© 1999 by European Society of Cardiology
Copyright © 1999, European Society of Cardiology
Decreased inward rectifier current in adult rabbit ventricular myocytes maintained in primary culture: a single-channel study
aDepartment of Experimental Cardiology, Academic Medical Center, University of Amsterdam, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands
b Department of Physiology, Academic Medical Center, University of Amsterdam, Meibergdreef 11, 1105 AZ Amsterdam, The Netherlands
* Corresponding author. Tel: +31-20-5663-269; fax: +31-20-6975-458
Objective: Regulation of ion channel function in heart has been shown to be affected by changes in the cellular environment. Recently it was shown that rabbit ventricular myocytes kept in primary culture, show a strong reduction in inward rectifier current (IK1). The aim of the present study was to elucidate the mechanism underlying this decrease in IK1, using single-channel measurements. In addition, we studied the effects of primary culture on the ATP-regulated K+ (K.ATP) channel, also a member of the inwardly rectifying K+ channel family. Methods: Adult rabbit ventricular myocytes were cultured for up to 3 days in Ham’s F-10 medium complemented with 1% rabbit serum and 5% glutamine. IK1 and K.ATP channel activity was studied in the inside-out patch configuration of the patch–clamp technique with equimolar K+ concentrations (140 mM K+) on the intra- and extracellular side. Single channel characteristics were determined at various times during culture and compared to those present in freshly isolated myocytes. Results: IK1 channels in freshly isolated myocytes (day 0) had a single-channel conductance of 56.1±2.5 pS (mean±SEM) and an open probability of 0.64±0.05 (mean±SEM). Neither the single-channel conductance nor the open probability (Po) underwent significant changes during culture. The mean number of channels per patch, however, was drastically reduced from 1.2±0.3 (mean±SEM) at day 0 to 0.17±0.06 at day three. K.ATP channel density and open probability, on the other hand, were both increased with an optimum at day two. Po increased from 0.27±0.06 at day 0 to 0.63±0.06 at day three. The mean number of channels per patch was 2.29±0.57 and 3.25±0.48 at days 0 and 3 respectively. The unitary current amplitude at –50 mV remained unchanged, suggesting no change in the K.ATP single-channel conductance. Conclusions: The decrease in IK1 in rabbit ventricular myocytes as has been observed during primary culture is the result of a reduction in the number of active channels and not of altered kinetic or conductive channel properties. The increase in K.ATP channel activity under the same conditions suggests that gene expression of both channel types is differently regulated.
KEYWORDS Cell culture/isolation; Ion channels; K–channel; Membrane currents; Myocytes
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