Local Ca2+ transients and distribution of BK channels and ryanodine receptors in smooth muscle cells of guinea-pig vas deferens and urinary bladder

摘要

The relationship between Ca2+ sparks spontaneously occurring at rest and local Ca2+ transients elicited by depolarization was analysed using two-dimensional confocal Ca2+ images of single smooth muscle cells isolated from guinea-pig vas deferens and urinary bladder. The current activation by these Ca2+ events was also recorded simultaneously under whole-cell voltage clamp.Spontaneous transient outward currents (STOCs) and Ca2+ sparks were simultaneously detected at −40 mV in approximately 50 % of myocytes of either type. Ca2+ sparks and corresponding STOCs occurred repetitively in several discrete sites in the subplasmalemmal area. Large conductance Ca2+-dependent K+ (BK) channel density in the plasmalemma near the Ca2+ spark sites generating STOCs was calculated to be 21 channels μm−2.When myocytes were depolarized from −60 to 0 mV, several local Ca2+ transients were elicited within 20 ms in exactly the same peripheral sites where sparks occurred at rest. The local Ca2+ transients often lasted over 300 ms and spread into other areas. The appearance of local Ca2+ transients occurred synchronously with the activation of Ca2+-dependent K+ current (IK,Ca).Immunofluorescence staining of the BK channel α-subunit (BKα) revealed a spot-like pattern on the plasmalemma, in contrast to the uniform staining of voltage-dependent Ca2+ channel α1C subunits along the plasmalemma. Ryanodine receptor (RyR) immunostaining also suggested punctate localization predominantly in the periphery. Double staining of BKα and RyRs revealed spot-like co-localization on/beneath the plasmalemma.Using pipettes of relatively low resistance, inside-out patches that included both clustered BK channels at a density of over 20 channels μm−2 and functional Ca2+ storage sites were obtained at a low probability of ∼5 %. The averaged BK channel density was 3–4 channels μm−2 in both types of myocyte.These results support the idea that a limited number of discrete sarcoplasmic reticulum (SR) fragments in the subplasmalemmal area play key roles in the control of BK channel activity in two ways: (i) by generating Ca2+ sparks at rest to activate STOCs and (ii) by generating Ca2+ transients presumably triggered by sparks during an action potential to activate a large IK,Ca and also induce a contraction. BK channels and RyRs may co-localize densely at the junctional areas of plasmalemma and SR fragments, where Ca2+ sparks occur to elicit STOCs.