fig7: Voltage dependence of deactivation time constants predicted by fitting the data for mutant I781T to the backward activation pathway: O⇄A⇄R. Deactivation time constant (solid line) was calculated by Eq. 2.2 (rate constants are indicated in Tables II and III). The model predicts an increase of the deactivation time constants, whereas the experimentally measured time constants decreased with increasing hyperpolarization (open circles connected by broken line).

Mentions: The backward pathway of Scheme 1 failed, however, to describe the acceleration of deactivation at more negative voltages. In such a scenario, the channel would have to close by transition O→A before returning to the resting state (A→R). If strong hyperpolarization accelerates the downward movement of voltage sensor (y(V)→∞), then voltage-independent pore closure serves as the rate-limiting stage preventing acceleration of deactivation. At negative potentials (V < −80 mV) this predicts a time constant τ(V)→1β (deduced from Eq. 2.2) that does not reproduce our experimental findings (see Fig. 7).

Beyl S, Kügler P, Kudrnac M, Hohaus A, Hering S, Timin E - J. Gen. Physiol. (2009)

Bottom Line: Rate constants were determined for 16-channel constructs assuming that pore mutations in IIS6 do not affect the activating transition of the voltage-sensing machinery (x(V) and y(V)).The model failed to reproduce current kinetics of mutation A780P that was, however, accurately fitted with individually adjusted x(V) and y(V).We speculate that structural changes induced by a proline substitution in this position may disturb the voltage-sensing domain.

Affiliation: Department of Pharmacology and Toxicology, University of Vienna, 1090 Vienna, Austria.

Abstract: Point mutations in pore-lining S6 segments of CaV1.2 shift the voltage dependence of activation into the hyperpolarizing direction and significantly decelerate current activation and deactivation. Here, we analyze theses changes in channel gating in terms of a circular four-state model accounting for an activation R-A-O and a deactivation O-D-R pathway. Transitions between resting-closed (R) and activated-closed (A) states (rate constants x(V) and y(V)) and open (O) and deactivated-open (D) states (u(V) and w(V)) describe voltage-dependent sensor movements. Voltage-independent pore openings and closures during activation (A-O) and deactivation (D-R) are described by rate constants alpha and beta, and gamma and delta, respectively. Rate constants were determined for 16-channel constructs assuming that pore mutations in IIS6 do not affect the activating transition of the voltage-sensing machinery (x(V) and y(V)). Estimated model parameters of 15 CaV1.2 constructs well describe the activation and deactivation processes. Voltage dependence of the "pore-releasing" sensor movement ((x(V)) was much weaker than the voltage dependence of "pore-locking" sensor movement (y(V)). Our data suggest that changes in membrane voltage are more efficient in closing than in opening CaV1.2. The model failed to reproduce current kinetics of mutation A780P that was, however, accurately fitted with individually adjusted x(V) and y(V). We speculate that structural changes induced by a proline substitution in this position may disturb the voltage-sensing domain.