Cooperative effect of S4–S5 loops in domains D3 and D4 on fast inactivation of the Na⁺ channel

Abstract

Cytoplasmic S4-S5 loops have been shown to be involved in fast inactivation of voltagegated ion channels. We studied mutations in these loops and their potential cooperative effects in domains D3 (N1151C, A1152C, I1160C/A) and D4 (F1473C, L1482C/A) of the human skeletal muscle Na + channel α-subunit (hNa v 1.4) using expression in tsA201 cells and the whole cell patch-clamp technique. All cysteine mutations were accessible to intracellularly applied sulfhydryl reagents which considerably destabilized fast inactivation. For different combinations of corresponding D3/D4 double mutations, fast inactivation could be almost completely removed. Thermodynamic cycle analysis indicated an additive effect for N1151C/F1473C and a significant cooperative effect for I1160/L1482 double mutations. Application of oxidizing reagents such as Cu-phenanthroline to link two cysteines via a disulfide bridge did not reveal evidence for a direct physical interaction of cysteines in D3 and D4. In addition to the pronounced alterations of fast inactivation, mutations of I1160 shifted steady-state activation in the hyperpolarizing direction and slowed the kinetics of both activation and deactivation. Sulfhydryl reagents had charge-dependent effects on I1160C suggesting interaction with negative charges in another protein region. We conclude that fast inactivation of the Na + channel involves both S4-S5 loops in D3 and D4 in a cooperative manner. D3/S4-S5 also plays an important role in activation and deactivation.

Mechanisms and models of cardiac sodium channel inactivation

“…40,41 Confoundingly, accessibility studies with Na v 1.4 showed that both the DIII S4-S5 and DIV S4-S5 linkers remain accessible when channels are inactivated. 40,42 Mutagenesis work also aided in demonstrating that the S6 segments of DI, DII, and DIV modulate fast inactivation. An alanine-mutagenesis study showed that mutations in the center 43 and in intracellular 44 end of DIV S6 segment in Na v 1.2 inhibited inactivation.…”

“…47 The C-terminus also plays an important role in modulating Na C channel inactivation. 48 Generally, From N-terminal to C-terminal, the intracellular end of DI S6 segment (DWCW window), 45,47 intracellular end of DII S6 segment, 45 DIII S4-S5 linker, 38,39,42 DIII-DIV linker, [25][26][27][28][29][30][31][32][33] DIV S4-S5 linker, [40][41][42] lower half of DIV S6 segment, 44 , 47 and C-terminus. 23,[48][49][50][51]53 each Na C channel homolog has distinct fast inactivation kinetics.…”

Distinct modulation of inactivation by a residue in the pore domain of voltage-gated Na+ channels: mechanistic insights from recent crystal structures

“…1C ) raises the question whether some of the gating perturbations resulted from a direct interaction between DIV-S6 and the selectivity filter. Thermodynamic mutant cycle analysis has previously been performed to identify direct molecular interactions during inactivation 20 – 23 . We applied this method to the mutant cycles consisting of wild type, K1237E, single cysteine replacements in DIV-S6 and combination of the latter two constructs.…”

The Voltage-Dependent Sodium Channel Family

“…The DIV VSM activates with the slowest kinetics (Bosmans, Martin-Eauclaire, & Swartz, 2008). Its movement frees an intracellular linker called the inactivation gate that connects DIII helix S6 to DIV helix S1 ( Figure 1A) (Capes, Goldschen-Ohm, Arcisio-Miranda, Bezanilla, & Chanda, 2013) The inactivation gate contains a cluster of hydrophobic residues containing the amino acid sequence IFMT (the IFMT motif) that can now bind to a corresponding inactivation particle receptor lying within the S4-S5 linkers of DII, DIII, and DIV (Popa, Alekov, Bail, Lehmann-Horn, & Lerche, 2004). As a result, the inactivation gate occludes the pore and inactivates the channel within a few milliseconds of opening.…”