Regulation of L-type calcium channel and delayed rectifier potassium channel activity by p21-activated kinase-1 in guinea pig sinoatrial node pacemaker cells.

Phosphorylation of ion channels plays an important role in the regulation of cardiac function, but signaling mechanisms controlling dephosphorylation are not well understood. We have tested the hypothesis that p(21)-activated kinase-1 (Pak1), a serine-threonine protein kinase regulated by Ras-related small G proteins, regulates sinoatrial node (SAN) ion channel activity through a mechanism involving protein phosphatase 2A. We report a novel role of Pak1-mediated signaling in attenuating isoproterenol-induced enhancement of L-type Ca(2+) current (I(CaL)) and delayed rectifier potassium current (I(K)) in guinea pig SAN pacemaker cells. We demonstrate that in guinea pig SAN: (1) there is abundant expression of endogenous Pak1 in pacemaker cells; (2) expression of constitutively active Pak1 depresses isoproterenol-induced upregulation of I(CaL) and I(K); (3) inhibition of protein phosphatase 2A increases the enhancement of I(K) and I(CaL) by isoproterenol in Ad-Pak1-infected cells; (4) protein phosphatase 2A coimmunoprecipitates with endogenous Pak1 in SAN tissue; and (5) expression of constitutively active Pak1 suppresses the chronotropic action of isoproterenol on pacemaker activity of intact SAN preparations. In conclusion, our data demonstrate that a Pak1 signaling pathway exists in cardiac pacemaker cells and that this novel pathway plays a role in the regulation of ion channel activity.

Modulation of the hyperpolarization-activated current (I(f)) by calcium and calmodulin in the guinea-pig sino-atrial node.

The aim of this study was to investigate possible regulation of the hyperpolarization-activated current (I(f)) by cytosolic calcium in guinea-pig sino-atrial (SA) node cells. Isolated SA node cells were superfused with physiological saline solution (36 degrees C) and the perforated patch voltage-clamp technique used to record I(f) activated by hyperpolarizing voltage steps. A 10-min loading of SA node cells with the calcium chelator BAPTA (using 10 microM BAPTA-AM) significantly reduced the amplitude of I(f) at all potentials studied (69+/-8% at -80 mV, n=6). BAPTA loading also shifted the voltage of half-activation (V(h)) of the conductance from -83+/-2 mV in control to -93+/-2 mV in BAPTA (n=6) without significantly altering the slope of activation. The calmodulin antagonists W-7 (10 microM), calmidazolium (25 microM) and ophiobolin A (20 microM) caused similar reductions in I(f) amplitude (73+/-4, 86+/-9 and 59+/-6% at -80 mV, n=6, 5 and 4, respectively) and shifts in V(h) (11+/-3, 14+/-3 and 8+/-2 mV). In cells pre-treated with W-7, exposure to BAPTA caused no further reduction in current amplitude (n=6). I(f) current amplitude was unaffected by the calmodulin dependent kinase (CaMKII) inhibitor KN-93 (1 microM) although this CaMKII inhibition did reduce L-type calcium by 48+/-19% at 0 mV (n=3). These results are consistent with a role for calcium and calmodulin in the regulation of I(f), via a mechanism that is independent of CaMKII. Alterations in intracellular calcium during the cardiac cycle may be involved in fine tuning the voltage-dependent properties of I(f) and may thus determine its relative contribution to pacemaking in the SA node.

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 microM), to block I(f) (funny current) channels, slowed but did not stop the spontaneous activity, and tetrodotoxin (10 microM), 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 microM). 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 microM) 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.