A reduction of peak inward L-type Ca²⁺ current (I_Ca) as well as acceleration of I_Ca inactivation (Pancrazio, 1996) have been implicated in the direct negative inotropic effect of volatile anaesthetics on the heart. Inactivation of I_Ca occurs via both Ca²⁺-dependent and voltage-dependent mechanisms. The latter consists of two kinetically distinct processes, a fast component occurring within milliseconds and an ultra-slow component occurring over seconds (Boyett et al. 1994). Using a mammalian heterologous expression system we sought to investigate the effects of halothane on ultra-slow inactivation of L-type Ca²⁺ currents.

HEK293 tsA201 cells were co-transfected with equimolar cDNA encoding cardiac specific isoforms of L-type Ca²⁺ subunits, α_1C and β_2a, together with green fluorescent protein (GFP). Expression of the α_1C subunit was confirmed by immunoblotting of cellular membrane fractions and by immunofluorescent imaging of fixed cells with anti-α_1C antibody. Cells were bathed in a Tris-based extracellular solution with 10 mM Ba²⁺ as the charge carrier (to block Ca²⁺-dependent inactivation). Fluorescent cells were voltage clamped (whole-cell configuration, holding potential -80 mV) and Ba²⁺ currents flowing through L-type Ca²⁺ channels (I_Ba) were elicited by 400 ms clamp pulses (from -70 to +60 mV in 10 mV increments) in the absence and presence of 0.6 mM halothane. To investigate the onset of ultra-slow inactivation, cells were clamped from -80 to +10 mV for 20 s during which I_Ba continued to inactivate. Experiments were carried out at 22-24 °C; data are expressed as means ± S.E.M. and P values result from paired t tests.

Peak inward I_Ba (at +10 mV) was 225 ± 19 pA under control conditions and this was reduced to 89 ± 7 pA by 0.6 mM halothane (P < 0.001, n = 6). During 20 s pulses, I_Ba inactivated with a time constant of 4180 ± 280 ms which was accelerated by 0.6 mM halothane (time constant of 1750 ± 80 ms; P = 0.02, n = 4). Peak I_Ba (during 400 ms pulses from -80 to +10 mV) following a 20 s pulse recovered back to control with a time constant of 3.2 s (n = 3) and recovery was slowed by halothane (time constant 7.8 s, n = 3). Application of 3.5 mg ml^-1 pronase to the bath solution to disrupt extracellular domains of the channel reduced the time constant of inactivation. These data suggest that ultra-slow inactivation is a property of α and β channel subunits and that this mechanism appears to involve extracellular domains (as seen in C-type inactivation), which are halothane sensitive.

This work was supported by The Wellcome Trust.