Endothelial K⁺channel lacks the Ca²⁺sensitivity‐regulating β subunit

Abstract

Hyperpolarizing large-conductance, Ca(2+)-activated K(+) channels (BK) are important modulators of vascular smooth muscle and endothelial cell function. In vascular smooth muscle cells, BK are composed of pore-forming alpha subunits and modulatory beta subunits. However, expression, composition, and function of BK subunits in endothelium have not been studied so far. In patch-clamp experiments we identified BK (283 pS) in intact endothelium of porcine aortic tissue slices. The BK opener DHS-I (0.05-0.3 micromol/l), stimulating BK activity only in the presence of beta subunits, had no effect on BK in endothelium whereas the alpha subunit selective BK opener NS1619 (20 micromol/l) markedly increased channel activity. Correspondingly, mRNA expression of the beta subunit was undetectable in endothelium, whereas alpha subunit expression was demonstrated. To investigate the functional role of beta subunits, we transfected the beta subunit into a human endothelial cell line (EA.hy 926). beta subunit expression resulted in an increased Ca(2+) sensitivity of BK activity: the potential of half-maximal activation (V(1/2)) shifted from 73.4 mV to 49.6 mV at 1 micromol/l [Ca(2+)](i) and an decrease of the EC(50) value for [Ca(2+)](i) by 1 microM at +60 mV was observed. This study demonstrates that BK channels in endothelium are composed of alpha subunits without association to beta subunits. The lack of the beta subunit indicates a substantially different channel regulation in endothelial cells compared to vascular smooth muscle cells.

Large-conductance Ca2+-activated potassium channel in mitochondria of endothelial EA.hy926 cells

“…However, when expressed in freshly isolated or cultured endothelial cells, BK Ca channels that are characterized by calcium and voltage dependence, iberiotoxin sensitivity, and a conductance between 190 and 300 pS are detected (6,17,26,28). Interestingly, the properties of the BK Ca channel in the human endothelial cell line EA.hy926 (26) are similar to those of the mitoBK Ca channel described in this study. The BK Ca channel of EA.hy926 cells has a single-channel conductance of 270 pS, which is the same as that of its mitoBK Ca channel counterpart.…”

“…In some freshly isolated endothelial cells, BK Ca channels are poorly expressed and iberiotoxin-sensitive currents are observed only at very positive potentials (15). This has been attributed to the absence of BK Ca regulatory ␤-subunits that increase the sensitivity to Ca 2ϩ in these cells (26). However, when expressed in freshly isolated or cultured endothelial cells, BK Ca channels that are characterized by calcium and voltage dependence, iberiotoxin sensitivity, and a conductance between 190 and 300 pS are detected (6,17,26,28).…”

“…This has been attributed to the absence of BK Ca regulatory ␤-subunits that increase the sensitivity to Ca 2ϩ in these cells (26). However, when expressed in freshly isolated or cultured endothelial cells, BK Ca channels that are characterized by calcium and voltage dependence, iberiotoxin sensitivity, and a conductance between 190 and 300 pS are detected (6,17,26,28). Interestingly, the properties of the BK Ca channel in the human endothelial cell line EA.hy926 (26) are similar to those of the mitoBK Ca channel described in this study.…”

“…The BK Ca channel of EA.hy926 cells has a single-channel conductance of 270 pS, which is the same as that of its mitoBK Ca channel counterpart. Its activity also strongly depends on membrane potential, and Ca 2ϩ concentration and is markedly increased by NS1619 (26).…”

Control and Coordination of Vasomotor Tone in the Microcirculation

“…The main K + channels expressed in resistance vessels, from a functional point of view, are: the ATP-sensitive K + channels (K ATP ), inward rectifying K + channels (K ir ) and Ca 2+ -activated K + channels (K Ca ) of small (SK Ca ), intermediate (IK Ca ) and large (BK Ca ) conductance (Jackson, 2000(Jackson, , 2005. K ATP and K ir are expressed in both endothelial and smooth muscle cells (Quayle et al, 1996;Jackson, 2000Jackson, , 2005Ko et al, 2008), whereas BK Ca are mostly found in smooth muscle cells (Jackson, 2005;Ko et al, 2008), but, on occasion, these K + channels have also been described in endothelial cells (Papassotiriou et al, 2000;Wang et al, 2005). In contrast, SK Ca and IK Ca are expressed exclusively in endothelial cells (Jackson, 2000;Kohler et al, 2000;Nilius & Droogmans, 2001;Eichler et al, 2003;Taylor et al, 2003;Brahler et al, 2009).…”