Ca2+-activated non-selective cation current in rabbit ventricular myocytes

Oscillatory currents (OCs) were studied in isolated rabbit ventricular myocytes with whole cell mode voltage clamp using Na+-free intracellular and extracellular solutions under conditions where K+ currents were anticipated to be eliminated or minimized. All OCs were dependent on release of Ca2+ from the sarcoplasmic reticulum (SR) because they were associated with intracellular Ca2+ ([Ca2+]i) transients, and were suppressed by high concentrations of BAPTA (20 mmol l−1) or pretreatment with the SR antagonist agents ryanodine (10 μmol l−1) or thapsigargin (1 μmol l−1). The reversal potential (Vrev) for OCs shifted with changes in the calculated Vrev for Cl− (ECl) but was between ECl and the calculated Vrev for elemental monovalent cations (ECat), indicating that more than one Ca2+-activated current contributed to OCs. Addition of the Ca2+-activated Cl− current (ICl(Ca)) antagonist, niflumic acid, shifted the OC Vrev to ECat, suggesting that ICl(Ca) and a Ca2+-activated non-selective cation current (ICAN) contributed to the observed OCs. A reduced niflumic acid-insensitive Ca2+-activated OC persisted following marked symmetrical reduction of Cl− in the intracellular and extracellular solutions. Subsequent removal of all extracellular monovalent cations, by N-methyl-D-glucamine (NMDG) substitution, eliminated OCs and the inward holding current suggesting that ICAN and ICl(Ca) accounted for all or most of the Ca2+-activated OC in the absence of Na+. The OC Vrev was equal to ECl in the absence of monovalent elemental cations. Under these conditions niflumic acid eliminated all OCs. Macroscopic OC is partially due to ICAN in rabbit ventricular myocytes. Ca2+-activated oscillatory currents (OCs) in cardiac muscle are likely to be important in the genesis of cardiac arrhythmias. Using physiological solutions, most observed OC seems to be accounted for by the electrogenic Na+-Ca2+ exchanger and by the Ca2+-activated chloride current (ICl(Ca)) (Zygmunt et al. 1998; Wu et al. 1999). Other potential sources of Ca2+-activated OC include inactivation of the L-type Ca2+ current (Sipido et al. 1995), inhibition of the inward rectifier current (IK1) (Zaza et al. 1998) and a Ca2+-activated non-selective cation current (ICAN) (Colquhoun et al. 1981; Ehara et al. 1988; Magishi et al. 1996). ICAN was first described in cultured rat cardiomyocytes using single channel recordings where it was found to lack voltage-dependent gating and to be equally permeant to Na+ and K+ (Colquhoun et al. 1981). Later workers using whole cell mode voltage clamp techniques also found evidence for ICAN in Purkinje fibres (Ehara et al. 1988) and ventricular myocytes (Giles & Shimoni, 1989). Another non-selective cation current has also been recorded in ventricular myocytes subjected to oxidative stress (Jabr & Cole, 1995) or following lysophosphatidyl choline-induced elevation of intracellular Ca2+ concentration ([Ca2+]i) (Magishi et al. 1996). However, some whole cell mode voltage clamp studies in ventricular myocytes have not supported the existence of ICAN (Sipido et al. 1995; Laflamme & Becker, 1996). Failure to implicate ICAN in some previous studies (Sipido et al. 1995; Laflamme & Becker, 1996) may be partially due to the lack of a specific antagonist, or differences in species or other experimental conditions. The present voltage clamp studies were performed in the absence of Na+ and after blockade of K+ currents in rabbit ventricular myocytes in whole cell mode. The hypothesis that ICAN is evident as a macroscopic current was tested by independently manipulating ECat and ECl.