, it was impossible to determine the exact values of V1/2 and the gating charge (z) for WT4 and WT3E71A1. However, the plots of normalized open probability as a function of membrane potential of both channels can be fit to Boltzmann distributions with similar slopes (i.e., similar gating charges), in which the V1/2 of WT3E71A1 is shifted to a more negative potential (Fig. 9 B). These results suggest that the presence of even one E71A subunit destabilizes the inactive state of the selectivity filter in the tetrameric channel.
Heteromers with additional E71A subunits (i.e., WT2E71A2 and WT1E71A3 channels) had open probability values of the same order of magnitude as E71A4 channels (Fig. 9 A and Table I). Although it is difficult to make a precise comparison of the open probability values of WT2E71A2, WT1E71A3, and E71A4 tetramers because of their high variability, it is clear that in all of these channels, the open probability is no longer voltage dependent (Fig. 9 A and Table I). These results suggest that the presence of two or more E71A subunits in a KcsA tetramer provides maximal suppression of the transition of the selectivity filter into the inactivated state.
The potassium channel KcsA has been used extensively as a model for the understanding of structure–function relationships in ion channels. Here, it has been demonstrated that functional KcsA can be expressed in vitro by cell-free coupled IVTT by using an E. coli extract. The tetrameric proteins formed in the IVTT mix are stable in SDS and therefore can be purified by SDS-PAGE. The extracted protein inserts spontaneously into planar lipid bilayers. By using single-channel recording, it was shown that the activity of KcsA extracted from gels is similar to the activity of KcsA purified by conventional means.
Cell-free expression and purification of heteromeric KcsA channels
Cell-free protein expression and purification by SDS-PAGE eliminates the time-consuming steps of traditional membrane protein purification protocols. In addition, in vitro synthesis provides numerous useful options, including the labeling of amino acids and the incorporation of unnatural amino acids (Spirin, 2002; Wang et al., 2006). Here, we have exploited one such application, namely, the production of heteromers of an oligomeric protein. Natural KcsA is a homotetramer. Therefore, it is difficult to analyze the contributions to protein function of individual subunits and the interactions between them. To this end, heterooligomers formed from WT and mutated subunits can be used. Two major techniques have been used previously for the production of heteromeric ion channels. The first is in vivo coexpression of WT and mutated polypeptides. This technique, combined with data-fitting analyses of macroscopic currents, was useful in determining the subunit stoichiometry of channels such as Shaker Kv (MacKinnon, 1991). The second technique uses tandemly fused polypeptides connected by flexible linkers. The genes are built from WT and mutant genes fused tail to head. This technique has been used in analyses of subunit cooperativity in processes including the activation of Kv channels (Zandany et al., 2008). One of the limitations of the first approach is the stochastic (or otherwise) sorting of the WT and mutant subunits in the heteromers. The second method addresses this problem. However, the fact that the subunits are fused tail to head can affect their ability to function normally or to insert correctly into membranes (McCormack et al., 1992; Sack et al., 2008).
In the study presented here, an IVTT system was used to produce heteromers of WT and mutant KcsA subunits with peptide extensions at their N termini. These extensions allowed heteromers with different ratios of WT and mutant subunits to be separated on the basis of size by electrophoresis. In this way, our technique overcomes the limitations of the other approaches; heteromers are produced in which the subunits are not covalently connected and the stoichiometry of the subunits is known. In recent years, this method has been used extensively to form heteroheptamers of α-hemolysin pores (Bayley and Jayasinghe, 2004). In the study presented here, the peptide extensions were removed by proteolysis, so that the subunits in the tetramers differed only by the desired mutation at position 71. This was an important step because the extensions were up to about one quarter of the mass of each monomer and situated close to the channel entrance. They might therefore have influenced channel activity. Both KcsA and α-hemolysin form stable oligomers in SDS polyacrylamide gels and therefore can be purified by electrophoresis. "Native" gel electrophoresis might be suitable for the separation and purification of oligomeric proteins that are not stable to SDS-PAGE.
The E71A mutation affects KcsA channel stability and ion conduction
E71A, the mutation used throughout this work, was shown to affect the stability of KcsA channels and their gating and conductance properties. The source of these effects is likely to be the elimination of the carboxyl–carboxylate interaction between Asp80 and Glu71 (Cordero-Morales et al., 2006b). As a result, the side chain of Asp80 becomes more flexible, which disrupts its interaction with Trp67. Consequently, Trp67 is able to switch from one rotameric state to another (Cordero-Morales et al., 2006b). In WT channels, the side chain of Trp67 is turned toward the adjacent protomer and lies in close proximity to several residues, including Arg89, which has been implicated in the stability of the tetramers (Irizarry et al., 2002). In E71A4, one of the rotamers of Trp67 is orientated away from the contact plane with the adjacent protomer (whereas the other is located as found in the WT channel). Therefore, the flexibility of Trp67 in E71A channels weakens the stability of the tetramer. Inorganic cations known to permeate or strongly block K+ channels confer stability to the KcsA channel (Krishnan et al., 2005), and indeed we found that the addition of 10 mM KCl to SDS polyacrylamide gels increased the stability of the E71A homotetramer.
Our results also show that the inward conductance of E71A KcsA channels is much higher than that of WT channels and is reduced at low pH. In the crystal structure of the E71A homotetramers (in the flipped conformation; Cordero-Morales et al., 2006b), Asp80 residues are exposed at the extracellular entrance. Therefore, our findings suggest that the exposed side chains create a ring of negative charge that decreases the height of the energy barrier for ion entry into the selectivity filter and therefore increases the inward current. At low pH, the Asp80 side chains become protonated, and their effect on the electrostatic potential at the entrance is reduced. By using the WT-E71A heteromers, it was shown that the addition of each E71A subunit to the tetrameric channel makes a cumulative contribution to the elevation of the inward conductance. In the flipped crystal structure of E71A tetramers, the distance between the carboxyl groups of the Asp80 side chains is ∼9 Å. Because the Debye length in 200 mM KCl is ∼7 Å, the independence of the contributions of each carboxyl group makes sense. Therefore, the stability of the intraprotomeric network of interactions between the pore helix and the external entrance of the channel is a major factor in the outward rectification of the WT KcsA channel.
The inactivation of KcsA as an allosteric process
