Substituted cysteine scanning in D1-S6 of the sodium channel hNav1.4 alters kinetics and structural interactions of slow inactivation.

Slow inactivation in voltage-gated Na+ channels (Navs) plays an important physiological role in excitable tissues (muscle, heart, nerves) and mutations that disrupt Nav slow inactivation can result in pathophysiologies (myotonia, arrhythmias, epilepsy). While the molecular mechanisms responsible for slow inactivation remain elusive, previous studies have suggested a role for the pore-lining D1-S6 helix. The goals of this research were to determine if (1) cysteine substitutions in D1-S6 affect gating kinetics and (2) methanethiosulfonate ethylammonium (MTSEA) accessibility changes in different kinetic states. Site-directed mutagenesis in the human skeletal muscle isoform hNav1.4 was used to substitute cysteine for eleven amino acids in D1-S6 from L433 to L443. Mutants were expressed in HEK cells and recorded from with whole-cell patch clamp. All mutations affected one or more baseline kinetics of the sodium channel, including activation, fast inactivation, and slow inactivation. Substitution of cysteine (for nonpolar residues) adjacent to polar residues destabilized slow inactivation in G434C, F436C, I439C, and L441C. Cysteine substitution without adjacent polar residues enhanced slow inactivation in L438C and N440C, and disrupted possible H-bonds involving Y437:D4 S4-S5 and N440:D4-S6. MTSEA exposure in closed, fast-inactivated, or slow-inactivated states in most mutants had little-to-no effect. In I439C, MTSEA application in closed, fast-inactivated, and slow-inactivated states produced irreversible reduction in current, suggesting I439C accessibility to MTSEA in all three kinetic states. D1-S6 is important for Nav gating kinetics, stability of slow-inactivated state, structural contacts, and state-dependent positioning. However, prominent reconfiguration of D1-S6 may not occur in slow inactivation.

Time- and state-dependent effects of methanethiosulfonate ethylammonium (MTSEA) exposure differ between heart and skeletal muscle voltage-gated Na(+) channels.

The substituted-cysteine scanning method (SCAM) is used to study conformational changes in proteins. Experiments using SCAM involve site-directed mutagenesis to replace native amino acids with cysteine and subsequent exposure to a methanethiosulfonate (MTS) reagent such as methanethiosulfonate ethylammonium (MTSEA). These reagents react with substituted-cysteines and can provide functional information about relative positions of amino acids within a protein. In the human heart voltage-gated Na(+) channel hNav1.5 there is a native cysteine at position C373 that reacts rapidly with MTS reagents resulting in a large reduction in whole-cell Na(+) current (I(Na)). Therefore, in order to use SCAM in studies in this isoform, this native cysteine is mutated to a non-reactive residue, e.g., tyrosine. This mutant, hNav1.5-C373Y, is resistant to the MTS-mediated decrease in I(Na). Here we show that this resistance is time- and state-dependent. With relatively short exposure times to MTSEA (<4min), there is little effect on I(Na). However, with longer exposures (4-8min), there is a large decrease in I(Na), but this effect is only found when hNav1.5-C373Y is inactivated (fast or slow) - MTSEA has little effect in the closed state. Additionally, this long-term, state-dependent effect is not seen in human skeletal muscle Na(+) channel isoform hNav1.4, which has a native tyrosine at the homologous site C407. We conclude that differences in molecular determinants of inactivation between hNav1.4 and hNav1.5 underlie the difference in response to MTSEA exposure.

Relative resistance to slow inactivation of human cardiac Na+ channel hNav1.5 is reversed by lysine or glutamine substitution at V930 in D2-S6.

Transmembrane segment 6 is implicated in slow inactivation (SI) of voltage-gated Na(+) channels (Na(v)s). To further study its role and understand differences between SI phenotypes of different Na(v) isoforms, we analyzed several domain 2-segment 6 (D2-S6) mutants of the human cardiac hNa(v)1.5, which is relatively resistant to SI. Mutants were examined by transient HEK cell transfection and patch-clamp recording of whole cell Na(+) currents. Substitutions with lysine (K) included N927K, V930K, and L931K. We show recovery from short (100 ms) depolarization to 0 mV in N927K and L931K is comparable to wild type, whereas recovery in V930K is delayed and biexponential, suggesting rapid entry into a slow-inactivated state. SI protocols confirm enhanced SI phenotype (rapid development, hyperpolarized steady state, slowed recovery) for V930K, contrasting with the resistant phenotype of wild-type hNa(v)1.5. This enhancement, not found in N927K or L931K, suggests that the effect in V930K is site specific. Glutamine (Q) substituted at V930 also exhibits an enhanced SI phenotype similar to that of V930K. Therefore, K or Q substitution eliminates hNa(v)1.5 resistance to SI. Alanine (A) or cysteine (C) substitution at V930 shows no enhancement of SI, and in fact, V930A and V930C, as well as L931K, exhibit a resistance to SI, demonstrating that characteristics of specific amino acids (e.g., size, hydrophobicity) differentially affect SI gating. Thus V930 in D2-S6 appears to be an important structural determinant of SI gating in hNa(v)1.5. We suggest that conformational change involving D2-S6 is a critical component of SI in Na(v)s, which may be differentially regulated between isoforms by other isoform-specific determinants of SI phenotype.

Slow-inactivation induced conformational change in domain 2-segment 6 of cardiac Na+ channel.

To examine conformational changes during slow inactivation involving domain 2-segment 6 (D2-S6) of human cardiac Na(+) channel (hNav1.5), we applied the substituted-cysteine accessibility method (SCAM) using methanethiosulfonate ethylammonium (MTSEA). We substituted cysteine (C) for native valine (V) at position 930 of D2-S6 in the MTSEA-resistant hNav1.5 mutant C373Y to produce the double mutant C373Y-V930C. Whole-cell Na(+) currents were recorded using patch-clamp techniques in transiently transfected HEK cells. In C373Y-V930C, we find that MTSEA (1.5 mM) applied in the closed state (-160 mV) has no significant effect on whole-cell Na(+) current, while MTSEA applied in the slow-inactivated state (prolonged depolarization at 0 mV) decreases current. We propose that D2-S6 in hNav1.5 undergoes molecular rearrangement during slow inactivation exposing the side chain of residue 930 such that it becomes accessible to modification by MTSEA.