INTERACTIONS BETWEEN MULTIPLE PHOSPHORYLATION SITES IN THE INACTIVATION PARTICLE OF A K- INSIGHTS INTO THE MOLECULAR MECHANISM OF PROTEIN-KINASE-C ACTION( CHANNEL )

Protein kinase C inhibits inactivation gating of Kv3.4 K+ channels, an d at least two NH2-terminal serines (S15 and S21) appeared involved in this interaction (Covarrubias et al. 1994. Neuron. 13:1403-1412). Her e we hale investigated the molecular mechanism of this regulatory proc ess. Site-directed mutagenesis (serine --> alanine) revealed two addit ional sites at S8 and S9. The mutation S9A inhibited the action of PKC by similar to 85%, whereas S8A, S15A, and S21A exhibited smaller redu ctions (41, 35, and 50%, respectively). In spite of the relatively lar ge effects of individual S --> A mutations, simultaneous mutation of t he four sites was necessary to completely abolish inhibition of inacti vation by PKC. Accordingly, a peptide corresponding to the inactivatio n domain of Kv3.4 was phosphorylated by specific PE;C isoforms, but th e mutant peptide (S[8,9,15,21]A) was not. Substitutions of negatively charged aspartate (D) for serine at positions 8, 9, 15, and 21 closely mimicked the effect of phosphorylation on channel inactivation. S --> D mutations slowed the rate of inactivation and accelerated the rate of recovery from inactivation. Thus, the negative charge of the phosph oserines is an important incentive to inhibit inactivation. Consistent with this interpretation, the effects of S8D and S8E (E = Glu) were v ery similar, yet S8N (N = Asn) had little effect on the onset of inact ivation but accelerated the recovery from inactivation. Interestingly, the effects of single S --> D mutations were unequal and the effects of combined mutations were greater than expected assuming a simple add itive effect of the free energies that the single mutations contribute to impair inactivation. These observations demonstrate that the inact ivation particle of Kv3.4 does not behave as a point charge and sugges t that the NH2-terminal phosphoserines interact in a cooperative manne r to disrupt inactivation. Inspection of the tertiary; structure of th e inactivation domain of Kv3.4 revealed the topography of the phosphor ylation sites and possible interactions that can explain the action of PKC on inactivation gating.