Based on electrophysiological measurements of mutated channels and molecular dynamics simulations, it has been proposed that an intraprotomeric network of interactions between Glu71 and Trp67 in the pore helix and Asp80 in the external entrance of KcsA (Fig. 1) promotes a distortion of the filter into a nonconductive configuration, leading to inactivation (Cordero-Morales et al., 2006b). In addition, because the open probability of the channel is increased at high positive applied potentials, it has been suggested that membrane depolarization relieves the inactivation of the channel by pulling Glu71 toward the intracellular side of the bilayer, thereby disrupting the network (Cordero-Morales et al., 2006a). The open probability of E71A4 is much higher than that of WT4, and it is voltage independent. Based on these results, it was proposed that the E71A mutation essentially abolishes inactivation because the presence of Ala in position 71 cannot support the network of interactions (Cordero-Morales et al., 2006b, 2007).
As mentioned earlier, our measurements of the unitary conductance of WT/E71A heteromers suggest that the identity of the residue at position 71 (Glu or Ala) in a protomer determines whether Asp80 in the same subunit is buried in the protein or exposed at the external entrance. Therefore, the network of interactions that is described above only exists in WT subunits and not in E71A subunits, whether they are present in homotetramers or heterotetramers. From our examination of inactivation, we surmise that the tetrameric selectivity filter can be in only one of two configurations: active (conductive) or inactive (nonconductive). For example, we did not observe intermediate current steps between the closed and the open levels (although we cannot reject the possibility that very short-lived states exist that were not acquired in our measurements). In other words, the transition of the four selectivity filter sequences (TVGYG) between their active and inactive conformations is concerted. Therefore, we can describe the inactivation of KcsA as an allosteric process, where the permeability of the selectivity filter (the active site) is affected by the state of the four independent networks of interactions between the pore helices and the external entrance (together: the regulatory sites). Each WT subunit in a tetramer contains a functional regulatory site that stabilizes the inactive conformation of the selectivity filter. In an E71A protomer, this regulatory site is defective.
Our results show that under strongly depolarizing potentials (above +50 mV), the open probabilities of WT/E71A heteromers with one or more E71A subunits are similar to the open probability of E71A homotetramers (Fig. 9 A and Table I). Therefore, we suggest that under these conditions, the transition of the selectivity filter sequences into the inactive state in all E71A-containing heteromers is defective. On the other hand, channels with four WT subunits have a much lower open probability in this voltage range, reflected by their greater tendency to inactivate. The network of interactions that stabilizes the inactivated state is relatively weak under a strong depolarizing potential, which pulls Glu71 toward the intracellular side of the bilayer, disrupting the network of interactions between Glu71, Trp67, and Asp80. We propose that, under these conditions, the concerted transition of the selectivity filter sequences of all four subunits into their inactive conformation will happen only when all of the subunits are stabilized in that conformation by the regulatory network (Fig. 10).
A decrease in the membrane potential does not affect the open probability of channels that contain two or more E71A subunits. In contrast, the open probabilities of E71A1WT3 and WT4 are reduced as the membrane potential becomes more negative (Fig. 9, A and B, and Table I). It has been suggested that at more negative potentials, the network of interactions in WT subunits that stabilizes inactivation becomes stronger (Cordero-Morales et al., 2006a). We propose that, under these conditions, the concerted transition of the selectivity filter sequences of all the subunits of a tetramer into the inactive conformation occurs even when the regulatory network is broken in one of the subunits (Fig. 10). However, because the open probability of channels with one E71A protomer is higher than WT4 across the entire measured voltage range (Fig. 9 A), we also propose that at hyperpolarized potentials (when the network of interactions is relatively strong), the destabilization of the regulatory network in one protomer still exerts a negative effect on the concerted transition of the selectivity filter sequences into the inactive conformation.
These studies of WT/E71A KcsA heteromers have offered a glimpse into the mechanism of inactivation of the KcsA channel. It was previously shown that the C-type inactivation of Kv channels is a cooperative process that involves a concerted conformational change in all four protomers (Ogielska et al., 1995; Panyi et al., 1995). Superimposition of the KcsA crystal structure (PDB entry 1K4C) with the crystal structures of the Kv1.2 channel from the Shaker family (PDB entries 2A79 and 2R9R) and the KvAP channel (PDB entry 1ORQ) suggests that in these channels there also exist networks of interactions between the pore helix and the external entrance of the channel. In addition, mutation of the residue corresponding to Glu71 of KcsA in the Shaker Kv (Val438) (Yifrach and MacKinnon, 2002) and Kv2.1 (Ile369) channels (De Biasi et al., 1993) affects the C-type inactivation of these channels. Therefore, we can speculate that C-type inactivation of voltage-gated channels, like KcsA, is also regulated by an allosteric site. In contrast with the KcsA channel, it has been shown that C-type inactivation in Shaker Kv channels is voltage independent, at least in the range of −25 to +50 mV (Hoshi et al., 1994). As proposed earlier (Chakrapani et al., 2007a), the absence of a charged residue in "position 71" of these channels might be the basis for the difference in the voltage dependency. Further, it was shown that when the conserved Val in position 370 (equivalent to position 71 in KcsA) of the Shaker channel Kv1.2 was mutated to Glu, the inactivation of channel was accelerated (Cordero-Morales et al., 2007).
In conclusion, comparison of the single-channel activity of homomeric and heteromeric channels formed from WT and E71A KcsA subunits shows that the stability of the intraprotomeric network of interactions between the pore helix and the external entrance of the channel makes a major contribution to both the outward rectification of the channel and to its transition into an inactive state. The effect of the stability of the network on the ion conductance is graded: the more protomers with a stabilized network, the greater the reduction in the inward current. On the other hand, channel inactivation, which is a concerted process, occurs only when, depending on the membrane polarization, the network of interactions is stabilized in three or four protomers.
This work was supported by a grant from the Medical Research Council. H. Bayley is the holder of a Royal Society Wolfson Research Merit Award.
Christopher Miller served as editor.
Tzitzilonis
Kwiatkowski
Choe
Riek
Conformational dynamics of the KcsA potassium channel governs gating properties
Nat. Struct. Mol. Biol.
. doi:
https://doi.org/10.1038/nsmb1311
Jayasinghe
Functional engineered channels and pores (Review)
Mol. Membr. Biol.
