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Kv4.3, with its complex open- and closed-state inactivation (CSI) characteristics, is a primary contributor to early cardiac repolarization. The two alternatively spliced forms, Kv4.3-short (Kv4.3-S) and Kv4.3-long (Kv4.3-L), differ by the presence of a 19-amino acid insert downstream from the sixth transmembrane segment. The isoforms are similar kinetically; however, the longer form has a unique PKC phosphorylation site. To test the possibility that inactivation is differentially regulated by phosphorylation, we expressed the Kv4.3 isoforms in Xenopus oocytes and examined changes in their inactivation properties after stimulation of PKC activity. Whereas there was no difference in open-state inactivation, there were profound differences in CSI. In Kv4.3-S, PMA reduced the magnitude of CSI by 24% after 14.4 s at -50 mV. In contrast, the magnitude of CSI in Kv4.3-L increased by 25% under the same conditions. Mutation of a putatively phosphorylated threonine (T504) to aspartic acid within a PKC consensus recognition sequence unique to Kv4.3-L eliminated the PMA response. The change in CSI was independent of the intervention used to increase PKC activity; identical results were obtained with either PMA or injected purified PKC. Our previously published 11-state model closely simulated our experimental data. Our data demonstrate isoform-specific regulation of CSI by PKC in Kv4.3 and show that the carboxy terminus of Kv4.3 plays an important role in regulation of CSI.

The molecular and biophysical mechanisms by which voltage-sensitive K(+) (Kv)4 channels inactivate and recover from inactivation are presently unresolved. There is a general consensus, however, that Shaker-like N- and P/C-type mechanisms are likely not involved. Kv4 channels also display prominent inactivation from preactivated closed states [closed-state inactivation (CSI)], a process that appears to be absent in Shaker channels. As in Shaker channels, voltage sensitivity in Kv4 channels is thought to be conferred by positively charged residues localized to the fourth transmembrane segment (S4) of the voltage-sensing domain. To investigate the role of S4 positive charge in Kv4.3 gating transitions, we analyzed the effects of charge elimination at each positively charged arginine (R) residue by mutation to the uncharged residue alanine (A). We first demonstrated that R290A, R293A, R296A, and R302A mutants each alter basic activation characteristics consistent with positive charge removal. We then found strong evidence that recovery from inactivation is coupled to deactivation, showed that the precise location of the arginine residues within S4 plays an important role in the degree of development of CSI and recovery from CSI, and demonstrated that the development of CSI can be sequentially uncoupled from activation by R296A, specifically. Taken together, these results extend our current understanding of Kv4.3 gating transitions.

In cardiac-specific Na(+)-Ca(2+) exchanger (NCX) knockout (KO) mice, the ventricular action potential (AP) is shortened. The shortening of the AP, as well as a decrease of the L-type Ca(2+) current (I(Ca)), provides a critical mechanism for the maintenance of Ca(2+) homeostasis and contractility in the absence of NCX (Pott C, Philipson KD, Goldhaber JI. Excitation-contraction coupling in Na(+)-Ca(2+) exchanger knockout mice: reduced transsarcolemmal Ca(2+) flux. Circ Res 97: 1288-1295, 2005). To investigate the mechanism that underlies the accelerated AP repolarization, we recorded the transient outward current (I(to)) in patch-clamped myocytes isolated from wild-type (WT) and NCX KO mice. Peak I(to) was increased by 78% and decay kinetics were slowed in KO vs. WT. Consistent with increased I(to), ECGs from KO mice exhibited shortened QT intervals. Expression of the I(to)-generating K(+) channel subunit Kv4.2 and the K(+) channel interacting protein was increased in KO. We used a computer model of the murine AP (Bondarenko VE, Szigeti GP, Bett GC, Kim SJ, and Rasmusson RL. Computer model of action potential of mouse ventricular myocytes. Am J Physiol Heart Circ Physiol 287: 1378-1403, 2004) to determine the relative contributions of increased I(to), reduced I(Ca), and reduced NCX current (I(NCX)) on the shape and kinetics of the AP. Reduction of I(Ca) and elimination of I(NCX) had relatively small effects on the duration of the AP in the computer model. In contrast, AP repolarization was substantially accelerated when I(to) was increased in the computer model. Thus, the increase in I(to), and not the reduction of I(Ca) or I(NCX), is likely to be the major mechanism of AP shortening in KO myocytes. The upregulation of I(to) may comprise an important regulatory mechanism to limit Ca(2+) influx via a reduction of AP duration, thus preventing Ca(2+) overload in situations of reduced myocyte Ca(2+) extrusion capacity.