No abstract

The Na+/Ca2+ exchanger's family of membrane transporters is widely distributed in cells and tissues of the animal kingdom and constitutes one of the most important mechanisms for extruding Ca2+ from the cell. Two basic properties characterize them. 1) Their activity is not predicted by thermodynamic parameters of classical electrogenic countertransporters (dependence on ionic gradients and membrane potential), but is markedly regulated by transported (Na+ and Ca2+) and nontransported ionic species (protons and other monovalent cations). These modulations take place at specific sites in the exchanger protein located at extra-, intra-, and transmembrane protein domains. 2) Exchange activity is also regulated by the metabolic state of the cell. The mammalian and invertebrate preparations share MgATP in that role; the squid has an additional compound, phosphoarginine. This review emphasizes the interrelationships between ionic and metabolic modulations of Na+/Ca2+ exchange, focusing mainly in two preparations where most of the studies have been carried out: the mammalian heart and the squid giant axon. A surprising fact that emerges when comparing the MgATP-related pathways in these two systems is that although they are different (phosphatidylinositol bisphosphate in the cardiac and a soluble cytosolic regulatory protein in the squid), their final target effects are essentially similar: Na+-Ca2+-H+ interactions with the exchanger. A model integrating both ionic and metabolic interactions in the regulation of the exchanger is discussed in detail as well as its relevance in cellular Cai2+ homeostasis.

Values for ionized [Ca] in squid axons were obtained by measuring the light emission from a 0.1-mul drop of aequorin confined to a plastic dialysis tube of 140-mum diameter located axially. Ionized Ca had a mean value of 20 x 10(-9) M as judged by the subsequent introduction of CaEGTA/EGTA buffer (ratio ca. 0.1) into the axoplasm, and light measurement on a second aequorin drop. Ionized Ca in axoplasma was also measured by introducing arsenazo dye into an axon by injection and measuring the Ca complex of such a dye by multichannel spectrophotometry. Values so obtained were ca. 50 x 10(-9) M as calibrated against CaEGTA/EGTA buffer mixtures. Wth a freshly isolated axon in 10 mM Ca seawater, the aequorin glow invariably increased with time; a seawater [Ca] of 2-3 mM allowed a steady state with respect to [Ca]. Replacement of Na+ in seawater with choline led to a large increase in light emission from aequorin. Li seawater partially reversed this change and the reintroduction of Na+ brought light levels back to their initial value. Stimulation at 60/s for 2-5 min produced an increase in aequorin glow about 0.1% of that represented by the known Ca influx, suggesting operationally the presence of substantial Ca buffering. Treatment of an axon with CN produced a very large increase in aequorin glow and in Ca arsenazo formation only if the external seawater contained Ca.

A B S T R A C T A method has been developed to measure Ca influx in internally dialyzed squid axons. This was achieved by controlling the dialyzed segment of the axon exposed to the external radioactive medium. The capacity of EGTA to buffer all the Ca entering the fiber was explored by changing the free EGTA at constant 14 --+ 0.012 pmol.cm-2-s -l (n = 12). Removal of ATP drops the Ca influx to 0.085 -+ 0.007 pmol-cm-2.s -1 (n = 12). Ca influx increased to 0.35 pmol.cm-2.s -1 when Nao was removed. This increment was completely abolished by removing Na~ and (or) ATP from the dialysis medium. At nominal zero [Ca++]t, no Nat-dependent Ca influx was observed. In the presence of ATP and Na~, [Ca++], activates the Ca influx along a sigmoid curve without saturation up to 1 gM [Ca++]v Removal of Nai ~ always reduced the Ca influx to a value similar to that observed in the absence of [Ca++]~ (0.087 -+ 0.008 pmol.cm-Z.s-t; n = 11). Under the above standard conditions, 50-60% of the total Ca influx was found to be insensitive to Na~, Cai v+, and ATP, sensitive to membrane potential, and partially inhibited by external Co ++ .

The mechanisms governing the return of intracellular calcium (Ca2+i) to baseline levels following depolarization‐evoked [Ca2+]i rises were investigated in Purkinje cell somata using tight‐seal whole‐cell recordings and fura‐2 microfluorometry, for peak [Ca2+]i ranging from 50 nM to 2 μM. Ca2+i decay was well fitted by a double exponential with time constants of 0.6 and 3 s. Both time constants were independent of peak [Ca2+]i but the contribution of the faster component increased with [Ca2+]i. Thapsigargin (10 μM) and cyclopiazonic acid (50 μM) prolonged Ca2+i decay indicating that sarco‐endoplasmic reticulum Ca2+ (SERCA) pumps contribute to Purkinje cell Ca2+i clearance. A modest participation in clearance was found for the plasma membrane Ca2+ (PMCA) pumps using 5,6‐succinimidyl carboxyeosin (40 μM). The Na+‐Ca2+ exchanger also contributed to the clearance process, since replacement of extracellular Na+ by Li+ slowed Ca2+i decay. Carbonyl cyanide m‐chlorophenylhydrazone (CCCP, 2 μM) and rotenone (10 μM) increased [Ca2+]i and elicited large inward currents at −60 mV. Both effects were also obtained with CCCP in the absence of external Ca2+, suggesting that mitochondrial Ca2+ uptake uncouplers release Ca2+ from intracellular stores and may alter the membrane permeability to Ca2+. These effects were irreversible and impeded tests on the role of mitochondria in Ca2+i clearance. The relative contribution of the clearance systems characterized in this study varied as a function of [Ca2+]i. At 0.5 μM Ca2+i, SERCA pumps, PMCA pumps and the Na+‐Ca2+ exchanger contribute equally to removal and account for 78 % of the process. Only 45 % of the removal at 2 μM Ca2+i can be explained by these systems. In this high [Ca2+]i range the major contribution is that of SERCA pumps (21 %) and of the Na+‐Ca2+ exchanger (18 %), whereas the contribution of PMCA pumps is only 6 %.