https://doi.org/10.1080/09687680410001716853
Blunck
Cordero-Morales
J.F.
Perozo
Bezanilla
Detection of the opening of the bundle crossing in KcsA with fluorescence lifetime spectroscopy reveals the existence of two gates for ion conduction
J. Gen. Physiol.
Braha
Cheley
Kasianowicz
J.J.
Gouaux
J.E.
Designed protein pores as components for biosensors
Brelidze
A ring of eight conserved negatively charged amino acids doubles the conductance of BK channels and prevents inward rectification
Proc. Natl. Acad. Sci. USA.
https://doi.org/10.1073/pnas.1532257100
Carvacho
Y.P.
Brauchi
Gonzalez-Nilo
F.D.
Latorre
Intrinsic electrostatic potential in the BK channel pore: role in determining single channel conductance and block
Chakrapani
A quantitative description of KcsA gating I: macroscopic currents
A quantitative description of KcsA gating II: single-channel currents
Malghani
Hobaugh
Spontaneous oligomerization of a staphylococcal alpha-hemolysin conformationally constrained by removal of residues that form the transmembrane beta-barrel
Protein Eng.
https://doi.org/10.1093/protein/10.12.1433
Heginbotham
Functional influence of the pore helix glutamate in the KcsA K+ channel
Biophys. J.
Conlan
Biochemical and biophysical characterization of a monomeric porin, OmpG
PhD thesis. Texas A&M University
Voltage-dependent gating at the KcsA selectivity filter
Jogini
D.M.
Molecular determinants of gating at the potassium-channel selectivity filter
Molecular driving forces determining potassium channel slow inactivation
Molecular architecture of full-length KcsA: role of cytoplasmic domains in ion permeation and activation gating
https://doi.org/10.1085/jgp.117.2.165
J.G.
pH-dependent gating in the Streptomyces lividans K+ channel
https://doi.org/10.1021/bi972997x
De Biasi
H.A.
Drewe
Taglialatela
Inactivation determined by a single site in K+ pores
Pflugers Arch.
Morais Cabral
Pfuetzner
Gulbis
Chait
B.T.
The structure of the potassium channel: molecular basis of K+ conduction and selectivity
Science.
https://doi.org/10.1126/science.280.5360.69
Paajanen
Activation-coupled inactivation in the bacterial potassium channel KcsA
Principles of selective ion transport in channels and pumps
Odessey
Tetrameric stoichiometry of a prokaryotic K+ channel
https://doi.org/10.1021/bi970988i
Kolmakova-Partensky
Functional reconstitution of a prokaryotic K+ channel
LeMasurier
Single streptomyces lividans K+ channels: functional asymmetries and sidedness of proton activation
Direct introduction of single protein channels and pores into lipid bilayers
https://doi.org/10.1021/ja042470p
Zagotta
W.N.
Aldrich
R.W.
Shaker potassium channel gating. I: transitions near the open state
Howorka
Improved protocol for high-throughput cysteine scanning mutagenesis
Biotechniques.
768, 770 passim
Sequence-specific detection of individual DNA strands using engineered nanopores
Nat. Biotechnol.
https://doi.org/10.1038/90236
Imoto
Sakmann
Mishina
Nakai
Bujo
Rings of negatively charged amino acids determine the acetylcholine receptor channel conductance
https://doi.org/10.1038/335645a0
Irizarry
Kutluay
Opening the KcsA K+ channel: tryptophan scanning and complementation analysis lead to mutants with altered gating
https://doi.org/10.1021/bi026393r
PCR mutagenesis and recombination in vivo
PCR Primer: A Laboratory Manual
Dieffenbach
C.W.
Dveksler
Cold Spring Harbor Laboratory Press
Krishnan
M.N.
J.P.
Trombley
Moczydlowski
Functional role and affinity of inorganic cations in stabilizing the tetrameric structure of the KcsA K+ channel
KcsA: it's a potassium channel
Lockless
S.W.
Zhou
Structural and thermodynamic properties of selective ion binding in a K+ channel
PLoS Biol.
E.B.
Crystal structure of a mammalian voltage-dependent Shaker family K+ channel
López-Barneo
Effects of external cations and mutations in the pore region on C-type inactivation of Shaker potassium channels
Receptors Channels.
Determination of the subunit stoichiometry of a voltage-activated potassium channel
Potassium channels and the atomic basis of selective ion conduction (Nobel Lecture)
Angew. Chem. Int. Ed. Engl.
Zagnoni
Sandison
Binding of anionic lipids to at least three nonannular sites on the potassium channel KcsA is required for channel opening
https://doi.org/10.1529/biophysj.107.117507
Single-channel characterisation of potassium channels with high temperature studies
PhD thesis. University of Oxford
L.E.
Tanouye
Sigworth
F.J.
Tandem linkage of Shaker K+ channel subunits does not ensure the stoichiometry of expressed channels
Montal
Formation of bimolecular membranes from lipid monolayers and a study of their electrical properties
https://doi.org/10.1073/pnas.69.12.3561
Nimigean
Electrostatic tuning of ion conductance in potassium channels
https://doi.org/10.1021/bi0348720
Ogielska
Haab
Cooperative subunit interactions in C-type inactivation of K channels
Panyi
C-type inactivation of a voltage-gated K+ channel occurs by a cooperative mechanism
Leuther
W.G.
Release of proteins and peptides from fusion proteins using a recombinant plant virus proteinase
https://doi.org/10.1006/abio.1994.1060
Slo1 tail domains, but not the Ca2+ bowl, are required for the β1 subunit to increase the apparent Ca2+ sensitivity of BK channels
https://doi.org/10.1085/jgp.20028692
Shamotienko
How to validate a heteromeric ion channel drug target: assessing proper expression of concatenated subunits
Schrempf
Kümmerlen
Hinnah
Betzler
Steinkamp
A prokaryotic potassium ion channel with two predicted transmembrane segments from Streptomyces lividans
J.W.
L.-Q.
In vitro synthesis, tetramerization and single channel characterization of virus-encoded potassium channel Kcv
https://doi.org/10.1016/j.febslet.2007.02.005
Spirin
Cell-free protein synthesis
Cell-Free Translation Systems
Suh-Lailam
Hevel
Efficient cleavage of problematic tobacco etch virus (TEV)-protein arginine methyltransferase constructs
https://doi.org/10.1016/j.ab.2008.12.031
Posson
Parsa
P.V.
Molecular mechanism of pH sensing in KcsA potassium channels
Valiyaveetil
Lipids in the structure, folding, and function of the KcsA K+ channel
https://doi.org/10.1021/bi026215y
van Dalen
de Kruijff
Efficient membrane assembly of the KcsA potassium channel in Escherichia coli requires the protonmotive force
EMBO Rep.
https://doi.org/10.1093/embo-reports/kvd067
Hegger
Influence of lipids on membrane assembly and stability of the potassium channel KcsA
Xie
Expanding the genetic code
Annu. Rev. Biophys. Biomol. Struct.
https://doi.org/10.1146/annurev.biophys.35.101105.121507
Yellen
The moving parts of voltage-gated ion channels
Q. Rev. Biophys.
Yifrach
Energetics of pore opening in a voltage-gated K(+) channel
Cell.
Zandany
Ovadia
Direct analysis of cooperativity in multisubunit allosteric proteins
Morais-Cabral
J.H.
Chemistry of ion coordination and hydration revealed by a K+ channel-Fab complex at 2.0 Å resolution
data&figures
related&metrics
Related & Metrics
View large Download slide
Superposition of the selectivity filter region of the WT KcsA structure (PDB entry 1K4C; magenta) and the flipped E71A KcsA structure (PDB entry 2ATK; green). There is a network of interactions between the side chains of Asp80, Glu71, and Trp67 in the WT channel. This network is broken in the mutated channel, and the side chains of Asp80 are exposed at the extracellular entrance of the channel. K+ ion binding sites inside the selectivity filter are presented as blue spheres and water molecules as red spheres. One KcsA protomer is omitted for clarity. Both of these structures are believed to represent conductive forms of the selectivity filter exhibiting four K+ ion binding sites with an overall occupancy of two K+ ions. The superposition was created with PyMOL (http://www.pymol.org).
