β-Adrenoceptor stimulation activates large-conductance Ca2+-activated K+ channels in smooth muscle cells from basilar artery of guinea pig

Abstract

We studied the effect of isoproterenol on the Ca2+-activated K+ (BK) channel in smooth muscle cells isolated from the basilar artery of the guinea pig. Cells were studied in a whole-cell configuration to allow the clamping of intracellular Ca2+ concentration, [Ca2+]i. Macroscopic BK channel currents were recorded during depolarizing test pulses from a holding potential (VH) of 0 mV, which was used to inactivate the outward rectifier. The outward macroscopic current available from a VH of 0 mV was highly sensitive to block by external tetraethylammonium Cl (TEA) and charybdotoxin, and was greatly augmented by increasing [Ca2+]i from 0.01 to 1.0 microM. With [Ca2+]i between 0.1 and 1.0 microM, 0.4 microM isoproterenol increased this current by 58.6 +/- 17.1%, whereas with [Ca2+]i at 0.01 microM a sixfold smaller increase was observed. With [Ca2+]i > or = 0.1 microM, 100 microM dibutyryl -adenosine 3':5: cyclic monophosphate (cAMP) and 1 microM forskolin increased this current by 58.5 +/- 24.1% and 59.7 +/- 10.3%, respectively. The increase with isoproterenol was blocked by 4.0 microM propranolol extracellularly, and by 10 U/ml protein kinase inhibitor intracellularly. Single-channel openings during depolarizing test pulses from a VH of 0 mV recorded in the whole-cell configuration under the same conditions (outside-out-whole-cell recording) indicated a slope conductance of 260 pS. In conventional outside-out patches, this 260-pS channel was highly sensitive to block by external TEA, and in inside-out patches, its probability of opening was greatly augmented by increasing [Ca2+]i from 0.01 to 1.0 microM. Outside-out-whole-cell recordings with [Ca2+]i > or = 0.1 microM indicated that 100 microM dibutyryl-cAMP increased the probability of opening of the 260-pS channel by 152 +/- 115%. In inside-out patches, the catalytic subunit of protein kinase A increased the probability of opening, and this effect also depended on [Ca2+]i , with a 35-fold larger effect observed with 0.1-0.5 microM Ca2+ compared to 0.01 microM Ca2+. We conclude that the BK channel in cerebrovascular smooth muscle cells can be activated by beta-adrenoceptor stimulation, that the effect depends strongly on [Ca2+]i, and that the effect is mediated by cAMP-dependent protein kinase A with no important contribution from a direct G-protein or phosphorylation-independent mechanism. Our data indicate that the BK channel may participate in beta-adrenoceptor-mediated relaxation of cerebral vessels, although the importance of this pathway in obtaining vasorelaxation remains to be determined.

cAMP-Dependent Vasodilators Cross-Activate the cGMP-Dependent Protein Kinase to Stimulate BK Ca Channel Activity in Coronary Artery Smooth Muscle Cells

“…51 In the present study we used only freshly dissociated myocytes and did not observe PKA stimulation of BK Ca channel activity, nor did we observe an effect of selective PKA inhibitors on intact cells. Interestingly, Song and Simard 52 reported that PKA stimulated BK Ca channel activity in freshly dissociated cells from guinea pig basilar artery, but this stimulation was consistent only when [Ca 2ϩ ] i was Ն0.1 mol/L. Therefore, it is clear that both cAMP and cGMP can stimulate BK Ca channel activity in VSM; however, the identification of the specific kinase(s) involved in the response appears to be heterogeneous with respect to vessel and species.…”

Reconstitution of β-Adrenergic Modulation of Large Conductance, Calcium-activated Potassium (Maxi-K) Channels in Xenopus Oocytes

“…It should be noted that the degree of stimulation observed (30 -35%) is somewhat less than reported in mammalian cells. In smooth muscle cells, current stimulation varies from approximately 50% to 100%, and is somewhat dependent on the step voltage and [Ca 2ϩ ] i (7,38,39). Considerable controversy exists with respect to the mechanism of stimulatory coupling to maxi-K channels.…”

RACK1 is a BKCa channel binding protein

“…The channel's auxiliary ␤ 1 -subunit (primarily expressed in smooth muscle) contains a clear PKG consensus sequence and a potential PKA site (51). Whether these sites are phosphorylated in vivo, however, has yet to be determined.In general, PKA, PKG, and cSrc activate the BK Ca channel (10,24,28,33,45), although some inhibitory effects of PKA and cSrc have also been described (2, 50). PKC is inhibitory (5, 21).…”

“…In general, PKA, PKG, and cSrc activate the BK Ca channel (10,24,28,33,45), although some inhibitory effects of PKA and cSrc have also been described (2, 50). PKC is inhibitory (5, 21).…”