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We investigated the voltage dependence of nifedipine sensitivity of the ion channels formed by α1 subunits of the cardiac and smooth muscles (CM and SM, respectively) L-type Ca2+ channels stably expressed in Chinese hamster ovary (CHO) cells. Equilibrium inhibition of the α1 subunits, directing Ba2+ current ( I α1), by different concentrations of nifedipine was measured at the holding potentials ( V h ) of −100 mV and −50 mV. At V h =−100 mV, the SM α1 subunit was found to be 6-fold more sensitive for nifedipine than the subunit ( K −100=8.3 and 50.4 nM, respectively). Depolarization to −50 mV resulted in about sevenfold increase in the nifedipine potency for both subunits ( K −50=1.25 and 6.95 nM, respectively). The voltage dependence of steady-state inactivation could be fitted by a sum of two Boltzmann’s equations with slope factors of about 12 and 5 mV. The midpoints of both components in the CM α1 subunit (−75.6 and −42.8 mV) were more negative than those in the SM subunit (−63.7 and −37.7 mV). The relative contribution of the less sloped component in the control was rather low, being less pronounced in the CM (0.15) than in the SM (0.34) subunits. Nifedipine shifted the midpoints of inactivation curves to more negative potentials. The shift was more pronounced for the SM α1 subunit (−24.8 mV compared with −11.8 mV for the CM subunit in the presence of 10 nM nifedipine). Nifedipine differentially affected the two Boltzmann components of inactivation curves, more effectively inhibiting the steeper component. In the presence of 10 nM nifedipine, this component completely disappeared in the SM subunit, while its relative contribution in the CM subunit decreased from 0.85 to 0. 57, resulting in an apparent decrease in the steepness. These results are inconsistent with the receptor modulated hypothesis and suggest the existence of two mechanisms of inactivation characterized by different voltage dependence. [ABSTRACT FROM AUTHOR]

Barium currents through ion channels formed by α1-subunit of L-type Ca2+ channel ( I α1) were recorded from cultured chinese hamster ovary (CHO) cells. The cells were stably transfected with either a cardiac or a smooth muscle (SM) variant of α1-subunit. The I α1 in both cases exhibited similar fast voltage-dependent activation kinetics and slow apparent inactivation kinetics. With 10 mM Ba2+ in the bath solution, I α1 was activated at potentials more positive than −40 mV, peaked between 0 and +10 mV, and reversed at about +50 mV. In addition to slow apparent inactivation of inward current, both subunits provided an extremely slow voltage-dependent inactivation at potentials more positive than −100 mV, with half-maximum inactivation at −43.4 mV for cardiac and −41.4 mV for SM α1-subunits. The onset of inactivation as well as recovery from this process were within a time range of minutes. The voltage dependence of steady-state inactivation could be fitted by the sum of two Boltzmann's equations with slope factors of about 12 mV and 5 mV. A less sloped component has its midpoints at −75.6 and −63.7 mV, and a steeper component has its midpoints at −42.8 and −37.7 mV for cardiac and SM α1-subunits, respectively. Relative contribution of the steeper component was higher in both subunits (0.86 and 0.66 for cardiac and SM subunits, respectively). For comparison, the inactivation curves for 5-sec-long conditioning prepulses could be fitted by single Boltzmann's distribution with a 20 mV more positive midpoint and a slope factor of about 13 mV. In contrast to the steady-state inactivation curves, they showed considerable overlap with the steady-state activation curve. Our results reflect functional consequences of known sequence differences between α1-subunits of the cardiac and SM L-type Ca2+ channels and could be used in structural modeling of Ca2+ channel gating. In addition, they show that depolarization-induced window current has a transient nature and decays with the development of extremely slow inactivation. This is the first demonstration that slow inactivation of the L-type Ca2+ channel is an intrinsic property of its α1-subunits. [ABSTRACT FROM AUTHOR]