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Address correspondence to David J. Adams School of Biomedical Sciences, The University of Queensland, Queensland 4072, Australia. Fax: (07) 3365-4933; email: dadams{at}uq.edu.au
It has been shown that ß auxiliary subunits increase current amplitude in voltage-dependent calcium channels. In this study, however, we found a novel inhibitory effect of ß3 subunit on macroscopic Ba2+ currents through recombinant N- and R-type calcium channels expressed in Xenopus oocytes. Overexpressed ß3 (12.5 ng/cell cRNA) significantly suppressed N- and R-type, but not L-type, calcium channel currents at "physiological" holding potentials (HPs) of -60 and -80 mV. At a HP of -80 mV, coinjection of various concentrations (0–12.5 ng) of the ß3 with Cav2.2
1 and 2
enhanced the maximum conductance of expressed channels at lower ß3 concentrations but at higher concentrations (>2.5 ng/cell) caused a marked inhibition. The ß3-induced current suppression was reversed at a HP of -120 mV, suggesting that the inhibition was voltage dependent. A high concentration of Ba2+ (40 mM) as a charge carrier also largely diminished the effect of ß3 at -80 mV. Therefore, experimental conditions (HP, divalent cation concentration, and ß3 subunit concentration) approaching normal physiological conditions were critical to elucidate the full extent of this novel ß3 effect. Steady-state inactivation curves revealed that N-type channels exhibited "closed-state" inactivation without ß3, and that ß3 caused an 
40-mV negative shift of the inactivation, producing a second component with an inactivation midpoint of approximately -85 mV. The inactivation of N-type channels in the presence of a high concentration (12.5 ng/cell) of ß3 developed slowly and the time-dependent inactivation curve was best fit by the sum of two exponential functions with time constants of 14 s and 8.8 min at -80 mV. Similar "ultra-slow" inactivation was observed for N-type channels without ß3. Thus, ß3 can have a profound negative regulatory effect on N-type (and also R-type) calcium channels by causing a hyperpolarizing shift of the inactivation without affecting "ultra-slow" and "closed-state" inactivation properties.
Key Words: voltage-dependent calcium channel • Xenopus oocyte • ß3 auxiliary subunit • negative regulation • voltage-dependent inactivation
1 subunit interaction domain; HP, holding potential; HVI, high-voltage inactivation; LVI, low-voltage inactivation; VDCC, voltage-dependent calcium channel.
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