Members of a previously unidentified family of potassium channel subunits were cloned from rat and human brain. The messenger RNAs encoding these subunits were widely expressed in brain with distinct yet overlapping patterns, as well as in several peripheral tissues. Expression of the messenger RNAs in Xenopus oocytes resulted in calcium-activated, voltage-independent potassium channels. The channels that formed from the various subunits displayed differential sensitivity to apamin and tubocurare. The distribution, function, and pharmacology of these channels are consistent with the SK class of small-conductance, calcium-activated potassium channels, which contribute to the afterhyperpolarization in central neurons and other cell types.

The slow afterhyperpolarization that follows an action potential is generated by the activation of small-conductance calcium-activated potassium channels (SK channels). The slow afterhyperpolarization limits the firing frequency of repetitive action potentials (spike-frequency adaptation) and is essential for normal neurotransmission. SK channels are voltage-independent and activated by submicromolar concentrations of intracellular calcium. They are high-affinity calcium sensors that transduce fluctuations in intracellular calcium concentrations into changes in membrane potential. Here we study the mechanism of calcium gating and find that SK channels are not gated by calcium binding directly to the channel alpha-subunits. Instead, the functional SK channels are heteromeric complexes with calmodulin, which is constitutively associated with the alpha-subunits in a calcium-independent manner. Our data support a model in which calcium gating of SK channels is mediated by binding of calcium to calmodulin and subsequent conformational alterations in the channel protein.

An intermediate conductance calciumactivated potassium channel, hIK1, was cloned from human pancreas. The predicted amino acid sequence is related to, but distinct from, the small conductance calcium-activated potassium channel subfamily, which is Ϸ50% conserved. hIK1 mRNA was detected in peripheral tissues but not in brain. Expression of hIK1 in Xenopus oocytes gave rise to inwardly rectifying potassium currents, which were activated by submicromolar concentrations of intracellular calcium (K 0.5 ‫؍‬ 0.3 M). Although the K 0.5 for calcium was similar to that of small conductance calcium-activated potassium channels, the slope factor derived from the Hill equation was significantly reduced (1.7 vs. 3.5). Single-channel current amplitudes ref lected the macroscopic inward rectification and revealed a conductance level of 39 pS in the inward direction. hIK1 currents were reversibly blocked by charybdotoxin (K i ‫؍‬ 2.5 nM) and clotrimazole (K i ‫؍‬ 24.8 nM) but were minimally affected by apamin (100 nM), iberiotoxin (50 nM), or ketoconazole (10 M). These biophysical and pharmacological properties are consistent with native intermediate conductance calcium-activated potassium channels, including the erythrocyte Gardos channel.

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Background-It has been proposed that activation of endothelial SK3 (K Ca 2.3) and IK1 (K Ca 3.1) K ϩ channels plays a role in the arteriolar dilation attributed to an endothelium-derived hyperpolarizing factor (EDHF). However, our understanding of the precise function of SK3 and IK1 in the EDHF dilator response and in blood pressure control remains incomplete. To clarify the roles of SK3 and IK1 channels in the EDHF dilator response and their contribution to blood pressure control in vivo, we generated mice deficient for both channels. Methods and Results-Expression and function of endothelial SK3 and IK1 in IK1Ϫ/Ϫ /SK3 T/T mice was characterized by patch-clamp, membrane potential measurements, pressure myography, and intravital microscopy. Blood pressure was measured in conscious mice by telemetry. Combined IK1/SK3 deficiency in IK1Ϫ/Ϫ /SK3 T/T (ϩdoxycycline) mice abolished endothelial K Ca currents and impaired acetylcholine-induced smooth muscle hyperpolarization and EDHFmediated dilation in conduit arteries and in resistance arterioles in vivo. IK1 deficiency had a severe impact on acetylcholine-induced EDHF-mediated vasodilation, whereas SK3 deficiency impaired NO-mediated dilation to acetylcholine and to shear stress stimulation. As a consequence, SK3/IK1-deficient mice exhibited an elevated arterial blood pressure, which was most prominent during physical activity. Overexpression of SK3 in IK1Ϫ/Ϫ /SK3 T/T mice partially restored EDHF-and nitric oxide-mediated vasodilation and lowered elevated blood pressure. The IK1-opener SKA-31 enhanced EDHF-mediated vasodilation and lowered blood pressure in SK3-deficient IK1 ϩ/ϩ /SK3 T/T (ϩdoxy-cycline) mice to normotensive levels. Conclusions-Our study demonstrates that endothelial SK3 and IK1 channels have distinct stimulus-dependent functions, are major players in the EDHF pathway, and significantly contribute to arterial blood pressure regulation. Endothelial K Ca channels may represent novel therapeutic targets for the treatment of hypertension.

