Actions of myristyl-FRCRCFa, a cell-permeant blocker of the cardiac sarcolemmal Na-Ca exchanger, tested in rabbit ventricular myocytes

In cardiac muscle, the electrogenic Na-Ca exchanger plays important roles in determining action potential shape and in the beat-to-beat homeostasis of intracellular calcium. In this study we tested the actions of a putative cell-permeant blocker of the cardiac sarcolemmal Na-Ca exchange, "Myristyl- (Myr-) FRCRCFa". Experiments were performed using isolated rabbit right ventricular myocytes and whole-cell patch-clamp at 35-37 degrees C. The Na-Ca exchange current (INa-Ca), L-type calcium current (I-Ca,I-L), inward rectifier potassium current (I-K1) and delayed rectifier potassium current (I-K) were compared in untreated cells and cells incubated in a solution containing N-myristylated FRCRCFa. With other major currents blocked, INa-Ca was measured as the Ni-sensitive component of current during a voltage ramp applied from the holding potential of -40 mV, between +80 and -120 mV (ramp velocity 0.1 V s(-1)). In untreated cells, INa-Ca at +60 mV was 7.1+/-0.6 pA/pF and at -100 mV was -2.7+/-0.3 pA/pF (n=9). After a 15-min pre-incubation with 20 mu M Myr-FRCRCFa, INa-Ca was reduced to 4.2+/-0.3 pA/pF at +60 mV and -1.5+/-0.2 pA/pF at -100 mV (P<0.02; n=7). After incubation with 20 mu M Myr-FRCRCFa for 1 h, INa-Ca at both potentials was further reduced (2.3+/-0.8 pA/pF at +60 mV; -0.9+/-0.3 pA/pF at -100 mV; P<0.008 compared with control; n=4). Under selective recording conditions for I-Ca,I-L there was little difference in I-Ca,I-L density between untreated and cells incubated with Myr-FRCRCFa. A Boltzmann fit to the I-Ca,I-L/V relation showed no significant alteration of half-maximal activation potential or slope factor of activation. I-K1 was also largely unaffected by pre-incubation of cells with Myr-FRCRCFa. I-K, measured as deactivating tail current following 1-s test depolarisations to a range of test potentials, was also not significantly altered by Myr-FRCRCFa. The suppression of INa-Ca in cells incubated in Myr-FRCRCFa suggests that addition of the myristyl group to FRCRCFa peptide conveys cell permeancy to the peptide and that Myr-FRCRCFa applied externally to rabbit ventricular myocytes is moderately effective as an IN,ca blocker. I-Ca,I-L, I-K1 and I-K were largely unaffected by Myr-FRCRCFa. N-Myristylation of such conformationally constrained hexapeptides may, therefore, provide a means of producing cell-permeant inhibitors of the cardiac Na-Ca exchanger.