The combination of apamin (a specific inhibitor of small conductance calcium-activated K⁺ channels: SK_Ca) and charybdotoxin (a non-specific inhibitor of intermediate conductance calcium-activated K⁺ channels: IK_Ca) or of apamin and the non-peptide IK_Ca inhibitor (TRAM-34, Wulff et al., 2000), is required to abolish endothelium-dependent hyperpolarizations (EDHF; Busse et al., 2002; Gluais et al., 2004). The absolute requirement for inhibitors of SK_Ca and IK_Ca could be explained either by the simultaneous activation of the two populations of K_Ca channels during endothelial stimulation or by the expression of an heterotetramer composed of SK and IK alpha subunits. This study was designed to determine whether or not the expression of such an heterotetramer in native endothelial cells is a likely hypothesis.

Guinea-pigs were euthanized with a lethal dose of pentobarbital and the internal carotid arteries were dissected free and mounted in an organ bath. The smooth muscle cell membrane potential was recorded using sharp micro-electrodes. All the experiments were performed at 37°C in the presence of N -nitro-L-arginine (100 µM) and indomethacin (5 µM), respectively.

In control conditions (Krebs solution containing 2.5 mM Ca²⁺), acetylcholine (10 nM-10 µM) induced a concentration- and endothelium-dependent hyperpolarization. Apamin (0.5 µM) induced a partial inhibition of the hyperpolarization to acetylcholine (p<0.05) whereas charybdotoxin (0.1 µM) and TRAM-34 (10 µM) were completely ineffective. However, apamin + charybdotoxin, or apamin + TRAM-34 virtually abolished EDHF-mediated responses. In 0.5 mM Ca²⁺, the hyperpolarization to 1 µM acetylcholine (–14.6 ± 1.3 mV, n=7) was significantly lower than in 2.5 mM Ca²⁺ (-20.3 ± 1.1 mV, n=6, p < 0.05). Furthermore, the EDHF-mediated responses were predominantly sensitive to charybdotoxin or TRAM-34 but resistant to apamin (charybdotoxin: -21.2 ± 1.2 and –7.2 ± 1.1 mV, n=6, p < 0.05; TRAM-34: -17.5 ± 3.2 and –9.5 ± 1.4 mV, n=4 and 5, p < 0.05; apamin: -12.1. ± 2.6 and –10.7 ± 1.0 mV, n=5 and 6, p>0.05, in 2.5 and 0.5 mM calcium, respectively). In 0.5 mM calcium, the combination of the two toxins abolished the hyperpolarization to acetylcholine.

Thus, in the quiescent carotid artery of the guinea pig under control conditions both IK_Ca and SK_Ca are activated during the acetylcholine-induced EDHF response and activation of SK_Ca plays a predominant role (possibly because of a higher level of expression of this channel in the guinea-pig endothelial cells). However, under low-calcium conditions (0.5 mM Ca²⁺), IK_Ca channels are preferentially activated. This is consistent with the reported two- to five-fold higher calcium sensitivity for IK_Ca when compared with the SK_Ca channels (www.iuphar.org). The results do not support the existence of SK_Ca and IK_Ca heterotetramers in native endothelial cells. Instead, the EDHF response is triggered via the combined opening of homotetrameric IK_Ca and SK_Ca channels.

Busse , R., et al., (2002) Trends Pharmacol Sci.23, 374-380
Gluais, P., et al., (2004) Br. J. Pharmacol. Proc. Suppl., www.pa2online.org/ Vol1Issue4abst027P
Wulff , H., et al., (2000) Proc. Natl. Acad. Sci. USA.97, 8151-8158.