Characterization of specific allosteric effects of the Na⁺ channel β1 subunit on the Na_v1.4 isoform

The mechanism of inactivation of mammalian voltage-gated Na⁺ channels involves transient interactions between intracellular domains resulting in direct pore occlusion by the IFM motif and concomitant extracellular interactions with the β1 subunit. Na_vβ1 subunits constitute single-pass transmembrane proteins that form protein-protein associations with pore-forming α subunits to allosterically modulate the Na⁺ influx into the cell during the action potential of every excitable cell in vertebrates. Here, we explored the role of the intracellular IFM motif of rNa_v1.4 (skeletal muscle isoform of the rat Na⁺ channel) on the α-β1 functional interaction and showed for the first time that the modulation of β1 is independent of the IFM motif. We found that: (1) Na_v1.4 channels that lack the IFM inactivation particle can undergo a "C-type-like inactivation" albeit in an ultraslow gating mode; (2) β1 can significantly accelerate the inactivation of Na_v1.4 channels in the absence of the IFM motif. Previously, we identified two residues (T109 and N110) on the β1 subunit that disrupt the α-β1 allosteric modulation. We further characterized the electrophysiological effects of the double alanine substitution of these residues demonstrating that it decelerates inactivation and recovery from inactivation, abolishes the modulation of steady-state inactivation and induces a current rundown upon repetitive stimulation, thus causing a general loss of function. Our results contribute to delineating the process of the mammalian Na⁺ channel inactivation. These findings may be relevant to the design of pharmacological strategies, targeting β subunits to treat pathologies associated to Na⁺ current dysfunction.