Expression of functional KcsA channels by coupled IVTT. (A) Assembly of KcsA channels during IVTT. Products of IVTT were separated in a 12.5% SDS polyacrylamide gel supplemented with 10 mM KCl. Lane 1, WT KcsA; lane 2, E71A KcsA. The proteins were labeled with [35S]methionine and visualized by exposure of the dried gel to x-ray film. (B–D) Representative single-channel recordings of KcsA purified by SDS-PAGE. (B) WT KcsA in POPE/POPG (3:1) bilayers at +150 mV. (C) WT KcsA in DPhPC bilayers at ±100 mV. (D) E71A KcsA in DPhPC bilayers at ±100 mV. The measurements were made in 200 mM KCl, buffered with 10 mM HEPES, pH 7.0, in the cis chamber and 200 mM KCl, buffered with 10 mM succinic acid, pH 4.0, in the trans chamber. The traces were digitally filtered at 1 kHz for display.
The addition of a peptide extension to the N terminus of KcsA does not affect its activity. I-V curves of WT (squares) and E71A (triangles) KcsA without (filled symbols) or with the G8N extension at the N terminus (open symbols). Each data point represents the mean ± SD from at least three separate bilayers. All the measurements were done in 200 mM KCl, buffered with 10 mM HEPES, pH 7.0, in the cis chamber and 200 mM KCl, buffered with 10 mM succinic acid, pH 4.0, in the trans chamber.
Expression of heteromeric KcsA channels by coupled IVTT. The samples were separated in a 12.5% SDS polyacrylamide gel supplemented with 10 mM KCl. The proteins were labeled with [35S]methionine and visualized by exposure of the dried gel to x-ray film. Lanes 1–5: protein products from plasmid DNAs encoding WT-long and E71A-short in the ratios 4:0, 1:3, 2:2, 3:1, and 0:4. A schematic representation of the distribution of subunits in the heterotetramers is shown (right).
Electrophoretically purified KcsA heteromers. The five WT/E71A heteromers were purified by SDS-PAGE as described in Materials and methods and rerun in a 12.5% SDS polyacrylamide gel supplemented with 10 mM KCl, with or without treatment with TEV protease. The WT subunits had the long extension and the E71A subunits the short extension. Without treatment with the protease, the relative mobilities of the heteromers take on a staircase appearance because of the different total masses of the extensions. After proteolysis with TEV, all of the heteromers have the same mobility as a KcsA tetramer with an eight-Asn extension at the N terminus (right lane, M8N-KcsA; M, Met). The proteins were labeled with [35S]methionine and visualized by exposure of the dried gel to x-ray film. A schematic representation of the distribution of subunits in the heterotetramers is shown (right). The table at the bottom of the figure presents the ratios of WT KcsA and E71A KcsA subunits from spontaneously dissociated tetramers (not treated with TEV protease) on the same gel. The ratios were calculated with Quantity One software (Bio-Rad Laboratories).
Representative single-channel recordings of WT/E71A heteromers purified by SDS-PAGE. All measurements were done at ±100 mV in 200 mM KCl, buffered with 10 mM HEPES, pH 7.0, in the cis chamber and 200 mM KCl, buffered with 10 mM succinic acid, pH 4.0, in the trans chamber. The traces were digitally filtered at 1 kHz for display. All-points histograms of the traces are shown.
Currents through WT4 and E71A4 at pH 7.0 and 3.0 in the cis chamber. I-V curves of WT4 (squares) and E71A4 (triangles) at pH 7.0 (filled symbols) and 3.0 (open symbols) in the cis chamber. Each data point represents the mean ± SD from at least three separate bilayers. All the measurements were done in 200 mM KCl, buffered with 10 mM succinic acid, pH 4.0, in the trans chamber and with 200 mM KCl, buffered with 10 mM HEPES, pH 7.0, or with 10 mM succinic acid, pH 3.0, in the cis chamber.
(A) I-V relationships for homotetramers and heterotetramers of KcsA. Diamonds, WT4; squares, WT3E71A1; triangles, WT2E71A2; circles, WT1E71A3; crosses, E71A4. Each data point represents the mean ± SD from at least four separate bilayers. (B) Unitary conductance values of WT/E71A heteromers at −100 mV. Each data point represents the mean ± SD from at least 11 separate bilayers. The measurements were done in 200 mM KCl, buffered with 10 mM HEPES, pH 7.0, in the cis chamber and 200 mM KCl, buffered with 10 mM succinic acid, pH 4.0, in the trans chamber.
Voltage dependences of the open probabilities of WT/E71A heteromers. (A) Plots of open probability (Po) versus voltage. Diamonds, WT4; squares, WT3E71A1; triangles, WT2E71A2; circles, WT1E71A3; stars, E71A4. Each data point represents the mean ± SD from at least three separate bilayers. The measurements were done in 200 mM KCl, buffered with 10 mM HEPES, pH 7.0, in the cis chamber and 200 mM KCl, buffered with 10 mM succinic acid, pH 4.0, in the trans chamber. (B) Normalized values of the data shown in A for WT4 and WT3E71A1. Values were normalized to the maximum Po value, which was set at Po = 1. Diamonds, WT4; squares, WT3E71A1. The continuous curves are the best fits to Boltzmann functions for WT4 (black) and WT3E71A1 (red).
Figure 10.
A model for inactivation of KcsA channels as an allosteric process. Upon inactivation, there is a concerted transition of all four selectivity filter sequences (TVGYG) from the active conformation (green symbols) to the inactive conformation (red symbols). The conformation of the selectivity filter (the "active site" by analogy with allosteric enzymes) is regulated by networks of interactions between the pore helix and the external entrance of the channel that are formed independently within each subunit of the tetramer (the "regulatory sites" by analogy with allosteric enzymes). The selectivity filter sequence of each WT KcsA protomer is stabilized in its inactive conformation (striped symbols). On the other hand, the regulatory sites of E71A protomers are defective and do not support the stabilization of the inactive conformation of the selectivity filter sequences (open symbols). Under highly depolarizing potentials (top panel), the network of interactions is weakened (Cordero-Morales et al., 2006a). Under these conditions, the concerted transition of all four selectivity filter sequences into inactive conformations happens only when all four are stabilized by their regulatory site. When the membrane potential is negative (bottom panel), the network of interactions in the regulatory site becomes stronger. As a result, the concerted transition of the four selectivity filter sequences into inactive conformations occurs even when one of the sequences is not stabilized in the inactive conformation by its regulatory site. Pale red symbols correspond to unfavorable states of the KcsA channel.
Table I.
Open probabilities of WT/E71A heteromers
WT4 WT3E71A1 WT2E71A2 WT1E71A3 E71A4
V, mV Po n Po n Po n Po n Po n
−50 0.0036 ± 0.0018 11 0.047 ± 0.020 5 0.31 ± 0.17 9 0.46 ± 0.19 10 0.57 ± 0.26 9
50 0.025 ± 0.016 11 0.30 ± 0.19 7 0.27 ± 0.18 9 0.56 ± 0.22 11 0.58 ± 0.18 12
All measurements were made in 200 mM KCl, buffered with 10 mM HEPES, pH 7.0, in the cis chamber and 200 mM KCl, buffered with 10 mM succinic acid, pH 4.0, in the trans chamber. Values are mean ± SD.
J Gen Physiol (October,2006)
Tuning the tetraethylammonium sensitivity of potassium channel Kcv by subunit combination
J Gen Physiol (March,2012)
About JGP


