Ca2+ images and K+ current during depolarization in smooth muscle cells of the guinea-pig vas deferens and urinary bladder

1. 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 was deferens or urinary bladder. 2. 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 mu m diameter) 'hot spots', over 95% of which were located within 1.5 mu m of the cell membrane. Depolarization at 30 s intervals activated hot spots at the same places. 3. Cd2+ or verapamil abolished both hot spots and Ca2+-activated K+ current (I-K,I-Ca). Caffeine almost abolished hot spots and markedly reduced I-K,I-Ca. Cyclopiazonic acid, which raised basal global [Ca2+], decreased the rise in hot spot [Ca2+] and I-K,I-Ca amplitude during depolarization. These results suggest that Ca2+ entry caused Ca2+-induced Ca2+ release (CICR). 4. Under voltage clamp, hot spot [Ca2+] closely paralleled the rise in I-K,I-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. 5. It is concluded that the entry of Ca2+ in the early stages of an action potential evokes CICR from discrete subplasmalemma Ca2+ storage sites slid generates hot spots that spread to initiate a contraction. The activation of Ca2+-dependent K+ channels in the plasmalemma over hot spots initiates I-K,I-Ca and action potential repolarization.