Elevations of intracellular calcium reflect normal voltage-dependent behavior, and not constitutive activity, of voltage-dependent calcium channels in gastrointestinal and vascular smooth muscle.
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+) influx 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-field epifluorescence with near simultaneous total internal reflection fluorescence 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 reflected 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+)].
McCarron
John G
JG
Strathclyde Institute of Pharmacy and Biomedical Sciences, SUPA, University of Strathclyde, Glasgow G4 0NR, Scotland, UK. john.mccarron@strath.ac.uk
Olson
Marnie L
ML
Currie
Susan
S
Wright
Amanda J
AJ
Anderson
Kurt I
KI
Girkin
John M
JM
D000200:Action Potentials / Q000502:physiology; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000378:metabolism*; D041981:Gastrointestinal Tract / Q000378:metabolism*; D006168:Guinea Pigs; D007424:Intracellular Fluid / Q000378:metabolism*; D008297:Male; D009131:Muscle, Smooth, Vascular / Q000378:metabolism*
D015220:Calcium Channels; D002118:Calcium