Effects of amiodarone on mutant Na +/Ca 2+ exchangers expressed in CCL 39 cells

Abstract

Using the whole cell voltage clamp, we reported previously that amiodarone acutely inhibits Na +/Ca 2+ exchange current ( I NCX) in guinea pig cardiac ventricular myocytes. Intracellular application of trypsin via the patch pipette attenuated the blocking effect of amiodarone, suggesting that amiodarone affects the Na +/Ca 2+ exchanger (NCX) from the cytoplasmic side. Here, we attempted to detect the site of amiodarone inhibition using wild type NCX1, mutants, and NCX3 expressed in CCL39 fibroblasts. I NCX was recorded by ramp pulses. Amiodarone at 30 μM inhibited I NCX by 80% in cells expressing wild type NCX1. However, 30 μM amiodarone inhibited I NCX by about 55% in cells expressing mutant NCX1 with amino acids 217-671 (ΔXIP) or 247-671 (Δ247-671) deleted in the long intracellular loop between the transmembrane segments (TM) 5 and 6. I NCXs from NCX mutants deleted of cytoplasmic TM1-2, TM3-4 or the C-terminus were inhibited by amiodarone to a similar extent as the wild type. Amiodarone also inhibited I NCX of NCX3 by 76%. These results suggest that a long intracellular loop may be involved in the inhibition of NCX1 by amiodarone, but that other intracellular loops, XIP region or C terminus are not involved in the amiodarone inhibition of NCX1.