Determinants of Voltage-Dependent Gating and Open-State Stability in the S5 Segment of Shaker Potassium Channels

Abstract

The best-known Shaker allele of Drosophila with a novel gating phenotype, Sh 5, differs from the wild-type potassium channel by a point mutation in the fifth membrane-spanning segment (S5) (Gautam, M., and M.A. Tanouye. 1990. Neuron. 5:67–73; Lichtinghagen, R., M. Stocker, R. Wittka, G. Boheim, W. Stühmer, A. Ferrus, and O. Pongs. 1990. EMBO [Eur. Mol. Biol. Organ.] J. 9:4399–4407) and causes a decrease in the apparent voltage dependence of opening. A kinetic study of Sh 5 revealed that changes in the deactivation rate could account for the altered gating behavior (Zagotta, W.N., and R.W. Aldrich. 1990. J. Neurosci. 10:1799–1810), but the presence of intact fast inactivation precluded observation of the closing kinetics and steady state activation. We studied the Sh 5 mutation (F401I) in ShB channels in which fast N-type inactivation was removed, directly confirming this conclusion. Replacement of other phenylalanines in S5 did not result in substantial alterations in voltage-dependent gating. At position 401, valine and alanine substitutions, like F401I, produce currents with decreased apparent voltage dependence of the open probability and of the deactivation rates, as well as accelerated kinetics of opening and closing. A leucine residue is the exception among aliphatic mutants, with the F401L channels having a steep voltage dependence of opening and slow closing kinetics. The analysis of sigmoidal delay in channel opening, and of gating current kinetics, indicates that wild-type and F401L mutant channels possess a form of cooperativity in the gating mechanism that the F401A channels lack. The wild-type and F401L channels' entering the open state gives rise to slow decay of the OFF gating current. In F401A, rapid gating charge return persists after channels open, confirming that this mutation disrupts stabilization of the open state. We present a kinetic model that can account for these properties by postulating that the four subunits independently undergo two sequential voltage-sensitive transitions each, followed by a final concerted opening step. These channels differ primarily in the final concerted transition, which is biased in favor of the open state in F401L and the wild type, and in the opposite direction in F401A. These results are consistent with an activation scheme whereby bulky aromatic or aliphatic side chains at position 401 in S5 cooperatively stabilize the open state, possibly by interacting with residues in other helices.

Molecular Compatibility of the Channel Gate and the N Terminus of S5 Segment for Voltage-gated Channel Activity

“…9B, bottom) and Leu 371 in S4. This structural arrangement is consistent with the strong impact on voltage sensitivity of mutating these amino acids, and with the assigned role of S5 in regulating the final steps in activation that leads to channel opening (16,48). Thus, our functional findings on a hybrid Kv/KcsA channel also question the structural arrangement that emerged from the KvAP crystals, and favor a more conventional type of conformation for Kv channels.…”

An Intersubunit Interaction between S4-S5 Linker and S6 Is Responsible for the Slow Off-gating Component in Shaker K+ Channels

“…dues Arg-394, Glu-395, Leu-398, and Tyr-485, which we will abbreviate as RELY interaction, stabilizing the activated state of the channel. Nevertheless, concerns that the slow off-gating component may not develop at physiological conditions need to be addressed as it had been suggested that the non-conducting phenotype of W434F Shaker channels is due to C-type inactivation and that the slow off-gating component arises from this feature (40,41), although results of other authors are more consistent with an open state stabilization than with C-type inactivation (11,13). In addition, it had been proposed that different permeant ions variably alter the kinetics of the off-gating currents (11,12,42).…”

“…The slow off-gating component has also been proposed to be dependent on the permeant ion (6,11,12) and was thought to be linked to C-type inactivation (11). Other authors, however, view it rather as a stabilization of the open state (13). Although theoretical descriptions of off-gating currents have been proposed (14 -16), the correlating structural determinants underlying these patterns remain unclear.…”

“…Several mutants of the Shaker K ϩ channel have been described previously to suppress the slow off-gating component (13,(17)(18)(19). In most cases, pore opening did not occur in the experimental range, confirming that the slow component is linked to pore opening (6,14).…”

Molecular Diversity and Regulation of Renal Potassium Channels

“…The S4 segment stands out by virtue of having 6 -9 positively charged amino acids (either lysine or arginine) out of ϳ20. Membrane depolarization is believed to open the channel through an S4-mediated conformational change (256,302,318,364,425,434,514). The recent X-ray structure of the voltage-gated K ϩ channel has shown that S1-S4 helices are attached to the pore region and the latter half of S3 and S4 form the voltage sensor (245,246).…”