Ultra-slow inactivation in μ1 Na⁺ channels is produced by a structural rearrangement of the outer vestibule

While studying the adult rat skeletal muscle Na⁺ channel outer vestibule, we found that certain mutations of the lysine residue in the domain III P region at amino acid position 1237 of the α subunit, which is essential for the Na⁺ selectivity of the channel, produced substantial changes in the inactivation process. When skeletal muscle α subunits (μ1) with K1237 mutated to either serine (K1237S) or glutamic acid (K1237E) were expressed in Xenopus oocytes and depolarized for several minutes, the channels entered a state of inactivation from which recovery was very slow, i.e. the time constants of entry into and exit from this state were in the order of ~100 s. We refer to this process as 'ultra-slow inactivation.' By contrast, wild-type channels and channels with the charge-preserving mutation K1237R largely recovered within ~60 s, with only 20-30% of the current showing ultra-slow recovery. Coexpression of the rat brain β1 subunit along with the K1237E α subunit tended to accelerate the faster components of recovery from inactivation, as has been reported previously of native channels, but had no effect on the mutation-induced ultra-slow inactivation. This implied that ultra-slow inactivation was a distinct process different from normal inactivation. Binding to the pore of a partially blocking peptide reduced the number of channels entering the ultra-slow inactivation state, possibly by interference with a structural rearrangement of the outer vestibule. Thus, ultra-slow inactivation, favored by charge-altering mutations at site 1237 in μ1 Na⁺ channels, may be analogous to C-type inactivation in Shaker K⁺ channels.