Allosteric effects of calcium and voltage on the gating of a calcium sensitive potassium channel.

다국어 초록 (Multilingual Abstract)

The cloned potassium channel <italic>mSlo</italic> is sensitive to intracellular calcium as well as to changes in membrane voltage. In this dissertation, I have examined the effects of both calcium and voltage on the gating of single molecules of <italic>mSlo</italic> heterologously expressed in <italic>Xenopus laevis</italic> oocytes. Using the inside out voltage clamp recording technique, I have examined the single channel kinetics of <italic>mSlo</italic> to determine the complexity of its gating in the absence of any intracelluar calcium. I have found that the intrinsic voltage dependence of <italic>mSlo</italic> is quite complex, and that even in the absence of calcium the channel is able to occupy multiple closed and open conformational states. I extended this study of channel gating in zero calcium by examining gating behavior in single channels composed of subunits with non-identical voltage sensors. I was able to form these heteromeric channels by coexpressing wildtype <italic>mSlo</italic> channels with the R207Q point mutant. This point mutation neutralizes a residue in the S4 transmembrane region of the subunit, and its result on gating is to shift the equilibrium of voltage sensor activation toward negative voltages. By examining the behavior of channels composed of both wildtype and R207Q subunits, I was able to test the possibility of cooperative interactions between voltage sensors in this channel. My results indicate that the voltage sensors of <italic>mSlo</italic> do not show a strong evidence of cooperativity during channel gating. Similarly, I investigated the possibility of cooperative interactions between the calcium sensors of <italic>mSlo</italic> by studying the gating of single molecules of <italic>mSlo</italic> with non-identical calcium sensors. I formed these heteromers by coexpressing wildtype subunits with chimeric subunits. The chimeric subunits were formed from the core of <italic>mSlo1 </italic> and the C-terminus of <italic>mSlo3</italic>, a large conductance potassium channel that is voltage dependent but shows no sensitivity to changes in intracellular calcium. My results with these calcium sensor heteromultimers show that the calcium sensors of <italic>mSlo</italic> also act independently during channel gating.