Elevations of intracellular calcium refl ect normal voltage-dependent behavior, and not constitutive activity, of voltage-dependent calcium channels in gastrointestinal and vascular smooth muscle

Abstract

In smooth muscle, the gating of dihydropyridine-sensitive Ca 2+ 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 Ca 2+ infl ux and determining the bulk average cytoplasmic Ca 2+ concentration. Here, the contribution of voltage-dependent and constitutively active channel behavior to Ca 2+ signaling has been studied in voltage-clamped single vascular and gastrointestinal smooth muscle cells using wide-fi eld epifl uorescence with near simultaneous total internal refl ection fl uorescence microscopy. Depolarization ( - 70 to +10 mV) activated a dihydropyridine-sensitive voltage-dependent Ca 2+ current (I Ca) and evoked a rise in [Ca 2+ ] in each of the subplasma membrane space and bulk cytoplasm. In various regions of the bulk cytoplasm the [Ca 2+ ] increase ([Ca 2+ ] c ) was approximately uniform, whereas that of the subplasma membrane space ([Ca 2+ ] PM ) had a wide range of amplitudes and time courses. The variations that occurred in the subplasma membrane space presumably refl ected an uneven distribution of active Ca 2+ channels (clusters) across the sarcolemma, and their activation appeared consistent with normal voltage-dependent behavior. Indeed, in the present study, dihydropyridine-sensitive Ca 2+ channels were not normally constitutively active. The repetitive localized [Ca 2+ ] PM rises ( " persistent Ca 2+ sparklets " ) that characterize constitutively active channels were observed rarely (2 of 306 cells). Neither did dihydropyridine-sensitive constitutively active Ca 2+ channels regulate the bulk average [Ca 2+ ] c. A dihydropyridine blocker of Ca 2+ channels, nimodipine, which blocked I Ca and accompanying [Ca 2+ ] c rise, reduced neither the resting bulk average [Ca 2+ ] c (at - 70 mV) nor the rise in [Ca 2+ ] 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 Ca 2+ channels appear clustered in certain regions of the plasma membrane, constitutive activity is unlikely to play a major role in [Ca 2+ ] c regulation. The stochastic, voltage-dependent activity of the channel provides the major mechanism to generate rises in [Ca 2+ ]. © 2009 McCarron et al.