Voltage-Gated Sodium Channel Activity in Mouse Skeletal Muscle Fibers: Normal Gating and Defects Associated with Periodic Paralysis Mutants

dc.contributor.advisorCannon, Stephen C.en
dc.creatorFu, Yuen
dc.description.abstractMutations in SCN4A, the gene encoding the skeletal muscle Na+ channel (NaV1.4) α-subunit, cause several disorders related to skeletal muscle excitability. The functional consequences of these NaV1.4 mutations have been extensively characterized in heterologous expression systems. These studies have significantly advanced our understanding of the pathophysiology of these disorders. The in vivo functional consequences on channel activity, however, have yet to be defined. Animal models are now available in genetically engineered mice, which provide an opportunity to examine channel function in mature skeletal muscle. We optimized a two-electrode voltage clamp protocol to improve the fidelity of Na+ current recording from acutely dissociated intact muscle fibers. Computer simulation, incorporating measured capacitance and ionic current densities, was used to confirm sufficient voltage control and distortion-free Na+ currents. The gating properties of endogenous Na+ currents were measured and compared between two mouse strains, C57BL/6 and 129-E. The most dramatic finding was a hyperpolarized shift in the voltage dependence of activation (-25 mV) and fast inactivation (-18 mV) as compared to the studies in HEK293 cells expressing NaV1.4 plus the accessory β1-subunit. A possible contribution from NaV1.5 channels in the mouse muscle preparation was excluded by RT-PCR and TTX sensitivity. There was no significant difference in voltage dependence of fast gating between C57BL/6 and 129-E. The entry rate into slow inactivation was slower for Na+ channel in 129-E fibers; while the recovery from slow inactivation was similar between two mouse stains. Two NaV1.4 missense mutations associated with divergent clinical phenotypes - NaV1.4-M1592V in hyperkalemic periodic paralysis (HyperPP) and NaV1.4-R663H (homolog of human R669H) in hypokalemic periodic paralysis (HypoPP) - were characterized with voltage-clamp recordings in fully differentiated fibers from knock-in mutant mice. The NaV1.4-M1592V mutation produced gain-of-function defects, with the major changes being a slightly increased persistent current and moderately disrupted slow inactivation. In contrast, the HypoPP knock-in mutant R663H resulted in loss-of-function changes, due to an enhancement of inactivation, both fast and slow, and impaired activation. These observations provide important validation of prior findings using heterologous expression systems and yield quantitative information on the severity of the gating defects in mammalian skeletal muscles.en
dc.subjectParalysis, Hyperkalemic Periodicen
dc.subjectParalysis, Hypokalemic Periodicen
dc.subjectSodium Channelsen
dc.subjectMuscle, Skeletalen
dc.subjectMice, Knockouten
dc.titleVoltage-Gated Sodium Channel Activity in Mouse Skeletal Muscle Fibers: Normal Gating and Defects Associated with Periodic Paralysis Mutantsen
thesis.degree.departmentGraduate School of Biomedical Sciencesen
thesis.degree.grantorUT Southwestern Medical Centeren
thesis.degree.nameDoctor of Philosophyen


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