Fundamental importance of Na + –Ca 2+ exchange for the pacemaking mechanism in guinea‐pig sino‐atrial node

Na + –Ca 2+ exchange (NCX) current has been suggested to play a role in cardiac pacemaking, particularly in association with Ca 2+ 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 + –Ca 2+ 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 μ m ), to block I f (funny current) channels, slowed but did not stop the spontaneous activity, and tetrodotoxin (10 μ 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 Ca 2+ chelators BAPTA and EGTA, and by exposure to the NCX inhibitor KB‐R7943 (5 μ m ). When rapid switch to low‐Na + solution caused cessation of spontaneous activity, this was found (using confocal microscopy, with fluo‐4 as the Ca 2+ probe) to be accompanied by an initial fall in cytosolic [Ca 2+ ], with subsequent appearance of Ca 2+ waves. Inhibition of SR Ca 2+ uptake with cyclopiazonic acid (CPA, 30 μ m ) slowed but did not stop spontaneous activity. Rapid switch to low‐Na + solution in the presence of CPA caused abolition of spontaneous Ca 2+ transients and a progressive rise in cytosolic [Ca 2+ ]. With ratiometric fluorescence methods (indo‐5F as the Ca 2+ probe), the minimum level of [Ca 2+ ] between beats was found to be approximately 225 n m , and abolition of beating with nifedipine, acetylcholine or adenosine caused a fall in cytosolic [Ca 2+ ] 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 [Ca 2+ ] that normally does not fall below 225 n m between beats) and/or from a novel, recently suggested role of the NCX protein to allow a Na + leak pathway.