Identification, cloning and expression of rabbit vascular smooth muscle Kv1.5 and comparison with native delayed rectifier K+ current

1. The molecular basis of voltage-gated, delayed rectifier K+ (K-DR) channe ls in vascular smooth muscle cells is poorly defined. In this study we empl oyed (i) an antibody against Kv1.5 and (ii) a cDNA clone encoding Kv1.5 der ived from rabbit portal vein (RPV) to demonstrate Kv1.5 expression in RPV a nd to compare the properties of RPVKv1.5 expressed in mammalian cells with those of native RPV K-DR current. 2. Expression of Kv1.5 channel protein in RPV was demonstrated by (i) immun ocytolocalization of an antibody raised against a C-terminal epitope of mou se cardiac Kv1.5 in permeabilized, freshly isolated RPV smooth muscle cells and (ii) isolation of a cDNA clone encoding RPVKv1.5 by reverse transcript ion-polymerase chain reaction (RT-PCR) using mRNA derived from endothelium- denuded and adventitia-free RPV. 3. RPVKv1.5 cDNA was expressed in mammalian L cells and human embryonic kid ney (HEK293) cells and the properties of the expressed channels compared wi th those of native K-DR channels of freshly dispersed myocytes under identi cal conditions. 4. The kinetics and voltage dependence of activation of L cell-expressed RP VKv1.5 and native K-DR current were identical, as were the kinetics of reco very from inactivation and single channel conductance. In contrast, there w as little similarity between HEK293 cell-expressed RPVKv1.5 and native Ii c urrent. 5. Inactivation occurred with the same voltage for half-maximal availabilit y, but the kinetics and slope constant for the voltage dependence of inacti vation for L cell-expressed RPVKv1.5 and the native current were different: slow time constants were 6.5 +/- 0.6 and 3.5 +/- 0.4 s and slope factors w ere 4.7 +/- 0.2 and 7.0 +/- 0.8 mV respectively. 6. This study provides immunofluorescence and functional evidence that Kv1. 5 alpha-subunits are a component of native K-DR channels of vascular smooth muscle cells of RPV However, the differences in kinetics and voltage sensi tivity of inactivation between L cell- and HEK293 cell-expressed channels a nd native K-DR channels provide functional evidence that vascular K-DR curr ent is not due to homomultimers of RPV Kv1.5 alone. The channel structure m ay be more complex, involving heteromultimers and modulatory Kv beta-subuni ts, and/or native K-DR current may have other components involving Kv alpha -subunits of other families.