INTRACELLULAR AND EXTRACELLULAR AMINO-ACIDS THAT INFLUENCE C-TYPE INACTIVATION AND ITS MODULATION IN A VOLTAGE-DEPENDENT POTASSIUM CHANNEL

The rate of C-type inactivation of the cloned voltage-gated potassium channel, Kv1.3, measured in membrane patches from Xenopus oocytes, inc reases when the patch is detached from the cell; the structural basis for this on-cell/off-cell change was examined. First, four serine and threonine residues, that are putative sites for phosphorylation by pro tein kinases A and C, were mutated to alanines. Mutating any one of th ese residues, or two or three of them simultaneously, does not elimina te the change in C-type inactivation. However, the basal rate of C-typ e inactivation in the cell-attached patch is markedly slower in the tr iple phosphorylation site mutant. Second; a homologous potassium chann el, Kv 1.6, does not exhibit the on-cell/off-cell change. When an extr acellular histidine at position 401 of Kv1.3 is replaced with tyrosine , the residue at the equivalent position (430) in Kv1.6, the resulting Kv1.3 H401Y mutant channel does not undergo the on-cell/off-cell chan ge. The results indicate that several potentially phosphorylatable int racellular amino acids influence the basal rate of C-type inactivation , but are not essential for the on-cell/off-cell change in inactivatio n kinetics. In contrast, an extracellular amino acid is critical for t his on-cell/off-cell change.