Abstract-The endothelium is a critical regulator of vascular tone, and dysfunction of the endothelium contributes to numerous cardiovascular pathologies. Recent studies suggest that apamin-sensitive, small-conductance, Ca 2ϩ -activated K ϩ channels may play an important role in active endothelium-dependent vasodilations, and expression of these channels may be altered in disease states characterized by vascular dysfunction. Here, we used a transgenic mouse (SK3 T/T ) in which SK3 expression levels can be manipulated with dietary doxycycline (DOX) to test the hypothesis that the level of expression of the SK subunit, SK3, in endothelial cells alters arterial function and blood pressure. SK3 protein was elevated in small mesenteric arteries from SK3 T/T mice compared with wild-type mice and was greatly suppressed by dietary DOX. SK3 was detected in the endothelium and not in the smooth muscle by immunohistochemistry. In whole-cell patch-clamp experiments, SK currents in endothelial cells from SK3 T/T mice were almost completely suppressed by dietary DOX. In intact arteries, SK3 channels contributed to sustained hyperpolarization of the endothelial membrane potential, which was communicated to the arterial smooth muscle. Pressure-and phenylephrine-induced constrictions of SK3 T/T arteries were substantially enhanced by treatment with apamin, suppression of SK3 expression with DOX, or removal of the endothelium. In addition, suppression of SK3 expression caused a pronounced and reversible elevation of blood pressure. These results indicate that endothelial SK3 channels exert a profound, tonic, hyperpolarizing influence in resistance arteries and suggest that the level of SK3 channel expression in endothelial cells is a fundamental determinant of vascular tone and blood pressure. Key Words: endothelium Ⅲ potassium channels Ⅲ vascular tone Ⅲ blood pressure B lood pressure and flow are regulated by the constriction and dilation of resistance arteries, generally with internal diameters Ͻ300 m. 1 Physiological stimulation through elevations in intravascular pressure or increased sympathetic activity promotes smooth muscle depolarization, intracellular Ca 2ϩ influx, and vasoconstriction. The resulting increase in total peripheral resistance within the vasculature increases blood pressure. 2 The endothelium exerts a dilating influence that opposes arterial constriction. Activation of K ϩ channels is thought to contribute to this influence through increased release of relaxing factors such as NO and prostacyclin (PGI 2 ) and through smooth muscle hyperpolarization. [3][4][5] The smallconductance Ca 2ϩ -activated K ϩ (SK) channel has received considerable attention as a potential mediator of these responses. SK channels are opened by intracellular Ca 2ϩ via an association with calmodulin 6 and are believed to play a role in the modulation of tissue excitability. 7 Of the three characterized SK channel isoforms (SK1, SK2, and SK3), 8 mRNA for SK2 and SK3 has been identified in endothelial cells. 9 Apamin, a toxin blocker of S...

Small conductance Ca(2+)-activated potassium channels (SK channels) are coassembled complexes of pore-forming SK alpha subunits and calmodulin. We proposed a model for channel activation in which Ca2+ binding to calmodulin induces conformational rearrangements in calmodulin and the alpha subunits that result in channel gating. We now report fluorescence measurements that indicate conformational changes in the alpha subunit after calmodulin binding and Ca2+ binding to the alpha subunit-calmodulin complex. Two-hybrid experiments showed that the Ca(2+)-independent interaction of calmodulin with the alpha subunits requires only the C-terminal domain of calmodulin and is mediated by two noncontiguous subregions; the ability of the E-F hands to bind Ca2+ is not required. Although SK alpha subunits lack a consensus calmodulin-binding motif, mutagenesis experiments identified two positively charged residues required for Ca(2+)-independent interactions with calmodulin. Electrophysiological recordings of SK2 channels in membrane patches from oocytes coexpressing mutant calmodulins revealed that channel gating is mediated by Ca2+ binding to the first and second E-F hand motifs in the N-terminal domain of calmodulin. Taken together, the results support a calmodulin- and Ca(2+)-calmodulin-dependent conformational change in the channel alpha subunits, in which different domains of calmodulin are responsible for Ca(2+)-dependent and Ca(2+)-independent interactions. In addition, calmodulin is associated with each alpha subunit and must bind at least one Ca2+ ion for channel gating. Based on these results, a state model for Ca2+ gating was developed that simulates alterations in SK channel Ca2+ sensitivity and cooperativity associated with mutations in CaM.

T-type Ca2+ channels (T channels) underlie rhythmic burst discharges during neuronal oscillations that are typical during sleep. However, the Ca2+-dependent effectors that are selectively regulated by T currents remain unknown. We found that, in dendrites of nucleus reticularis thalami (nRt), intracellular Ca2+ concentration increases were dominated by Ca2+ influx through T channels and shaped rhythmic bursting via competition between Ca2+-dependent small-conductance (SK)-type K+ channels and Ca2+ uptake pumps. Oscillatory bursting was initiated via selective activation of dendritically located SK2 channels, whereas Ca2+ sequestration by sarco/endoplasmic reticulum Ca2+-ATPases (SERCAs) and cumulative T channel inactivation dampened oscillations. Sk2-/- (also known as Kcnn2) mice lacked cellular oscillations, showed a greater than threefold reduction in low-frequency rhythms in the electroencephalogram of non-rapid-eye-movement sleep and had disrupted sleep. Thus, the interplay of T channels, SK2 channels and SERCAs in nRt dendrites comprises a specialized Ca2+ signaling triad to regulate oscillatory dynamics related to sleep.