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J. Gen. Physiol.,
Volume 112, Number 1, July 1, 1998 71-84
From the Department of Pathology, Anatomy and Cell Biology, Jefferson Medical College, Philadelphia, Pennsylvania 19107
Protein kinase C inhibits inactivation gating of Kv3.4 K+ channels, and at least two NH2-terminal
serines (S15 and S21) appeared involved in this interaction (. Neuron. 13:1403-1412). Here
we have investigated the molecular mechanism of this regulatory process. Site-directed mutagenesis (serine 
alanine) revealed two additional sites at S8 and S9. The mutation S9A inhibited the action of PKC by ~85%, whereas
S8A, S15A, and S21A exhibited smaller reductions (41, 35, and 50%, respectively). In spite of the relatively large
effects of individual S A mutations, simultaneous mutation of the four sites was necessary to completely abolish
inhibition of inactivation by PKC. Accordingly, a peptide corresponding to the inactivation domain of Kv3.4 was
phosphorylated by specific PKC isoforms, but the 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 phosphoserines is an important incentive to inhibit inactivation.
Consistent with this interpretation, the effects of S8D and S8E (E = Glu) were very similar, yet S8N (N = Asn) had
little effect on the onset of inactivation 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 additive effect of the free energies that the single mutations contribute to impair inactivation. These
observations demonstrate that the inactivation particle of Kv3.4 does not behave as a point charge and suggest
that the NH2-terminal phosphoserines interact in a cooperative manner to disrupt inactivation. Inspection of the
tertiary structure of the inactivation domain of Kv3.4 revealed the topography of the phosphorylation sites and
possible interactions that can explain the action of PKC on inactivation gating.
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