Gracing the corner of 8th and Nueces, in an established neighborhood near downtown Austin, the Gilfillan House emits a silhouette of craftsmanship from the past. Designed by the legendary Austin architect Charles H. Page, this historic home was built in 1905, and is marked at the corner by a far-reaching heritage oak tree. Named for the original owner, businessman and banker William L. Gilfillan, the property has passed through many hands during its long-standing residence in Austin. The Prairie School style home has been cherished, rented out, subdivided, neglected, restored, and celebrated in elegant galas. By the elaborate plaster details framing the entrance and stretching views from the balconies, one can see why the Gilfillan House should treat the Austin community for another 100 years.
The work of O'Connell Architecture at the Gilfillan House includes exterior restoration of the historic house, interior rehabilitation for commercial office use, and preservation of character-defining features. Oversight of masonry restoration, window restoration and conservation, repair of the wood railings and deep overhanging eaves and brackets, and reconstruction of a few missing features are part of the focus.


text:
Our Pokemon Quiz
What has the local scene been like in the recent weeks? People on the streets are with their noses in their phones. Playing Pokemon Go is ubiquitous. We are in too. Augmented reality game is likely the going forward of many games. Though Pokemon Go was never intended for very young children, we know how it attracts when the parents play.
I welcomed my kids on board to play WITH us, with parental guidance. Good playing habits start early. We warned of safety being the utmost concern, followed by playing with a sensible mind. In our sense, we don't do in-app purchases. Sorry, Niantic.
So yeap, we've been Pokemon-ing (not addictive-ly) and sharing our adventures with like minders. Over food and drinks one day, we came up with Pokemon riddles! Mostly kids invented, so it gotta be really easy. No bounty for getting all correct, but maybe some fun moments with kids.
Try it, without scrolling to answers.
1. Which Pokemon always wins?
2. Which Pokemon needs nebulizer?
3. Which Pokemon is always scared?
4. Which Pokemon likes to eat onions?
5. Which Pokemon is a brave knight?
6. Which Pokemon likes to eat ikan billies?
7. Which Pokemon likes to do the dishes?
8. Which Pokemon can break free?
9. Which Pokemon sneezes alot?
10. Which Pokemon attracts?
1. Machamp
2. Koffing
3. Fearow
4. Onix
5. Dragonite
6. Ekan
7. Oddish
8. Butterfree
9. Pikachu
10. Magnemite
How did you fare?
10/10 - Get back to work
7/10 - Moderate player
5/10 - Try hanging out at MBS
1/10 - Where have you been?
All in the good name of Fun. Happy mid-week, everyone!
Ps: Special Thanks to Uncle Snorlax and kids for contributions.
Posted by Mum's calling at 07:46 2 comments :
Labels: Community , Family
Roald Dahl books your child will love!
It would have have been Roald Dahl's 100th birthday this month. A familiar story writer if you grew up reading his books.
In the eyes of many, Roald Dahl is one great storyteller since history. His stories have been fancifully imagined and ever captivating to the little readers out there. If your kids are between the ages of 6 to 12, his books are likely to work through the heart of theirs. It's a shame that not all his books made it into my childhood reading list. I am however, very much glad that Jazz is taking pleasure in reading almost of all his books.
Jazz may be an avid reader, but she is also one picky reader who selects her books with an interesting synopsis. Once she gets hold of a good read, she is likely to dive into it repeatedly. I find it amusing at how she can read and re-read a book more than once. I guess, good things are meant to be savoured over and over again.
In midst of her huge Roald Dahl collection, I asked her for 4 best titles that will delight any child in devouring from one page to another. Here's what she recommended:
1. Charlie and the Chocolate Factory
We know right! This has got to top the list.
Willy Wonka's famous chocolate factory is opening at last! But only five lucky children will be allowed inside. Mr Wonka devises a plan to hide 5 golden tickets beneath the wrappers of his famous chocolate bars. Charlie Bucket can't believe his luck when he finds the very last of Mr Willy Wonka's Golden Tickets inside his chocolate bar. He wins a magical tour around Willy Wonka's famous chocolate factory and sees the most amazing creations. One by one, the other four children on the tour cause some major trouble and are carried away. Charlie is the only child who doesn't cause trouble, and Willy Wonka tells him he won! What did he win? The whole chocolate factory. Mr Wonka wants someone to take over for him when he gets old.
It's a very sweet and heart-warming book to devour.
After reading this book, you may want to hunt down its movie. There are two adaptions of it, we caught the 2005 released, directed by Tim Burton. A nice and heart-warming watch. So good, that we caught it more than once.
Matilda Wormwood is only five years old, but she is an exceptional genius. Unfortunately her parents failed to notice that. During her toddlerhood, Matilda asks her Dad for a book and is pressured to watch television instead. She discovers the library and reads the classics, not simple children's books. She is however, chastised by her parents for her intelligence. Worse, her horrible headmistress Miss Trunchbull is a bully who makes life difficult for Matilda's teacher, Miss Honey, and her friends. But what Miss Trunchbull doesn't know is that Matilda has a trick or two up her sleeve, she has supernatural powers that can manipulate objects. Matilda uses this to her full advantage both with her parents and Miss Trunchbull. Just as her parents are caught in a cheating business scheme, and about to move, Matilda moves in with Miss Honey, where the two live happily and smartly together.
You will be very amazed at the wits Matilda possesses, as you read along. Matilda won the Children's Book Award shortly after it was published in 1988, and it has continued to delight audiences ever since.
Again, do hunt down its movie after reading. A lovely watch that our family enjoyed tremendously. Released in 1996 and driected by Danny DeVito. There's a musical on it too! We haven't got an opportunity to catch it.
3. The BFG
The Big Friendly Giant is unlike other giants. For a start, he doesn't like to eat people and it's not long before he becomes orphan Sophie's very best friend. He is one giant who captured dreams and kept them in bottles for children to enjoy while they were asleep. The dream-hunting giant takes orphan Sophie back to his cave in Giant Country, where he lives surrounded by nine other fearsome giants who spend every night guzzling down humans. Sophie learns that the giant is called the Big Friendly Giant (The BFG) because he does not eat humans. Sophie isn't able to go back to the orphanage because the BFG is afraid she'll tell others about the existence of giants and he'll be forced to live in a zoo.
The two devise a plan to mix up a nightmare for the Queen of England so that they can stop the giants from ever eating another human being. Or, as the giants call them, human beans. The BFG speaks in quite a turned-around way, but we always understand him. He tells Sophie, "Words...is oh such a twitch-tickling problem to me all my life. So you must simply try to be patient and stop squibbling. As I am telling you before, I know exactly what words I am wanting to say, but somehow or other they is always getting squiff-squiddled around."
The name, Sophie was inspired by Roald Dahl's granddaughter, Sophie Dahl. And as you can read, speaking in proper language is not BFG's forte. So if you are one fussy parent about reading perfect English, this may be a book you want to delay or avoid. Just in case, he begins speaking and writing like one. But it really is one great story!
And yes, the movie adaption is in the cinemas! Oh wait, is it still showing?
4. The Witches
The witches is an award-winning story by Roald Dahl about a boy, his grandmother and a hotel full of witches. It's not a fairy tale, but about real witches.
This is a book about 'real witches', the ones that absolutely loathe children and are always plotting to get rid of them. The Grand High Witch of all the World has gathered together the witches of Inkland for an annual conference at the Hotel Magnificent in Bournemouth. Their agenda is the elimination of all the children in the country and the prospects for their young victims look bleak. But the Grand High Witch and her cronies have reckoned without the spark and ingenuity of a young guest at the hotel and his rather brilliant grandmother.
