SUMMARY1. Experiments are described in which the [Ca]o-dependent component of 22Na efflux is monitored under conditions of membrane potential control by voltage clamp.2. The apparent affinity of the efflux system for external Ca is very low in choline sea water (apparent KD 50 mM); but increases dramatically when choline is replaced isosmotically by Li or K (apparent KD -1-2 mM). Ca influx changes in a parallel fashion. Tris behaves much like choline and guanidinium is about two-thirds as effective as Li.3. Replacement of Li by K has little effect on the apparent affinity for external Ca but brings about a small (30-40 %) increase in the maximal flux. The increase in maximum flux can be removed by electrical hyperpolarization to the potential before application ofK and, in the absence ofK, can be mimicked by electrical depolarization. These experiments suggest that the stimulatory effect of K on the Ca-dependent Na efflux into Li sea water is electrical in origin.4. Partial replacement of choline by K stimulates the Ca-dependent Na efflux; but only part of this stimulation can be removed by electrical hyperpolarization and, in the absence of K, electrical depolarization only brings about a relatively small stimulation. This is because only part of the stimulation that follows addition of K to choline sea waters is electrical in origin: the rest reflects an increase in the apparent affinity for external Ca that is brought about by K acting chemically. The maximum efflux into K is about 40 % higher than that into choline. That this may reflect an electrical effect is supported by the observation that electrical depolarization increases the flux into choline sea water containing 110 mm-Ca where the Ca-binding site is close to saturation.5. The voltage clamp was used to determine the voltage dependence of the Ca-dependent Na efflux into Li sea water, choline sea water and choline sea water containing 100 mM-Na. In all three cases the flux increased with depolarization and was still rising at + 70 mV. The dependence on potential was not very steep, an e-fold increase occurred over approximately 50 mV.

Until recently, intracellular free calcium has been amenable to measurement and investigation only in cells large enough to permit either microinjection of a suitable Ca sensor such as a aequorin or arsenazo III or insertion of a Ca-sensitive microelectrode. This constraint on cell size was removed by the development of the fluorescent Ca2+ -sensitive dye Quin-2 and its acetoxymethyl ester, which can be introduced into a wide range of cell types. A major requirement of any intracellular Ca2+ indicator is that it should not disturb intracellular Ca2+ homeostasis and Quin-2 is generally considered to be satisfactory in this respect. We now report that injection of Quin-2 into squid (Loligo forbesi) axons can almost completely abolish one component of Ca2+ entry--intracellular Na+ (Nai)-dependent Ca2+ inflow, which occurs via Na/Ca exchange. Mixtures of Ca and Quin-2 that buffer an ionized Ca2+ at close to physiological concentrations also block Nai-dependent Ca2+ influx but these same mixtures fail to block the extracellular Na+ (Na0)-dependent extrusion of Ca2+, showing that Quin-2 acts specifically on Ca2+ inflow.

Temperature normally affects peak L-type Ca2+ channel (CaCh) current with a temperature coefficient (Q10) of between 1.8 and 3.5; in cardiomyocytes attenuating protein kinase A activity increases Q10 whilst activating it lowers Q10. We examine temperature effects using cloned human cardiac CaChs expressed in Xenopus oocytes. Peak inward currents (IBa) through expressed CaChs (i.e. alpha1C alpha2/deltaa beta1b) exhibited a Q10 of 5.8+/-0.4 when examined between 15 and 25 degreesC. The nifedipine-sensitive IBa exhibited a higher Q10 of 8.7+/-0.5, whilst the nifedipine-insensitive IBa exhibited Q10 of 3.7+/-0.3. Current/voltage (I/V) relationships shifted to negative potentials on warming. Using instead a different CaCh beta subunit isoform, beta2c, gave rise to an IBa similar to those expressed using beta1b. We utilized a carboxyl deletion mutant, alpha1C-Delta1633, to determine the temperature sensitivity of the pore moiety in the absence of auxiliary subunits; IBa through this channel exhibited a Q10 of 9.3+/-0.3. However, the Q10 for macroscopic conductance was reduced compared to that of heteromeric channels; decreasing from 5.0 (i.e. alpha1C alpha2/deltaa beta1b) and 3.9 (i.e. alpha1C alpha2/deltaa beta2c) to 2.4 (alpha1C-Delta1633). These observations differ markedly from those made in studies of cardiomyocytes, and suggest that enhanced sensitivity may depend on the membrane environment, channel assembly or other regulatory factors.

SUMMARY1. Experiments are described in which Ca efflux is monitored in axons under voltage clamp.2. As Ca efflux consists of more than one component, conditions were sought where one component predominates. Thus external Na-dependent Ca efflux can be studied in relative isolation either at pH 9 0 or in fully poisoned axons immersed in Ca-free media; external Ca-dependent Ca efflux can be studied in fully poisoned axons immersed in Na-free media and the Na-independent, energy requiring, pump is best examined in Na and Ca-free sea waters.3. Both in unpoisoned axons at pH 9 0 and fully poisoned axons at pH 7-8, the external Na-dependent Ca efflux is activated by hyperpolarization and inhibited by depolarization. Depolarizations achieved either electrically or by exposure to high K are roughly comparable and the inhibition brought about by high K can largely be removed by electrical hyperpolarization to the initial resting potential. In both Na sea waters and choline sea waters containing 100 mM-Na, Ca efflux is increased e-fold over approximately 50 mV.4. In choline sea water, external Ca-dependent Ca efflux from fully poisoned axons is unaffected by voltage over the range -80 to -30 mV. But addition of K or Li activates the flux and this activation is increased by hyperpolarization and decreased by depolarization, suggesting that the activating cation may also be transported into the axon. 5. The Na-independent, energy-requiring, flux is inhibited by electrical hyperpolarization and stimulated by electrical depolarization. External K also stimulates the flux and part of this stimulation can be removed by electrical hyperpolarization. These data show that the energy-dependent pump is sensitive to membrane potential in the physiological range and suggest that it may be an electrogenic process.6. The finding that voltage affects the energy-dependent (uncoupled) pump and external Na-dependent fluxes in opposite directions may help explain why the total Ca efflux from intact axons responds to potential in a very variable manner.