Effects of amiodarone on mutant Na+/Ca2+ exchangers expressed in CCL 39 cells.
Using the whole cell voltage clamp, we reported previously that amiodarone acutely inhibits Na+/Ca2+ exchange current (INCX) 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+/Ca2+ 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. INCX was recorded by ramp pulses. Amiodarone at 30 microM inhibited INCX by 80% in cells expressing wild type NCX1. However, 30 microM amiodarone inhibited INCX by about 55% in cells expressing mutant NCX1 with amino acids 217-671 (DeltaXIP) or 247-671 (Delta247-671) deleted in the long intracellular loop between the transmembrane segments (TM) 5 and 6. INCXs 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 INCX 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.
Watanabe
Yasuhide
Y
Department of Ecology and Clinical Therapeutics, School of Nursing, Fukushima Medical University, Fukushima 960-1295, Japan. w-yasu@tm.hama-med.ac.jp
Iwamoto
Takahiro
T
Matsuoka
Isao
I
Ono
Tomoyuki
T
Shigekawa
Munekazu
M
Kimura
Junko
J
D000638:Amiodarone / Q000494:pharmacology*; D000818:Animals; D006224:Cricetinae; D004285:Dogs; D005786:Gene Expression Regulation / Q000187:drug effects / Q000502:physiology; D009154:Mutation / Q000187:drug effects / Q000502:physiology*; D051381:Rats; D019831:Sodium-Calcium Exchanger / Q000096:biosynthesis* / Q000235:genetics*
D019831:Sodium-Calcium Exchanger; D000638:Amiodarone