Gating behaviour of sodium currents in adult mouse muscle recorded with an improved two-electrode voltage clamp

The availability of knock-in mutant mouse models for channelopathies of skeletal muscle has generated the need for improved methods to record ionic currents under voltage clamp in fully differentiated adult muscle fibres. A two-electrode voltage clamp has been optimized for recording Na⁺ currents in small fibres dissociated from the footpad. Clamp speed and spatial homogeneity were achieved by using short fibres (<600 μm) that were detubulated with hyperosmolar glycerol. Series resistance errors were reduced by limiting current amplitude with low [Na⁺]. The quality of the voltage clamp was explored with computer simulations of a finite cable model with active conductances. Simulations quantitatively defined the range of conditions for which clamp control can be maintained, and provided estimates for the errors in the determination of gating parameters from standard pulse protocols. Sodium currents recorded from short fast-twitch muscles revealed a hyperpolarized shift in the voltage dependence of activation (V_1/2-52 mV) and fast inactivation (V_1/2-88 mV) compared to expression studies of Na_V1.4 in mammalian cell lines. Slow inactivation occurred at depolarized potentials (V_1/2-69 mV) relative to fast inactivation. These data reveal a marked divergence in the voltage dependence of fast and slow inactivation and provide normative values of Na⁺ channel behaviour for mouse skeletal muscle that will serve as a reference for the investigation of muscle ion channelopathies using genetically engineered mice or computer simulation.