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Mechanism of voltage sensing in Shaker potassium channels

The S4 region is the main voltage sensing region in the 6TM (six transmembrane segment) voltage-gated channel family. Baker et al. (1998) introduced a cysteine residue into the middle of S4 (F370C) of the Shaker channel. They found that modification of this residue with MTSET resulted in the loss of channel currents. Did this mean that the voltage sensor was locked in the "deactivated" position? We have examined ionic currents and gating currents in channels with cysteine mutations in positions 370 to 374 of Shaker. In none of these are gating currents entirely abolished by MTSET applied to the intracellular membrane surface, but the voltage dependence of gating charge is shifted.

- Youshan Yang and Yangyang Yan
 
 


Conformational changes in the BK channel

The BK channel is a calcium-activated potassium channel.  Jiang et al. (2002) have hypothesized that a large conformational change in the "gating ring" formed by RCK domains in the MthK channel is produced by Ca2+ binding and channel activation. It is likely that the human BK channel has a similar structure. Using transposon-mediated insertions, we have inserted GFP variants into 52 different positions of the human BK cDNA. Of these 52 fluorescent fusion protein constructs, 20 result in surface-expressed, functional channels. We plan to use these constructs to test, through FRET, for large changes in distances between domains. - Teresa Giráldez, in a collaboration with the labs of Thom Hughes and David Zenisek.

 
Structure of BK channels

Negative stain is a technique in which macromolecules are trapped in a layer of dried heavy-element salt, for visualization in the electron microscope. The most popular stain is uranyl acetate, but it has a low pH of about 4.5. We have embedded purified, detergent-solubilized BK channel protein in ammonium molybdate. This stain allows the pH to be in the neutral range and Ca2+ concentration to be controlled. The resulting images have low contrast, but may allow a 3D reconstruction to be obtained.   - Christoph Cantener, in a collaboration with the lab of Ed Moczydlowski.

Structure of an IP3 receptor

The Type 1 IP3 receptor is an intracellular Ca2+-release channel that is abundant in the purkinje cells of the cerebellum, among other places. We have used single-particle, cryo-EM techniques to image the unliganded IP3 receptor from mouse cerebellum. The result was the first 3D structure of this protein, at a resolution of 24 Å. We are now pursuing a higher-resolution structure of the unliganded (closed) receptor, and also structures of the receptor with ligands bound that open the channel. - Qiu-Xing Jiang and David Chester, in a collaboration with the lab of Barbara Ehrlich.

 

Last modified: June 21, 2004 ns.

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