Amino acid residues required for fast Na(+)-channel inactivation: charge neutralizations and deletions in the III-IV linker.

The cytoplasmic linker connecting domains III and IV of the voltage-gated Na+ channel is thought to be involved in fast inactivation. This linker is highly conserved among the various Na+ channels that have been cloned. In the rat brain IIA Na+ channel, it consists of 53 amino acids of which 15 are charged. To investigate the role of this linker in inactivation, we mutated all 15 of the charged residues in various combinations. All but one of these mutants expressed functional channels, and all of these inactivated with kinetics similar to the wild-type channel. We then constructed a series of deletion mutations that span the III-IV linker to determine if any region of the linker is essential for fast inactivation. Deletion of the first 10 amino acids completely eliminated fast inactivation in the channel, whereas deletion of the last 10 amino acids had no substantial effect on inactivation. These results demonstrate that some residues in the amino end of the III-IV linker are critical for fast Na(+)-channel inactivation, but that the highly conserved positively charged and paired negatively charged residues are not essential.