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

The relationship between Ca2+ sparks spontaneously occurring at rest and lo cal Ca2+ transients elicited by depolarization was analysed using two-dimen sional confocal Ca2+ images of single smooth muscle cells isolated from gui nea-pig vas deferens and urinary bladder. The current activation by these C a2+ events was also recorded simultaneously under whole-cell voltage clamp. 2. Spontaneous transient outward currents (STOCs) and Ca2+ sparks were simu ltaneously detected at -40 mV in approximately 50% of myocytes of either ty pe. Ca2+ sparks and corresponding STOCs occurred repetitively in several di screte sites in the subplasmalemmal area. Large conductance Ca2+-dependent K+ (BK) channel density in the plasmalemma near the Ca" spark sites generat ing STOCs was calculated to be 21 channels mum(-2). 3. When myocytes wore depolarized from -60 to 0 mV, several local Ca2+ tran sients were elicited within 20 ms; in exactly the same peripheral sites whe re sparks occurred at rest. The local Ca2+ transients often lasted over 300 ms and spread into other areas. The appearance of local Ca2+ transients oc curred synchronously with the activation of Ca2+-dependent K+ current 4. Immunofluorescence staining of the BK channel alpha -subunit (BK alpha) revealed a spot-like pattern on the plasmalemina, in contrast to the unifor m staining of voltage-dependent Ca2+ channel alpha 1C subunits along the pl asmalemma. Ryanodine receptor (RyR) immunostaining also suggested punctate localization predominantly in the periphery. Double staining of BKa and RyR s revealed spot-like co-localization on/beneath the plasmalemma. 5. Using pipettes of relatively low resistance, inside-out patches that inc luded both clustered BK channels at a density of over 20 channels mum(-2) a nd functional Ca2+ storage sites were obtained -2 at a low probability of s imilar to5%. The averaged BK channel density was 3-4 channels mum(-2) in bo th types of myocyte. 6. These results support the idea that a limited number of discrete sarcopl asmic reticulum. (SR) fragments in the subplasmalemmal area play key roles in the control of BK channel activity in two ways: (i) by generating Ca2+ s parks at rest to activate STOCs and (ii) by generating Ca2+ transients pres umably triggered by sparks during an action potential to activate a large a nd 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.