It does sound a tad creepy, but if my not so brave girl can handle, I'm sure it would delight any child. At least we know, Roald Dahl's stories are always with a beautiful end. "Read how the witches schemed to turn the children into mice, but failed" said Jazz.
Once your child gets onto these books, read on to these other titles by Roald Dahl. Highly recommended by Jazz too.
Fantastic Mr. Fox
Are any of these your child's favourite too?
A good storybook helps encompass reading as part of a lifestyle, and a good book is any book your child likes. Reading is probably the best tutor for any child.
If your child has not started on chapter books, read a chapter a day to your child nightly. It will send your child sweet into dreams. And of course, use that animated voice when reading the BFG. It will be fun!
Books are gifts that you can open again and again. Here's the dessert - We love that children read, we love that children visit the library and we want to advocate the love for reading. We don't have the riches of the World, but we would like to bless 2 little readers with the titles "Charlie and The Chocolate Factory" and "The BFG" with our pocket money. Not from Jazz's collection, as she really treasures them.
To join the giveaway, we will be happy and appreciative that you:
1. Like Mum's calling on Facebook
2. Leave a comment on this Facebook post to tell us which title your child will like, Charlie and the Chocolate Factory OR The BFG
That's it! And of course, we wouldn't mind that you share this post with your friends, because reading is for everyone, and sharing is caring!
Giveaway ends on 10 October, 2359hrs.
This giveaway has ended, Congrats Chong Swee S. and Jessie L!
Labels: Books , Reading
The Magic Paintbrush [Giveaway]
This October, watch an old-time classic unveil live on stage, as itheatre brings through an endearing Chinese folk the tale of The Magic Paintbrush!
The Magic Paintbrush will be a musical treat, where any child over the age of three can bring their parents, and older brothers and sisters, aunties and uncles. Basically a whole kampong of people will enjoy it. Marvellous music, puppets and lively action will enthral the youngest audience members, whilst a witty script will entrance grownups and older children.
Toni Lee, a youngster from present-day Singapore is struggling with the pressures and stress of schoolwork and parental expectations. What a familiar local scene! With an accidental wish, Toni quite unexpectedly meets a Magical Paintbrush, and is transported back in time to a Southern Province of China, where Toni becomes Ma Liang. In a series of magical adventures, Ma Liang's story unfolds.
Meet the tuneful Phoenix who guides and helps Ma Liang with his quest. Watch out for the comical Palace Guards, Sotong and Shrimp, as they try to capture Ma Liang for the cruel, greedy Emperor and his wicked schemes.
Through much fun and laughter, a clear and strong moral message about the value of creativity, friendship, and the dangers of using, or abusing power is presented in this magical, musical family show.
Suitable for kids above 3 and definitely a great treat for the after exam kids!
27 0ct 2016 till 30 Oct 2016
Thu to Fri: 10.30am and 2.30pm
Sat and Sun: 11am and 2.30pm
1 Nov 2016 till 12 Nov 2016
Tue and Thu: 10.30am and 2.30pm
Wed and Fri: 10.30am
Sat: 11am, 2.30pm and 7.30pm
Sun: 11am and 5pm
Venue: Drama Centre Theatre
Ticket price: $32 (Exclude booking fee)
We have 3 pairs of tickets for giveaway to 3 lucky winners. This Magic Paintbrush treat will be on 29 October (Saturday), 11am. To take part, all you had need to do is:
Facebook Contest (2 winners)
2. Like itheatre on Facebook
3. Tag 3 friends on this Facebook post, whom you think will be keen on this giveaway.
Instagram Contest (1 winner)
1. Follow @mumscalling on Instagram
2. Tag 3 friends in the comment section of this post, whom you think will be keen in this giveaway.
You don't have to, but sharing is caring! And yes, you may try your luck with both handlers. Giveaway closes on 03 Oct 2016, 2359hrs. Best of luck!
Winners will be picked by random.org
Winners will be announced on Facebook and this blog post
Winners will have 24 hours to respond, failing which a new winner will be drawn
Date and Time of show is firm
All incomplete entries will be disqualified.
All entries will be verified before the winners are announced.
Giveaway is no way sponsored, endorsed, administered or associated with Facebook.
Little Llama: Open ended toys for play
Open ended play is always the best play for any child. Being parents, we love toys that allow free play and endless imagination. Little Llama sent our kids a couple of MOLUK toys, and we were thrilled by its simplicity. MOLUK, founded in Switzerland strives to create innovative and sustainable products that don't just entertain kids on a superficial level, but invite real interaction.
We had the opportunity to explore the Bilibo, Oogi and Plui rain cloud.
I really don't know what this is, but I know the kids are having some crazy fun with it. Along with the endless exploration, they sit, rock and spin in it, play games and hold stuff in it. Free play means there is no one way of dealing with it, whether indoor or outdoor.
This multi-award winning Bilibo is widely regarded as one of the most innovative and versatile open-ended toys. Deceptively simple and magically attractive, it's iconic shell shape and bright colors immediately appeal to kids. Bilibo arouses curiosity and playfully trains basic motor skills and balance.
It is highly durable and shock resistant for the hands of any kids. For choosing the colour green, I am coincidentally converting one of the kids to a turtle.
There really are endless possibilities to playing it, we've tried filling it with smaller toys and spin it so quickly until all of the the toys fly out. Not my favorite for clean up, but it sure was fun to watch how delighted the kids were to watch their toys being strewn everywhere. We also used it in the bathroom as a mini tub for play.
Starting to look more of a human toy. Oogi is an irresistibly tactile and wonderfully expressive new figure toy. With suction cup head, hands and feet, and long stretchy arms, it connects to any smooth surface. The suction is mild, hence it does fall off any smooth surface after some while.
The kids would fix Oogis on their Bilibo or even bring it into the bathroom for little brother's shower entertainment. They play quite a fair bit of it in the kitchen's fridge while I'm whipping meals up. These Oogis love doing yoga and crazy acrobatic tricks. Whatever the kids can think of, they will flex. I love how easily these can fit in any bag for bringing entertainment out. It goes anywhere and everywhere easily.
Mama's best deal is that they are made from high-quality, food-grade silicone rubber that's safe and durable. This is sounding like something chew-able for our baby! Bet he will love this play.
Plui rain cloud
I found out, this is my favourite toy of all. For the learning of science out of it.
Sink the toy cloud into a tub of water and spot bubbles oozing out of the top hole as it allows water in. We explained that the cloud is removing air to make room for water to come in. Kids then become little rainmakers by bringing the water filled cloud up to create rain! We are also in command to stop the rain by covering the top hole with our finger. By doing this, air is prevented from pushing the water out. Learning through play is exemplified!
Don't you think Science should come in as a subject in lower primary? Young kids are often so filled with curiosity.
This little rain cloud is quite an indispensable accessory during our bath time now. I love too that there's a hidden mechanism that allows parents to open the Cloud for cleaning. No worry about mould growing.
This is another high quality toy that's free of phathalates, BPA or lead.
I love how these toy selections come with no small parts and are safe for very young toddlers. It's evident that the simplest toys can bring endless entertainment. There are no ON and OFF switches, batteries or complicated instructions, they are all powered simply by a child's imagination!
MOLUK toys can be purchased at Little Llama, with free shipping over $60. They do have bundle deals which can be great as gifts too.
And here's the good news, Little Llama is giving away 2 Plui Rain Cloud to 2 lucky readers. 1 for Facebook and the other for Instagram participation. For Facebook participation, simply enter the following steps into the Rafflecopter widget below:
2. Like Little Llama on Facebook
Do ensure that you have entered the above steps in the Rafflecopter widget below as winner will be picked by Rafflecopter
And if you are on Instagram, you may hop over to @mumscalling participate too!
Giveaway ends on 21 September, 12:00am.
Facebook or Instagram accounts set up purely to take part in contests will be deemed ineligible to win
Giveaway is no way sponsored, endorsed, administered or associated with Facebook
Labels: Giveaway , Review


