Mexiletine block of disease-associated mutations in S6 segments of the human skeletal muscle Na+ channel

Masanori P. Takahashi, Stephen C. Cannon

Research output: Contribution to journalArticle

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Abstract

1. Over twenty different missense mutations in the α-subunit of the adult skeletal muscle Na+ channel (hSkM1) have been identified as a cause of myotonia or periodic paralysis. We examined state-dependent mexiletine block for mutations involving the putative binding site in S6 segments (V445M, S804F, V1293I, V1589M and M1592V). Whole-cell Na+ currents were measured from wild-type (WT) and mutant channels transiently expressed in HEK cells. 2. Use-dependent block (10 ms pulses to - 10 mV, at 20 Hz) in 100 μM mexiletine was reduced modestly by mutations in IVS6 (V1589M, M1592V) and enhanced by the mutation in IS6 (V445M). For mutations in IIS6 (S804F) and IIIS6 (V1293I) use-dependent block was not statistically different from that of wild-type channels. 3. Resting-state block (10 ms pulses to - 10 mV from -150 mV, at 0.1 Hz) of S6 mutants was comparable to that of WT (dissociation constant for resting channels, KR = 650 ± 40 μM, n = 9). The S6 mutant with the greatest change in KR was V445M (KR = 794 ± 45 μM, n = 5), but this difference was only marginally significant (P = 0.047). 4. A modified technique for estimating local anaesthetic affinity of inactivated channels was developed to reduce errors due to slow inactivation and to failure of drug binding to reach equilibrium. Mexiletine affinity for inactivated channels was reduced by mutations in IVS6 (V1589M: dissociation constant for the inactivated state (KI) = 44.7 μM; M1592V: KI = 40.0 μM) and increased by the mutation in IS6 (V445M: KI = 15.0 μM), compared to wild-type channels (KI = 28.3 μM). 5. We conclude that the disease-associated S6 mutations in domains I-IV cause at most a 2-fold change in inactivated state affinity and have even less of an effect on resting block. Model simulations show that the reduced use-dependent block of IVS6 mutants derives primarily from an increased off-rate at hyperpolarized potentials, whereas the enhanced use-dependent block of the IS6 mutant was due to a higher affinity for inactivated V445M channels.

Original languageEnglish (US)
Pages (from-to)701-714
Number of pages14
JournalJournal of Physiology
Volume537
Issue number3
DOIs
StatePublished - Dec 15 2001

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Mexiletine
S 6
Skeletal Muscle
Mutation
Myotonia
Missense Mutation
Local Anesthetics
Paralysis
Binding Sites

ASJC Scopus subject areas

  • Physiology

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Mexiletine block of disease-associated mutations in S6 segments of the human skeletal muscle Na+ channel. / Takahashi, Masanori P.; Cannon, Stephen C.

In: Journal of Physiology, Vol. 537, No. 3, 15.12.2001, p. 701-714.

Research output: Contribution to journalArticle

Takahashi, Masanori P. ; Cannon, Stephen C. / Mexiletine block of disease-associated mutations in S6 segments of the human skeletal muscle Na+ channel. In: Journal of Physiology. 2001 ; Vol. 537, No. 3. pp. 701-714.
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N2 - 1. Over twenty different missense mutations in the α-subunit of the adult skeletal muscle Na+ channel (hSkM1) have been identified as a cause of myotonia or periodic paralysis. We examined state-dependent mexiletine block for mutations involving the putative binding site in S6 segments (V445M, S804F, V1293I, V1589M and M1592V). Whole-cell Na+ currents were measured from wild-type (WT) and mutant channels transiently expressed in HEK cells. 2. Use-dependent block (10 ms pulses to - 10 mV, at 20 Hz) in 100 μM mexiletine was reduced modestly by mutations in IVS6 (V1589M, M1592V) and enhanced by the mutation in IS6 (V445M). For mutations in IIS6 (S804F) and IIIS6 (V1293I) use-dependent block was not statistically different from that of wild-type channels. 3. Resting-state block (10 ms pulses to - 10 mV from -150 mV, at 0.1 Hz) of S6 mutants was comparable to that of WT (dissociation constant for resting channels, KR = 650 ± 40 μM, n = 9). The S6 mutant with the greatest change in KR was V445M (KR = 794 ± 45 μM, n = 5), but this difference was only marginally significant (P = 0.047). 4. A modified technique for estimating local anaesthetic affinity of inactivated channels was developed to reduce errors due to slow inactivation and to failure of drug binding to reach equilibrium. Mexiletine affinity for inactivated channels was reduced by mutations in IVS6 (V1589M: dissociation constant for the inactivated state (KI) = 44.7 μM; M1592V: KI = 40.0 μM) and increased by the mutation in IS6 (V445M: KI = 15.0 μM), compared to wild-type channels (KI = 28.3 μM). 5. We conclude that the disease-associated S6 mutations in domains I-IV cause at most a 2-fold change in inactivated state affinity and have even less of an effect on resting block. Model simulations show that the reduced use-dependent block of IVS6 mutants derives primarily from an increased off-rate at hyperpolarized potentials, whereas the enhanced use-dependent block of the IS6 mutant was due to a higher affinity for inactivated V445M channels.

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