Electrical events and intracellular calcium concentration ([Ca2+]) imaged using fluo‐3 and laser scanning confocal microscopy were simultaneously monitored in single smooth muscle cells freshly isolated from guinea‐pig vas deferens or urinary bladder.
Images obtained every 8 ms, during stepping from ‐60 to 0 or +10 mV for 50 ms under voltage clamp, showed that a rise in [Ca2+] could be detected within 20 ms of depolarization in five to twenty small (< 2 μm diameter) ‘hot spots’, over 95 % of which were located within 1.5 μm of the cell membrane. Depolarization at 30 s intervals activated hot spots at the same places.
Cd2+ or verapamil abolished both hot spots and Ca2+‐activated K+ current (IK,Ca). Caffeine almost abolished hot spots and markedly reduced IK,Ca. Cyclopiazonic acid, which raised basal global [Ca2+], decreased the rise in hot spot [Ca2+] and IK,Ca amplitude during depolarization. These results suggest that Ca2+ entry caused Ca2+‐induced Ca2+ release (CICR).
Under voltage clamp, hot spot [Ca2+] closely paralleled the rise in IK,Ca and reached a peak within 20 ms of the start of depolarization, but the rise in global [Ca2+] over the whole cell area was much slower. Step depolarization to potentials positive to ‐20 mV caused hot spots to grow in size and coalesce, leading to a rise in global [Ca2+] and contraction. Ca2+ hot spots also occurred during the up‐stroke of an evoked action potential under current clamp.
It is concluded that the entry of Ca2+ in the early stages of an action potential evokes CICR from discrete subplasmalemma Ca2+ storage sites and generates hot spots that spread to initiate a contraction. The activation of Ca2+‐dependent K+ channels in the plasmalemma over hot spots initiates IK,Ca and action potential repolarization.
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.
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