Regulation of Kv4.3 Closed-State Inactivation and Recovery by Extracellular Potassium and Intracellular KChIP2b

Abstract

Mechanisms underlying Kv4 channel inactivation and recovery are presently unclear, although there is general consensus that the basic characteristics of these processes are not consistent with Shaker (Kv1) N- and P/C-type mechanisms. Kv4 channels also differ from Shaker in that they can undergo significant inactivation from pre-activated closed-states (closed-state inactivation, CSI), and that inactivation and recovery kinetics can be regulated by intracellular KChIP2 isoforms. To gain insight into the mechanisms regulating Kv4.3 CSI and recovery, we have analyzed the effects of increasing [K(+)](o) from 2 mM to 98 mM in the absence and in the presence of KChIP2b, the major KChIP2 isoform expressed in the mammalian ventricle. In the absence of KChIP2b, high [K(+)](o) promoted Kv4.3 inactivated closed-states and significantly slowed the kinetics of recovery from both macroscopic and closed-state inactivation. Coexpression of KChIP2b in 2 mM [K(+)](o) promoted non-inactivated closed-states and accelerated the kinetics of recovery from both macroscopic and CSI. In high [K(+)](o), KChIP2b eliminated or significantly reduced the slowing effects on recovery. Attenuation of CSI by the S4 charge-deletion mutant R302A, which produced significant stabilization of non-inactivated closed-states, effectively eliminated the opposing effects of high [K(+)](o) and KChIP2b on macroscopic recovery kinetics, confirming that these results were due to alterations of CSI. Elevated [K(+)](o) therefore slows Kv4.3 recovery by stabilizing inactivated closed-states, while KChIP2b accelerates recovery by destabilizing inactivated closed-states. Our results challenge underlying assumptions of presently popular Kv4 gating models and suggest that Kv4.3 possesses novel allosteric mechanisms, which are absent in Shaker, for coupling interactions between intracellular KChIP2b binding motifs and extracellular K(+)-sensitive regulatory sites.

KV4.3 Expression and gating: S2 and S3 acidic and S4 innermost basic residues

“…11 While much is understood about basic mechanisms underlying activation, inactivation, and recovery in Shaker/K V 1.4, [12][13][14][15][16][17][18][19][20][21] corresponding mechanisms in K V 4 are presently undetermined. [3][4][5] There is nonetheless general [3][4][5][22][23][24] (although not universal [25][26][27] ) consensus that "conventional" Shaker N-and C-type inactivation processes [12][13][14][15][16][17] are not operative in K V 4. In particular, K V 4 displays prominent closed state inactivation (CSI) at hyperpolarized ("subthreshold") potentials, [3][4][5]22,24,[28][29][30][31][32][33] while CSI is minimal to absent in Shaker/K V 1.4.…”

KV4.3 N-terminal deletion mutant D2-39

“…In particular, K V 4 channels: (i) Display significant voltage-dependent closed state inactivation (CSI) (reviewed in refs. 2, 5 and 7); (ii) Are distinctly regulated by [ 30,31 and (iii) Are significantly modulated by KChIPs undergo subtle use-and/or state-dependent interactions with K V 4 channel domains that would not be detected in crystallographic studies. Prior evidence has also been presented 45,46 for putative KChIP binding sites existing in K V 4.2 C-terminal domains (see Discussion).…”

“…To begin reassessment of these issues, we used Xenopus oocytes and two-microelectrode voltage clamp to analyze effects of the K V 4.3 N-terminal deletion mutant Δ2-39 under basal conditions and in the presence of KChIP2b. 2,31,[47][48][49] Emphasis was placed upon characterizing effects of Δ2-39 on macroscopic (open state) versus CSI and recovery. In accord with some prior K V 4.2 studies, 35,36 our Δ2-39 results provide evidence suggestive of an N-type-like inactivation mechanism existing in K V 4.3.…”

The "structurally minimal" isoform KChIP2d modulates recovery of Kv4.3 N-terminal deletion mutant <meta charset="utf-8" /> <span class="s2">Δ</span>2-39

“…1, inset). Beginning at an interpulse interval of Dt = 60 msec (so as to "bypass" this initial complication of sigmoidicity 14 ), the constrained exponential best fit to the mean D2-39 + KChIP 2d data points resulted in an estimated recovery time constant (trec = 120 msec) that was very similar to that of D2-39 + KChiP studies 2,[9][10][11][12] indicates that deletion of the K v 4.3 proximal N-terminal, while mimicking the slowing effect of KChIP 2b on macroscopic inactivation, [15][16][17] does not mimic the acceleratory effects of KChIP 2b on recovery. The second result (which is in contrast to previous K v 4.2 studies 2,10,11 ) indicates that an intact N-terminal proximal domain is not obligatory for KChIP2 isoforms to exert acceleratory effects upon K v 4 channel recovery.…”

“…Based upon these prior studies, [14][15][16][17]23 we thus wondered: Is KChiP 2d also capable of accelerating D2-39 recovery?…”