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Department of Anesthesiology, Washington University School of Medicine, St Louis, MO 63110.
1. Currents elicited by acetylcholine (ACh) were studied in the rat phaeochromocytoma cell line, PC12, using patch-clamp techniques. 2. Whole-cell ACh-elicited currents are inwardly rectifying and intracellular Mg2+ can play a role in determining the extent of whole-cell current rectification. Increasing the intracellular Mg2+ concentration, [Mg2+]i, augmented the rectification. The effects of increased [Mg2+]i on the whole-cell current can be explained by the block of receptor channels by Mg2+. 3. In the nominal absence of internal divalent cations, however, a substantial degree of rectification remains. This rectification is probably not due to divalent cations, as buffering the external Mg2+ concentration to 50 microM and the internal concentration to nominally 0 Mg2+ did not reduce the rectification. The remanent rectification was not due to block by the main permeant cation, Na+. Using K+ or Cs+ as the main monovalent cation inside the cell did not diminish the rectification. Neither replacing the pH buffer, HEPES, with phosphate buffer nor increasing the intracellular pH removed the rectification. 4. For ACh receptor channels in excised patches, the voltage dependence of the probability of being open (Popen) stemmed mainly from the voltage dependence of the channel burst duration. The channel opening rate was relatively voltage independent. The weak voltage dependence displayed by the channel burst duration was insufficient to account for the reduced whole-cell outward current at positive potentials. The mean burst duration of the channel did not have a simple logarithmic relationship with voltage. 5. In the absence of intracellular Mg2+, the instantaneous current-voltage relationship for whole-cell currents was linear suggesting that the I-V relationship of single channels in perfused cells is linear and does not contribute to the rectification of the whole-cell current. 6. In perfused cells, receptor channels had a low steady-state probability of being open at positive potentials compared to channels in excised patches. Voltage jumps to positive potentials revealed a process in perfused cells which could account for the low Popen. Relaxations of agonist-induced current at +40 mV had a large, exponentially decaying component that quickly closed channels (rate constant, tau, approximately 400 microseconds). The mechanism responsible for this decay could explain the rectification that remains in the absence of intracellular divalent cations.(ABSTRACT TRUNCATED AT 400 WORDS)
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