Ca Influx Through Ca Channels in Rabbit Ventricular Myocytes During Action Potential Clamp Influence of Temperature

Ca influx via Ca current (ICa) during the action potential (AP) was determined at 25°C and 35°C in isolated rabbit ventricular myocytes using AP clamp. Contaminating currents through Na and K channels were eliminated by using Naand K-free solutions, respectively. DIDS (0.2 mmol/L) was used to block Ca-activated chloride current (ICl(Ca)). When the sarcoplasmic reticulum (SR) was depleted of Ca 21 by preexposure to 10 mmol/L caffeine, total Ca entry via ICa during the AP was '12 mmol/L cytosol (at both 25°C and 35°C). Similar Ca 21 influx at 35°C and 25°C resulted from a combination of higher and faster peak ICa, offset by more rapid ICa inactivation at 35°C. During repeated AP clamps, the SR gradually fills with Ca, and consequent SR Ca release accelerates ICa inactivation during the AP. During APs and contractions in steady state, total Ca influx via ICa was reduced by '50% but was again unaltered by temperature (5.660.2 mmol/L cytosol at 25°C, 6.060.2 mmol/L cytosol at 35°C). Thus, SR Ca release is responsible for sufficient ICa inactivation to cut total Ca 21 influx in half. However, because of the kinetic differences in ICa, the amount of Ca influx during the first 10 ms, which presumably triggers SR Ca release, is much greater at 35°C. ICa during a first pulse, given just after the SR was emptied with caffeine, was subtracted from ICa during each of 9 subsequent pulses, which loaded the SR. These difference currents reflect ICa inactivation due to SR Ca 21 release and thus indicate the time course of local [Ca] in the subsarcolemmal space near Ca channels produced by SR Ca release (eg, maximal at 20 ms after the AP activation at 35°C). Furthermore, the rate of change of this difference current may reflect the rate of SR Ca release as sensed by L-type Ca channels. These results suggest that peak SR Ca release occurs within 2.5 or 5 ms of AP upstroke at 35°C and 25°C, respectively. ICl(Ca) might also indicate local [Ca ], and at 35°C in the absence of DIDS (when ICl(Ca) is prominent), peak ICl(Ca) also occurred at a time comparable to the peak ICa difference current. We conclude that SR Ca 21 release decreases the Ca influx during the AP by '50% (at both 25°C and 35°C) and that changes in ICa (and ICl(Ca)), which depend on SR Ca 21 release, provide information about local subsarcolemmal [Ca]. The full text of this article is available at http://www.circresaha.org. (Circ Res. 1999;85:e7-e16.)