Cysteine Residues C489 and C1135 of NaV 1.5 Play a Critical Role in Producing Late Sodium Current through nNOS-Dependent S-Nitrosylation of NaV 1.5

Direct S-nitrosylation of cysteine residues (SNO) in the cardiac sodium channel (NaV1.5) has been shown to increase late sodium current (INa) and has been implicated as a key biophysical mechanism underlying prolongation of the action potential in Long QT Syndromes type 9 and 12. The specific SNO site(s) of NaV1.5 that regulate increased late INa have not been established and we sought to determine if specific cysteine residue(s) in NaV1.5 mediate SNO-dependent increase of arrhythmogenic late INa. Cysteine residues were identified as candidates for S-nitrosylation by similarity to consensus sequences for S-nitrosylation and individual residues in NaV1.5 were replaced with alanine using site-directed mutagenesis (C280A, C374A, C489A, C649A, C683A, C686A, C1135A, C1166A, C1171A, and C1849A). Mutant or wild-type NaV1.5 was co-expressed in transfected HEK293 cells with the neuronal nitric oxide synthase (nNOS), and the adaptor protein α1-syntrophin (SNTA1), to optimize NaV1.5 nitrosylation. Using the whole-cell patch clamp technique, INa was recorded and late INa measured between 600-700ms as a percentage of peak INa. Wild-type NaV1.5 showed 0.80±0.07% (n=99) late INa at −20 mV which was decreased by the nNOS inhibitor L-NMMA to 0.24±0.05% (n=9). Only two Cys mutants showed reduced nitrosylation-dependent late INa; C489A (0.48±0.06%, n=32, p = 0.009) and C1135A (0.45±0.07%, n=29, p = 0.007). When the nNOS inhibitor L-NMMA was added to cells expressing C489A or C1135A the level of late INa was similar to L-NMMA treated wild-type (0.28±0.14%, n=7; 0.28±0.06%, n=8, respectively). The double mutation of C489A and C1135A showed no significant nitrosylation-dependent late INa (0.26±0.04%, n=22, p = 0.0008). These data provide evidence that two specific cysteine residues (C489 and C1135) on NaV1.5 account for nitrosylation-dependent late INa.