Interactions between Multiple Phosphorylation Sites in the Inactivation Particle of a K+ Channel

Abstract

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 (Covarrubias et al. 1994. 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.

Tyrosine Phosphorylation of the Inactivating Peptide of the Shaker B Potassium Channel: A Structural−Functional Correlate

“…In such a hypothetical case, tyrosine phosphorylation on the ShB peptide would be equivalent to the reported serine phosphorylation on the inactivation peptide of the rapidly inactivating K v 3.4 channel (22,24), and thus, it could have important consequences on the regulation of neuronal excitability. Moreover, similarly to the observations reported here regarding the resemblance between the effects of tyrosine phosphorylation and the L7E mutation in the ShB peptide, mutation of serine residues to aspartates in the K v 3.4 channel was found to mimic the action of protein kinase C (23). In any case, there is increasing evidence to support the fact that certain potassium channels exist in vivo as a complex with the kinases and phosphatases that regulate them (48)(49)(50), and therefore, covalent modification of these channels by phosphorylation at different sites, including the ball domains, appears as a likely candidate to further increase the already diverse functional patterns exhibited by the complex K + channel superfamily.…”

Kv3.4 channel function and dysfunction in nociceptors

“…7,8,[12][13][14]17,18 Moreover, Kv3.4 siRNA nearly ablates the high voltageactivated K C current in small-diameter DRG neurons. 7 Although fast-inactivating slowing of macroscopic inactivation in DRG nociceptors under normal conditions (Fig.…”

“…[12][13][14] Early reports showed modulation of fast Kv3.4 channel inactivation by oxidation, phosphorylation and PIP2 in heterologous expression systems. [15][16][17][18] Demonstrating that inactivation modulation also occurs in neurons, we showed that PKC activation dramatically slows inactivation of the native Kv3.4 channel expressed in DRG nociceptors. 7 Under physiological conditions, however, Kv3.4 channel inactivation is not modulated by PIP2.…”

“…To investigate these possibilities, it will be necessary to precisely determine how SCI alters the ensemble of ionic conductances that regulate spiking and shape the AP in different compartments of DRG neurons. In addition, while the molecular mechanism that governs the conversion of the Kv3.4 channel from A-type to delayed rectifier-type is known, 18,32 more work is necessary to understand how SCI induces downregulation of the Kv3.4 channel in DRG nociceptors. These investigations are crucial steps toward exploiting the Kv3.4 channel as a promising therapeutic target for persistent neuropathic pain.…”

Reduced Expression of A-Type Potassium Channels in Primary Sensory Neurons Induces Mechanical Hypersensitivity

“…This would convert a normally innocuous tactile stimulus turns into a noxious stimulus. In addition to reduced expression, I A s can also be suppressed if the channel becomes phosphorylated (Beck et al, 1998;Birnbaum et al, 2004;Sergeant et al, 2005). Phosphorylation of Kv4.2 in dorsal horn neurons has been proposed to underlie the induction of central sensitization, a cellular mechanism of neuropathic pain (Ji et al, 2003).…”