SUPPRESSION OF NEURONAL AND CARDIAC TRANSIENT OUTWARD CURRENTS BY VIRAL GENE-TRANSFER OF DOMINANT-NEGATIVE KV4.2 CONSTRUCTS

To probe the molecular identity of transient outward (A-type) potassiu m currents, we expressed a truncated version of Kv4.2 in heart cells a nd neurons. The rat Kv4.2 coding sequence was truncated at a position just past the first transmembrane segment and subcloned into an adenov iral shuttle vector downstream of a cytomegalovirus promoter (pE1Kv4.2 ST). We hypothesized that this construct would act as a dominant-negat ive 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 ro bust transient outward currents (I-to) after two days (14.0 pA/pF at 5 0 mV, n = 5). Cotransfection with pE1Kv4.2ST markedly suppressed the K v4.2 currents (0.8 pA/pF, n = 6, p < 0.02; cDNA ratio of 2:1 Kv4.2ST:w ild type), but in parallel experiments, it did not alter the current d ensity of coexpressed Kv1.4 or Kv1.5 channels, Kv4.2ST also effectivel y suppressed rat Kv4.3 current when coexpressed in CHO-K1 cells. We th en engineered a recombinant adenovirus (AdKv4.2ST) designed to overexp ress Kv4.2ST in infected cells. A-type currents in rat cerebellar gran ule cells were decreased two days after AdKv4.2ST infection as compare d 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 grou p, p < 0.001). Likewise, I-to in adult rat ventricular myocytes was su ppressed by AdKv4.2ST but not by Ad beta-galactosidase (8.8 pA/pF vers us 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 subc ellular protein localization by confocal microscopy. The protein was d istributed throughout the surface membrane and intracellular membrane systems, We conclude that genes from the Kv4 family are the predominan t contributors to the A-type currents in cerebellar granule cells and I-to in rat ventricle. Overexpression of dominant-negative constructs may be of general utility in dissecting the contributions of various i on channel genes to excitability.