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¹ Strathclyde Institute of Pharmacy and Biomedical Sciences, and ² Institute of Photonics, SUPA, University of Strathclyde, Glasgow G4 0NR, Scotland, UK
³ Beatson Institute for Cancer Research, Glasgow G61 1BD, Scotland, UK

Correspondence to J.G. McCarron: john.mccarron{at}strath.ac.uk

In smooth muscle, the gating of dihydropyridine-sensitive Ca²⁺ channels may either be stochastic and voltage dependent or coordinated among channels and constitutively active. Each form of gating has been proposed to be largely responsible for Ca²⁺ influx and determining the bulk average cytoplasmic Ca²⁺ concentration. Here, the contribution of voltage-dependent and constitutively active channel behavior to Ca²⁺ signaling has been studied in voltage-clamped single vascular and gastrointestinal smooth muscle cells using wide-field epifluorescence with near simultaneous total internal reflection fluorescence microscopy. Depolarization (–70 to +10 mV) activated a dihydropyridine-sensitive voltage-dependent Ca²⁺ current (I_Ca) and evoked a rise in [Ca²⁺] in each of the subplasma membrane space and bulk cytoplasm. In various regions of the bulk cytoplasm the [Ca²⁺] increase ([Ca²⁺]_c) was approximately uniform, whereas that of the subplasma membrane space ([Ca²⁺]_PM) had a wide range of amplitudes and time courses. The variations that occurred in the subplasma membrane space presumably reflected an uneven distribution of active Ca²⁺ channels (clusters) across the sarcolemma, and their activation appeared consistent with normal voltage-dependent behavior. Indeed, in the present study, dihydropyridine-sensitive Ca²⁺ channels were not normally constitutively active. The repetitive localized [Ca²⁺]_PM rises ("persistent Ca²⁺ sparklets") that characterize constitutively active channels were observed rarely (2 of 306 cells). Neither did dihydropyridine-sensitive constitutively active Ca²⁺ channels regulate the bulk average [Ca²⁺]_c. A dihydropyridine blocker of Ca²⁺ channels, nimodipine, which blocked I_Ca and accompanying [Ca²⁺]_c rise, reduced neither the resting bulk average [Ca²⁺]_c (at –70 mV) nor 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, although voltage-dependent Ca²⁺ channels appear clustered in certain regions of the plasma membrane, constitutive activity is unlikely to play a major role in [Ca²⁺]_c regulation. The stochastic, voltage-dependent activity of the channel provides the major mechanism to generate rises in [Ca²⁺].

Abbreviations used in this paper: MEL, murine erythroleukemia; TIRF, total internal reflection fluorescence microscopy.

© 2009 McCarron et al.
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