ATP-sensitive potassium (K_ATP) and nucleotide-diphosphate regulated (K_NDP) channels are important in controlling smooth muscle tone. In particular, the potent vasoconstrictor angiotensin II has been demonstrated to act in part via a protein kinase C (PKC)-dependent inhibition of K_ATP and K_NDP currents in native vascular smooth muscles. The molecular basis of these phenomena has not been elucidated. We have investigated the regulation of the cloned counterparts of these currents in HEK293 cells.

Stable lines were generated, containing either Kir6.1 with SUR2B or Kir6.2 with SUR2B, as described by Cui et al. (2001). Membrane currents were studied using the whole-cell patch-clamp technique. Bath solutions (pH 7.4) contained (mM): 140 KCl, 5 Hepes, 1.2 MgCl₂ and 2.6 CaCl₂. Pipette solutions (pH 7.2) contained (mM): 140 KCl, 5 Hepes, 1.2 MgCl₂, 10 EGTA, 1 CaCl₂, 1 ATP and 0.5 UDP. Stably expressed Kir6.1/SUR2B currents were activated with 100 µM diazoxide. Current values are given as means ± S.E.M. recorded at a potential of -60 mV. Student’s t test was used to calculate statistical significance at the 5 % level.

Application of 1 µM phorbol-12-myristate-13-acetate (PMA) significantly decreased Kir6.1/SUR2B current from 66.1 ± 13.9 to 42.4 ± 6.5 pA pF^-1 (n = 10) and phorbol-12,13-dibutyrate (PdBu) significantly reduced Kir6.1/SUR2B current from 68.9 ± 18.3 to 48.4 ± 12.2 pA pF^-1 (n = 10). In contrast, application of 1 µM PMA led to a small, non-significant increase in Kir6.2/SUR2B current from 608.4 ± 157.6 to 620.7 ± 172.51 pA pF^-1 (n = 8) and application of 1 µM PdBu led to a small non-significant decrease in current from 763.0 ± 173.4 to 739.6 ± 162.5 pA pF^-1 (n = 7). The angiotensin II receptor (subtype AT-1) was then transiently transfected into both stable lines. Application of 1 µM angiotensin II to these cells resulted in a significant decrease in Kir6.1/SUR2B current from 54.0 ± 10.8 to 31.4 ± 6.1 pA pF^-1 (n = 13), but caused a non-significant decrease in Kir6.2/SUR2B current from 570.0 ± 83.1 to 429.6 ± 74.7 pA pF^-1 (n = 8). In the presence of the PKC-selective inhibitor GF 109203X (3 µM), only a small non-significant decrease in Kir6.1/ SUR2B current from 77.9 ± 30.6 to 66.0 ± 26.9 pA pF^-1 (n = 6) was seen with 1 µM angiotensin II.

These results show that the cloned counterparts of vascular K_ATP channels can be regulated by PKC and that Kir6.1/SUR2B but not Kir6.2/SUR2B currents are modulated. The study of these cloned channels in HEK293 cells may help elucidate these clinically important regulatory mechanisms.This work was supported by the BBSRC and The Wellcome Trust.

Cui, Y., Giblin, J.P., Clapp, L.H. & Tinker, A. (2001). Proc. Natl Acad. Sci. USA 98, 729-734.