Elucidation of the mechanisms of gating in the Kv4.3 voltage-sensitive potassium channel.

摘要

The molecular and biophysical mechanisms by which Kv4 voltage-sensitive K+ channels respond to adjustments in membrane voltage are presently unresolved. With respect to inactivation gating, there is strong evidence that Shaker-like N- and P/C-type mechanisms are not involved. Kv4 channels also display prominent inactivation from pre-activated closed-states (closed-state inactivation, CSI), a process which is absent in Shaker (Kv1) channels. As in Shaker, voltage sensitivity in Kv4 is thought to be conferred by positively charged residues localized to the fourth transmembrane segment (S4) of the voltage-sensing domain. Kv1 channels possess four basic arginine residues (R1 - R4) that are responsible for carrying the majority of gating charge. In Kv4 channels, however, R1 is replaced by a neutral valine at position 287. In the absence of confirmed mechanisms underlying several gating transitions in Kv4.3, I hypothesized that the S4 voltage sensor domain may serve a primary regulatory role, specifically for the processes of closed-state inactivation and recovery. To test this hypothesis I analyzed the effects of charge elimination at positions 290, 293, and 296 (R2 - R4 using Shaker nomenclature) by mutation to the uncharged residue alanine (A). The R to A mutants eliminated individual positive charge while significantly reducing side chain volume and hydrophilic character. Their novel effects on gating may thus have been the result of electrostatic and/or structural perturbations. To address this issue, I next comparatively analyzed arginine to glutamine (R to Q) mutations at the same three positions. This maneuver maintained positive charge elimination of the R to A mutants while partially restoring native side chain volume and hydrophilic properties. To test whether the lack of charge at position 287 was responsible for noted differences in voltage sensitivity between Kv1 and Kv4.3, I next examined the role of charge addition at the site by mutation to arginine. With all three studies implicating a primary role for the S4 voltage sensor in regulating CSI and recovery, I examined these processes in greater detail through application of elevated extracellular potassium in the presence or absence of KChIP2b. Lastly, I explored the importance of potential electrostatic interactions between S2 and S3 negatively charged residues and positively charged K299 and R302 in S4. Through these studies I conclude that the S4 domain in Kv4.3 is responsible for regulating not only activation and deactivation processes, but also those of closed-state inactivation and recovery. In contrast to Shaker channels, closed-state inactivation appears to possess inherent voltage-dependence, or is uniquely coupled to activation. With the kinetics of deactivation and recovery processes paralleled across the range of conditions analyzed, I suggest that these processes are likely coupled. Finally, it is suggested that S4 may be oriented in the transmembrane electrical field unique from its position in Shaker, so that the transmembrane electrical field resides across R290 in the resting state. Taken together, these results support the argument that a more complicated gating model exists in Kv4.3 as compared to Kv1 channels, and that the regulation of this gating is determined largely by the S4 voltage sensor domain.