We have investigated the effects of Ca2+ on Na+ influx through ATP-activated channels in pheochromocytoma PC12 cells using single channel current recordings. Under cell-attached patch-clamp conditions with 150 mM Na+ and 2 mM Ca2+ in the pipette, the unitary current activity showed an open level of about -4.3 pA at -150 mV. The channel opening was interrupted by flickery noise as well as occasional transition to a subconducting state of about -1.7 pA at -150 mV. The open level was decreased with increased external Ca2+, suggesting that external Ca2+ blocks Na+ permeation. We assessed the block by Ca2+ as the mean amplitude obtained with heavy filtration according to Pietrobon et al. (Pietrobon, D., B. Prod'hom, and P. Hess, 1989. J. Gen. Physiol. 94:1-21). The block was concentration dependent with a Hill coefficient of 1 and a half-maximal concentration of approximately 6 mM. A similar block was observed with other divalent cations, and the order of potency was Cd2+ > Mn2+ > Mg2+ not equal to Ca2+ > Ba2+. High Ca2+, Mg2+ and Ba2+ did not block completely, probably because they can carry current in the channel. The block by external Ca2+ did not exhibit voltage dependence between -100 and -210 mV. In the inside-out patch-clamp configuration, the amplitude of inward channel current obtained with 150 mM external Na+ was reduced by increased internal Ca2+. The reduction was observed at lower concentrations than that by external Ca2+. Internal Ba2+ and Cd2+ induced similar reduction in current amplitude. This inhibitory effect of internal Ca2+ was voltage dependent; the inhibition was relieved with hyperpolarization. The results suggest that both external and internal Ca2+ can block Na+ influx through the ATP-activated channel. A simple one-binding site model with symmetric energy barriers is not sufficient to explain the Ca2+ block from both sides.
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1 March 1993
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March 01 1993
Block by calcium of ATP-activated channels in pheochromocytoma cells.
K Nakazawa,
K Nakazawa
Department of Cellular and Molecular Physiology, Harvard Medical School, Boston, Masssachusetts 02115.
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P Hess
P Hess
Department of Cellular and Molecular Physiology, Harvard Medical School, Boston, Masssachusetts 02115.
Search for other works by this author on:
K Nakazawa
Department of Cellular and Molecular Physiology, Harvard Medical School, Boston, Masssachusetts 02115.
P Hess
Department of Cellular and Molecular Physiology, Harvard Medical School, Boston, Masssachusetts 02115.
Online ISSN: 1540-7748
Print ISSN: 0022-1295
J Gen Physiol (1993) 101 (3): 377–392.
Citation
K Nakazawa, P Hess; Block by calcium of ATP-activated channels in pheochromocytoma cells.. J Gen Physiol 1 March 1993; 101 (3): 377–392. doi: https://doi.org/10.1085/jgp.101.3.377
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