text:
Lavoûte-sur-Loire ist eine französische Gemeinde mit  Einwohnern (Stand ) im Département Haute-Loire in der Region Auvergne-Rhône-Alpes (vor 2016 Auvergne); sie gehört zum Arrondissement Le Puy-en-Velay und zum Kanton Emblavez-et-Meygal (bis 2015 Saint-Paulien).

Geographie
Lavoûte-sur-Loire liegt etwa neun Kilometer nordnordöstlich von Le Puy-en-Velay in der Landschaft Velay und an der Loire. Umgeben wird Lavoûte-sur-Loire von den Nachbargemeinden Saint-Vincent im Norden, Beaulieu im Osten und Nordosten, Malrevers im Südosten, Chaspinhac im Süden und Südosten, Polignac im Süden und Südwesten, Blanzac im Südwesten sowie Saint-Paulien im Westen und Nordwesten.

Bevölkerungsentwicklung

Verkehr
Lavoûte-sur-Loire hat einen Bahnhalt an der Bahnstrecke Saint-Georges-d'Aurac–Saint-Étienne-Châteaucreux und wird im Regionalverkehr von Zügen des TER Auvergne-Rhône-Alpes zwischen Le Puy-en-Velay und Saint-Étienne Châteaucreux bedient.

Sehenswürdigkeiten
 Kirche Saint-Maurice, seit 1944 Monument historique
 Schloss Lavoûte-Polignac,  seit 1967 Monument historique
 Ruine der alten Loire-Brücke

Persönlichkeiten
 Melchior de Polignac (1661–1741), Erzbischof von Auch (1725–1741), Kardinal (ab 1713)

Weblinks

Ort in Auvergne-Rhône-Alpes
Ort an der Loire

redpajama_set_name: RedPajamaWikipedia


text:
è un manga di Akira Toriyama edito nel 1979 su Weekly Shonen Jump.

In Italia è stato pubblicato da Star Comics nelle raccolte Toriyama World (volume 1) del 1999 e Akira Toriyama - Menu à la Carte (volume 2) nel 2012.

