Intracellular β-nicotinamide adenine dinucleotide inhibits the skeletal muscle ClC-1 chloride channel.
ClC-1 is the dominant sarcolemmal chloride channel and plays an important role in regulating membrane excitability that is underscored by ClC-1 mutations in congenital myotonia. Here we show that the coenzyme β-nicotinamide adenine dinucleotide (NAD), an important metabolic regulator, robustly inhibits ClC-1 when included in the pipette solution in whole cell patch clamp experiments and when transiently applied to inside-out patches. The oxidized (NAD(+)) form of the coenzyme was more efficacious than the reduced (NADH) form, and inhibition by both was greatly enhanced by acidification. Molecular modeling, based on the structural coordinates of the homologous ClC-5 and CmClC proteins and in silico docking, suggest that NAD(+) binds with the adenine base deep in a cleft formed by ClC-1 intracellular cystathionine β-synthase domains, and the nicotinamide base interacts with the membrane-embedded channel domain. Consistent with predictions from the models, mutation of residues in cystathionine β-synthase and channel domains either attenuated (G200R, T636A, H847A) or abrogated (L848A) the effect of NAD(+). In addition, the myotonic mutations G200R and Y261C abolished potentiation of NAD(+) inhibition at low pH. Our results identify a new biological role for NAD and suggest that the main physiological relevance may be the exquisite sensitivity to intracellular pH that NAD(+) inhibition imparts to ClC-1 gating. These findings are consistent with the reduction of sarcolemmal chloride conductance that occurs upon acidification of skeletal muscle and suggest a previously unexplored mechanism in the pathophysiology of myotonia.
Biota Structural Biology Laboratory, St. Vincent's Institute of Medical Research, Fitzroy, Victoria 3065, Australia. bbennetts@svi.edu.au
Brett
B
Bennetts
Yawei
Y
Yu
Tsung-Yu
TY
Chen
Michael W
MW
Parker
D000255:Adenosine Triphosphate / Q000494:pharmacology / Q000502:physiology; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D018118:Chloride Channels / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D003198:Computer Simulation; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007424:Intracellular Fluid; D007700:Kinetics; D008564:Membrane Potentials; D008958:Models, Molecular; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000166:cytology / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009243:NAD / Q000494:pharmacology / Q000502:physiology*; D009865:Oocytes / Q000378:metabolism / Q000502:physiology; D018408:Patch-Clamp Techniques; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D014981:Xenopus
C086321:CLC-1 channel; D018118:Chloride Channels; D009243:NAD; D000255:Adenosine Triphosphate