Effects of III-IV linker mutations on human heart Na+ channel inactivation gating.

Abstract

Na+ channel inactivation, a critical determinant of refractoriness, differs in cardiomyocytes and neurons. In rat brain type IIa (rB2a) Na+ channels, a critical residue in the cytoplasmic linker between domains III and IV regulates fast inactivation such that a Phe-->Gln substitution (F1489Q) inhibits inactivation by at least 85%. Since this residue is conserved in voltage-gated Na+ channels, we tested whether F1485Q, the analogous mutation in human heart (hH1a) Na+ channels, has a similar functional effect. We found that fast inactivation in wild-type (WT) channels expressed in Xenopus oocytes was complete within 15 milliseconds at a test potential of 0 mV, and its time course was biexponential with time constants of 0.4 and 2 milliseconds. But in contrast to rB2a, the FQ mutation inhibited inactivation by < 50% and increased mean single-channel open time by only twofold. Residual fast inactivation was monoexponential, with a time constant similar to that of the slower phase of normal inactivation (2 milliseconds). In the mutant channels, unlike WT, null tracings were absent at holding potentials in the range of -140 to -120 mV, and the voltage range of steady-state inactivation coincided exactly with that of activation, suggesting that residual inactivation was tightly coupled to the open state. As in rB2a, simultaneous mutations of I1484Q and M1486Q, in addition to mutation F1485Q, completely inhibited fast inactivation. Our results show that in heart Na+ channels, the IFM cluster controls the stability of both open- and closed-channel inactivation in a manner qualitatively similar to that in the brain. Structural differences in the putative inactivation receptor may explain the distinct gating patterns in channel subtypes.

Local anesthetics as effectors of allosteric gating. Lidocaine effects on inactivation-deficient rat skeletal muscle Na channels.

“…Whole-cell currents recorded from wildtype Na channels expressed in Xenopus oocytes decay rapidly, whereas FQ channels exhibit slower decay and a sizable persistent plateau current at the end of a 100 ms depolarizing pulse to Ϫ 20 mV (Fig. 1 A ), as shown previously (35) for the analogous human cardiac (hH1) Na channel mutant. The half-time of current decay (the interval from the current peak to 50% reduction of the current amplitude) was 5.9 Ϯ 2.3 ms in wild-type channels ( n ϭ 40) vs. 45.9 Ϯ 23.3 ms in FQ channels ( n ϭ 25, P Ͻ 0.0001).…”

“…Mean open times for FQ single channels were prolonged relative to wild-type (31,35,42), but were still well-described by a single exponential (31,42). Drug association with the open channel at a rate comparable to intrinsic wild-type inactivation should be apparent as a reduction in the mean open time.…”

Potent block of inactivation-deficient Na+ channels by n-3 polyunsaturated fatty acids

“…The mutation IFM/QQQ in the linker between the third and fourth domains disables inactivation of Na ϩ channels (6,16). IFM/QQQ expressed in human embryonic kidney (HEK)-293 cells has been used to test several clinically relevant Na ϩ channel blockers (6). However, we found that expression of this mutant (especially the persistent portion of I Na ) in HEK-293t cells was poor, which made it difficult to obtain meaningful results.…”

“…The intracellular linker between domains 3 and 4 is essential for the fast inactivation (i.e., closing) of the Na ϩ channel, and deletion of this region causes persistence of a Na ϩ current (I Na ) during depolarization (2). The mutation IFM/QQQ in the linker between the third and fourth domains disables inactivation of Na ϩ channels (6,16). IFM/QQQ expressed in human embryonic kidney (HEK)-293 cells has been used to test several clinically relevant Na ϩ channel blockers (6).…”

Enhancing effects of salicylate on quinidine-induced block of human wild type and LQT3 related mutant cardiac Na+ channels

“…The III-IV linker region that mediates the inactivation process is the most conserved region in various Na + channel isoforms (3,5,20,21,24), while the transmembrane segment S6 of domain IV is important for the resting state block by the local anesthetic etidocaine (11). In the present study, the salicylate-induced enhancement of the tonic block and UDB by quinidine showed voltage dependency but not isoform specificity, indicating that salicylate would increase the binding affinity of quinidine for inactivated Na + channel α subunits through the conserved region of the Na + channel.…”