Trama
Nel commissariato di Yoikora sono tutti felici perché è il primo giorno di Tomato Akai, una ragazza diciottenne che vuole fare l'agente. Dopo essere arrivata in ritardo non ha scuse ed anzi incolpa la propria sveglia perché dormiva anche lei. Il caporeparto le ordina di cambiarsi in uno spogliatoio e non davanti a tutti come stava per fare, suscitando così la rabbia degli altri agenti. Dando prova di essere una frana come agente  viene affidata all'agente Slump il quale anziché prendere parte ad un caso di omicidio deve farle da guida nel territorio di competenza. Tomato combina danni anche in strada fino a quando Slump non riconosce Kura Akukenta, un ricercato per varie rapine e dice a Tomato di non perderlo di vista mentre lui lo riferisce al capo. Mentre Tomato segue il ladro appiccicatagli, questi prende in ostaggio un bambino. Tomato prende allora in ostaggio un gatto facendo rimanere Kura attonito, che prende uno scarafaggio mentre lei prende anche una formica in ostaggio. Grazie al temporeggiare di Tomato il ladro viene catturato, nel commissariato si fa un brindisi alla nuova agente ed in prigione Kura spazientisce i poliziotti mostrandogli altre formiche.

Note di produzione
Per le continue richieste da parte del direttore della rivista settimanale Shōnen Jump, Torishima, Akira Toriyama di disegnare un manga con protagonista una ragazza, venne creato Tomato Police Woman che fu pubblicato il 15 agosto 1979 proprio sulla Shōnen Jump. Grazie al successo di questo manga un paio d'anni dopo è uscito Dr. Slump & Arale.

Akira Toriyama


text:
Home Articles Injuries & Absences Expose WWE's Lack Of Depth On Top
Injuries & Absences Expose WWE's Lack Of Depth On Top
On Saturday night at the WWE live event in Madison, Wisconsin, two WWE Superstars suffered injuries during their matches on the show. The biggest star in the company, John Cena, suffered a swollen-shut eye as the result of a headbutt from his opponent that evening, Bray Wyatt. There has also been some talk that Cena may have suffered a concussion during the bout as well. If that is true, given WWE's policy on concussions, it's within the realm of possibility that Cena would be unable to perform at the company's upcoming Money In The Bank pay-per-view, which may already have a glaring absence with Daniel Bryan's status still a mystery.
The other Superstar who was injured on Saturday night, in a less severe incident, was Adam Rose. Rose suffered what was described as a bad gash above his right eye during his match on the card with Heath Slater.
The point to all of this is that when one or two top guys go down with injuries in WWE these days, considering the lack of depth at the top of the card, it really can have a dramatic effect on the company. The fact that WWE keeps the same few guys in the top spots means that if they go down with injuries, they don't really have anyone else that can be moved into those spots.
Batista "quit" the company on last Monday's RAW, as he will be taking his hiatus from the company to do some movie work. CM Punk walked out on the company. Undertaker only works once a year, and might even be done with that. Triple H only laces his boots up for the bigger pay-per-view events. Brock Lesnar only works a part-time schedule. As does Rob Van Dam. Guys like Christian, Rey Mysterio and other mid-card level talents are currently on the shelf.
Hell, even Roman Reigns nearly took himself out of the picture when he suffered a bad collision with Randy Orton during the company's recent European tour. He "only" suffered a gruesome gash above his eye, but nearly wound up with a concussion as well.
If WWE doesn't start getting more out of guys like Dolph Ziggler, Damien Sandow, Titus O'Neil, etc., they really put themselves at the mercy of the health of their top few guys. If they don't start moving guys like Cesaro, Bad News Barrett, Sheamus and others into higher profile positions, they risk the same thing there.
If you look up-and-down the current, active WWE roster, you will find that they aren't lacking in actual talent. They have the horses needed to win the big races. The only problem is, they pigeon hole a lot of their talented guys into goofy characters, or too many "stop-and-starts" where fans simply refuse to get behind them because they've been burnt too many times in the past.
There is almost no reason why a guy like Dolph Ziggler, for example, shouldn't be given some legitimate swings at the plate. His level of talent is so clearly obvious to the fans in particular, that it's almost a guarantee that he would deliver at any level he is given the opportunity to perform at. Yet, he gets none of these type of opportunities.
If Cena did suffer a concussion, with Money In The Bank only being a few weeks away, he technically shouldn't perform. Of course since we're talking about Cena, it's almost a guarantee that he will, even if medically speaking he shouldn't. But if he were to go down with something more serious, and if Bryan isn't going to be ready anytime soon, it's really tough to picture what WWE would do.
When you look at the very top baby faces in the company, the list isn't that long. Taking a hit with Cena, Bryan, and with Punk no longer in the picture, you're really left with guys who either aren't in position to carry the company as the top "good guy" act, or you're forced to rush someone like Roman Reigns into the spot long before he's actually ready for it. Even if he rises to the occasion and everything works out in this hypothetical scenario, it's still a waste of a long, slow-build that the company has been doing.
The bottom line is this: WWE has the talent. They have a number of gifted performers who should be doing a whole hell-of-a-lot more than they are doing right now. If they keep holding down guys who are capable of more, for seemingly no good reason other than "they can," they really are risking a lot with the injury-rate growing in recent months.
At the end of the day, WWE is going to do what they are going to do. In the end, they'll likely be just fine. The only thing is — they shouldn't be settling for just "fine." Just because there's no strong number two breathing down their neck, forcing them to deliver their best possible shows on a week-to-week basis, like they were forced to do during the Monday Night Wars, doesn't mean they shouldn't be trying to do that anyways.
I've always been of the opinion that WWE should just pretend it's 1998. Pretend like WCW is kicking their ass. They should pretend like if they don't deliver their best possible show imaginable on Monday night, it could all be over for them. That sense of urgency that they had during the Monday Night Wars resulted in them putting forth arguably the most entertaining era in history. Just because the landscape has changed and they are "the only game in town" doesn't mean they should be on "cruise control" every week, and that's exactly what it feels like they're doing to me.
What do you guys think? Leave your feedback in the "Comments" section below. You can also hit me up at Facebook.com/MattBooneWZR, and/or follow me on Twitter @MBoone420.
John Cena Makes Appearance During Official WrestleMania Announcement (VIDEO)
Monday Night Wars
Bobby Lashley Calls Riddle A 'Wannabe' Brock Lesnar, Wants A Match With Brock
$20,000 Award Being Offered By Batista Over Florida Manatee Cruelty
CM Punk Posts Graphic Throwback Photo From WWE Elimination Chamber Match
David Michael Bautista Jr.
He is an American actor, semi-retired professional wrestler, former mixed martial artist, and bodybuilder. From 2000 to 2010 and 2013 to 2014, he was signed to WWE under the ...


The dress is customized with a big panel from a vintage silk Japanese Obi with gorgeous tropical Japanese pattern.
The sides are decorated with refined antique lace from France and the center with antique metallic artwork pieces from Rajasthan/ India.
Also the backside is decorated with the same style of Indian metallic artwork.
This Dream Design is one of a kind and created with Love by Chilli at her Amsterdam studio.