MOLECULAR ANALYSIS OF THE PUTATIVE INACTIVATION PARTICLE IN THE INACTIVATION GATE OF BRAIN-TYPE IIA NA+ CHANNELS

Fast Na+ channel inactivation is thought to involve binding of phenyla lanine 1489 in the hydrophobic cluster IFM in LIII-IV of the rat brain type IIA Na+ channel. We have analyzed macroscopic and single channel currents from Na+ channels with mutations within and adjacent to hpdr ophobic clusters in LIII-IV. Substitution of F1489 by a series of amin o acids disrupted inactivation to differ ent extents. The degree of di sruption was closely correlated with the hydrophilicity of the amino a cid at position 1489. These mutations dramatically destabilized the in activated state and also significantly slowed the entry into the inact ivated state, consistent with tile idea that F1489 forms a hydrophobic interaction with a putative receptor during the fast inactivation pro cess. Substitution of a phe residue at position 1488 or 1490 in mutant s lacking F1489 did not restore normal inactivation, indicating that p recise location of F1489 is critical for its function. Mutations of T1 491 disrupted inactivation substantially, with large effects on the st ability of the inactivated state and smaller effects on tile rate of e ntry into the inactivated state. Mutations of several other hydrophobi c residues did not destabilize the inactivated state at depolarized po tentials, indicating that the effects of mutations at F1489 and T1491 are specific. The double mutant YY1497/8QQ slowed macroscopic inactiva tion at all potentials and accelerated recovery from inactivation at n egative membrane potentials. Some of these mutations in LIII-IV also a ffected the latency to first opening, indicating coupling between LIII -IV and channel activation. Our results show that the amino acid resid ues of the IFM hydrophobic cluster and the adjacent T1491 are unique i n contributing to the stability of the inactivated state, consistent w ith the designation of these residues as components of the inactivatio n particle responsible for fast inactivation of Na+ channels.