Properties of Na+ currents conducted by a skeletal muscle L-type Ca2+ channel pore mutant (SkEIIIK)

摘要

Four glutamate residues residing at corresponding positions within the four conserved membrane-spanning repeat of L-type Ca²⁺ channels are important structural determinants for the passage of Ca²⁺ across the selectivity filter. Mutation of the critical glutamate in Repeat III in the α1S subunit of the skeletal L-type channel (Cav1.1) to lysine virtually eliminates passage of Ca²⁺ during step depolarizations. In this study, we examined the ability of this mutant Cav1.1 channel (SkEIIIK) to conduct inward Na⁺ current. When 150 mM Na⁺ was present as the sole monovalent cation in the bath solution, dysgenic (Cav1.1 null) myotubes expressing SkEIIIK displayed slowly-activating, non-inactivating, nifedipine-sensitive inward currents with a reversal potential (45.6 ± 2.5 mV) near that expected for Na⁺. Ca²⁺ block of SkEIIIK-mediated Na⁺ current was revealed by the substantial enhancement of Na⁺ current amplitude after reduction of Ca²⁺ in the external recording solution from 10 mM to near physiological 1 mM. Inward SkEIIIK-mediated currents were potentiated by either ±Bay K 8644 (10 µM) or 200-ms depolarizing prepulses to +90 mV. In contrast, outward monovalent currents were reduced by ±Bay K 8644 and were unaffected by strong depolarization, indicating a preferential potentiation of inward Na⁺ currents through the mutant Cav1.1 channel. Taken together, our results show that SkEIIIK functions as a non-inactivating, junctionally-targeted Na⁺ channel when Na⁺ is the sole monvalent cation present and urge caution when interpreting the impact of mutations designed to ablate Ca²⁺ permeability mediated by Cav channels on physiological processes that extend beyond channel gating and permeability.