Suppression of Neuronal and Cardiac Transient Outward Currents by Viral Gene Transfer of Dominant-Negative Kv4.2 Constructs

Abstract

To probe the molecular identity of transient outward (A-type) potassium currents, we expressed a truncated version of Kv4.2 in heart cells and neurons. The rat Kv4.2-coding sequence was truncated at a position just past the first transmembrane segment and subcloned into an adenoviral shuttle vector downstream of a cytomegalovirus promoter (pE1Kv4.2ST). We hypothesized that this construct would act as a dominant-negative suppressor of currents encoded by the Kv4 family by analogy to Kv1 channels. Cotransfection of wild-type Kv4.2 with a beta-galactosidase expression vector in Chinese hamster ovary (CHO)-K1 cells produced robust transient outward currents (Ito) after two days (14.0 pA/pF at 50 mV, n = 5). Cotransfection with pE1Kv4.2ST markedly suppressed the Kv4.2 currents (0.8 pA/pF, n = 6, p < 0.02; cDNA ratio of 2:1 Kv4.2ST:wild type), but in parallel experiments, it did not alter the current density of coexpressed Kv1.4 or Kv1.5 channels. Kv4.2ST also effectively suppressed rat Kv4.3 current when coexpressed in CHO-K1 cells. We then engineered a recombinant adenovirus (AdKv4.2ST) designed to overexpress Kv4.2ST in infected cells. A-type currents in rat cerebellar granule cells were decreased two days after AdKv4. 2ST infection as compared with those infected by a beta-galactosidase reporter virus (116.0 pA/pF versus 281.4 pA/pF in Ad beta-galactosidase cells, n = 8 each group, p < 0.001). Likewise, Ito in adult rat ventricular myocytes was suppressed by AdKv4.2ST but not by Adbeta-galactosidase (8.8 pA/pF versus 21.4 pA/pF in beta-galactosidase cells, n = 6 each group, p < 0.05). Expression of a GFP-Kv4.2ST fusion construct enabled imaging of subcellular protein localization by confocal microscopy. The protein was distributed throughout the surface membrane and intracellular membrane systems. We conclude that genes from the Kv4 family are the predominant contributors to the A-type currents in cerebellar granule cells and Ito in rat ventricle. Overexpression of dominant-negative constructs may be of general utility in dissecting the contributions of various ion channel genes to excitability.