Fundamental importance of Na+-Ca2+ exchange for the pacemaking mechanism in guinea-pig sino-atrial node

Na+-Ca2+ exchange (NCX) current has been suggested to play a role in cardiac pacemaking, particularly in association with Ca2+ release from the sarcoplasmic reticulum (SR) that occurs just before the action potential upstroke. The present experiments explore in more detail the contribution of NCX to pacemaking. Na+-Ca2+ exchange current was inhibited by rapid switch to low-Na+ solution (with Li+ replacing Na+) within the time course of a single cardiac cycle to avoid slow secondary effects. Rapid switch to low-Na+ solution caused immediate cessation of spontaneous action potentials. ZD7288 (3 mu M), to block I-f (funny current) channels, slowed but did not stop the spontaneous activity, and tetrodotoxin (10 mu M), to block Na+ channels, had little effect, but in the presence of either of these agents, rapid switch to low-Na+ solution again caused immediate cessation of spontaneous action potentials. Spontaneous electrical activity was also stopped following loading of the cells with the Ca2+ chelators BAPTA and EGTA, and by exposure to the NCX inhibitor KB-R7943 (5 mu M). When rapid switch to low-Na+ solution caused cessation of spontaneous activity, this was found (using confocal microscopy, with fluo-4 as the Ca2+ probe) to be accompanied by an initial fall in cytosolic [Ca2+], with subsequent appearance of Ca2+ waves. Inhibition of SR Ca2+ uptake with cyclopiazonic acid (CPA, 30 mu M) slowed but did not stop spontaneous activity. Rapid switch to low-Na+ solution in the presence of CPA caused abolition of spontaneous Ca2+ transients and a progressive rise in cytosolic [Ca2+]. With ratiometric fluorescence methods (indo-5F as the Ca2+ probe), the minimum level of [Ca2+] between beats was found to be approximately 225 nM, and abolition of beating with nifedipine, acetylcholine or adenosine caused a fall in cytosolic [Ca2+] below this level. These observations support the hypothesis that NCX current is essential for normal pacemaker activity under the conditions of our experiments. A continuous depolarizing influence of current through the NCX protein might result from maintained electrogenic NCX (with 3:1 stoichiometry, supported by a cytosolic [Ca2+] that normally does not fall below 225 nM between beats) and/or from a novel, recently suggested role of the NCX protein to allow a Na+ leak pathway.