Voltage-Dependent Ca2+ Channels Are Clustered But Not Constitutively-Active In Smooth Muscle

Abstract

The organization and distribution of Ca2+ signals derived from depolarization-evoked Ca2+ entry has been studied in voltage-clamped single vascular and gastrointestinal smooth muscle cells using widefield epi-fluorescence with near simultaneous (2 ms) total internal reflection fluorescence microscopy. Depolarization activated a voltage-dependent Ca2+ current (ICa) and evoked a rise in [Ca2+] in the subplasma membrane space and bulk cytoplasm. The rise which occurred in various regions of the bulk cytoplasm ([Ca2+]c) was approximately uniform; that of the subplasma membrane space ([Ca2+]PM) had a wide range of amplitudes and time courses. The [Ca2+]PM variations presumably reflected an uneven distribution of active Ca2+ channels (clusters) across the sarcolemma. Constitutive activity in clusters of voltage-dependent Ca2+ channels has been proposed to determine bulk average [Ca2+]c. In the present study, channels are not constitutively active. The repetitive localized [Ca2+]PM rises (‘Ca2+ sparklets’) which characterize constitutively-active channels were observed rarely (<1 in 50 cells). Nor did constitutively-active voltage-dependent Ca2+ channels regulate the bulk average [Ca2+]c. A dihydropyridine blocker of voltage-dependent Ca2+ channels, nimodipine, which blocked ICa and accompanying [Ca2+]c rise, reduced neither the resting bulk average [Ca2+]c (at -70 mV) or the rise in [Ca2+]c which accompanied an increased electrochemical driving force on the ion by hyperpolarization (-130 mV). Activation of protein kinase C with indolactam-V did not induce constitutive channel activity. Thus while voltage-dependent Ca2+ channels appear clustered on the plasma membrane, constitutive activity in the channel is unlikely to play a major role in regulation [Ca2+]c. The voltage-dependent activity of the clustered channels may enable selective activation of various cellular processes by generating a localized rises in subplasma membrane [Ca2+].Supported by the Wellcome Trust and British Heart Foundation