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°C. The Na-Ca exchange current (I(Na-Ca)), L-type calcium current (I(Ca,L)), inward rectifier potassium current (I(KI)) 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, I(Na- 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, I(Na-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 μM Myr-FRCRCFa, I(Na-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 μM Myr-FRCRCFa for 1 h, I(Na-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,L), there was little difference in I(Ca,L) density between untreated and cells incubated with Myr-FRCRCFa. A Boltzmann fit to the I(Ca,L)/V relation showed no significant alteration of half-maximal activation potential or slope factor of activation. I(KI) 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 I(Na-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 I(Na-Ca) blocker. I(Ca,L), I(KI) 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.