Voltage-dependent open-state inactivation of cardiac sodium channels: gating current studies with Anthopleurin-A toxin.

Abstract

Journal of General Physiology. 106:601-616), we studied Na channel gating currents (Ig) in voltage-clamped single canine cardiac Purkinje cells at ~ 12~ Comparison of Ig recorded in response to step depolarizations before and after modification by Ap-A toxin showed that toxin-modified gating currents decayed faster and had decreased initial amplitudes. The predominate change in the charge-voltage (Q-V) relationship was a reduction in gating charge at positive potentials such that Q~ax was reduced by 33%, and the difference between charge measured in Ap-A toxin and in control represented the gating charge associated with Na channels undergoing inactivation by O-oi. By comparing the time course of channel activation (represented by the gating charge measured in Ap-A toxin) and gating charge associated with the O---~I transition (difference between control and Ap-A charge), the influence of activation on the time course of inactivation could be accounted for and the inherent voltage dependence of the O--~I transition determined. The O--*Itransition for cardiac Na channels had a valence of 0.75 e-. The total charge of the cardiac voltage-gated Na channel was estimated to be 5 e-. Because charge is concentrated near the opening transition for this isoform of the channel, the time constant of the O---)I transition at 0 mV could also be estimated (0.53 ms, ~ 12~ Prediction of the mean channel open time-voltage relationship based upon the magnitude and valence of the O---)Cand O---)Irate constants from INa and Ig data matched data previously reported from single Na channel studies in heart at the same temperature.

Outward stabilization of the voltage sensor in domain II but not domain I speeds inactivation of voltage-gated sodium channels

“…Although there are fewer studies on the discrete transitions in the activation processes of Na channels, the S4's from domains I, II, and III are thought to be principally involved in channel activation (2) while inactivation has been associated with the S4 in DIV (8,10,11,21,22,37,43,44). Studies of individual florescent-tagged voltage sensors in rat skeletal muscle Na channels (rSkM1, Na V 1.4) showed that the S4's in domains I-III all translocated rapidly during a step depolarization consistent with their role in the activation process (10).…”

“…I Na decay in WT channels is critically dependent upon inactivation from the open state (8,11,14,21,22,37,43,44); consequently the more rapid decay of I Na following stabilization of the DII-S4 suggests that inactivation has also become faster. Furthermore, the large negative shift in the V ½ of the steady-state Na channel availability curve after DII-S4 was stabilized also suggests it has a role in closed state inactivation, presumably by helping form or helping control Table 2).…”

“…It is possible that the presence of either the bound avidin and/or MTSEA-biotincap might obstruct or electrostatically affect the external vestibule, thereby affecting single-channel conductance. Another possible explanation for the decrease in G max is that stabilization of the DI-S4 secondarily affected the position of the DIV-S4, a voltage sensor that has been shown to be critically involved in Na channel inactivation (8,10,11,14,21,22,37,39,43,44). It has been reported that S4 movements in different domains of Na channels are cooperative, particularly between the S4's in domains I and IV (9).…”

“…For example, the outward movement of the fourth transmembrane segment in the fourth domain (DIV-S4) of the Na channel during a step depolarization has been associated with the Na channel kinetic transition from an open to a fast-inactivated state (8,11,21,22,43,44). Furthermore, the movement of the DIV-S4 during a depolarization is slower than the other S4's (10,37), and it contributes a large component (nearly 32%) to the maximal gating charge (Q max ) of the Na channel (10,39) with the outermost extracellular arginine (DIV-R1) estimated to contribute the most charge, ϳ20%, to Q max , while DIV-R2 and R3 contribute the remaining 12% (39).…”

Discovery and characterization of NaV modulatory venom peptides

“…Site-specific fluorescent labeling of the S4 segment of Na V 1.4 demonstrated the effect of stabilizing S4 of DI and DIV through α-scorpion toxin binding [236]. Both sea anemone and α-scorpion toxins have been shown to enhance the recovery of inactivation [232,237]. As the S4 region is responsible for voltagedependant gating, toxins that interact with this region are referred to as gating modifier toxins.…”