Neuronal nicotinic acetylcholine receptor currents in phaeochromocytoma (PC12) cells: dual mechanisms of rectification.
1. Neuronal nicotinic acetylcholine receptor (nAChR) currents in PC12 cells were studied using single-channel and whole-cell gigaohm seal voltage-clamp techniques. Nicotinic AChR agonists were applied using external pipettes. 2. The single-channel conductance of neuronal nAChRs in outside-out patches under Mg(2+)-free ionic conditions was 48 pS. In the absence of internal Mg2+ single-channel currents (outside-out patches) did not rectify. 3. Whole-cell nAChR currents recorded with normal internal solution (lacking Mg2+ chelators as described below) were strongly inwardly rectifying. Current vs. voltage relations showed a sharp inflexion near 0 mV, and outward current was never observed. 4. Extensive dialysis with internal solution containing EDTA and Na2-ATP, chelators of intracellular Mg2+ (Mgi2+), relieved rectification of instantaneous nAChR currents during voltage jumps from negative potentials. The instantaneous I-V relation became linear with voltage, in agreement with the expectation from single-channel measurements made under similar ionic conditions. 5. Agonist-induced currents recorded under Mgi(2+)-free conditions relaxed towards zero current during depolarizing voltage steps. Rectification of ACh-induced currents at the steady state could be described by a Boltzmann relation, with one-half of channels available at +3.4 mV and an effective gating charge of -0.97. Channel availability was approximately 95% at the resting potential. Opening and closing relaxations under Mgi(2+)-free conditions could be fitted by single exponential functions whose time constants were weakly voltage dependent. 6. A model of rectification was constructed incorporating two voltage-dependent processes: block by Mgi2+ and intrinsic channel gating. The I-V relations predicted by this model for both normal and Mgi(2+)-free conditions were in good agreement with the experimental data. We suggest that rectification of nAChR currents in these cells is due to concurrent activity of these two voltage-dependent processes. The relative contributions of these two mechanisms are frequency dependent, with block by Mgi2+ dominant for fast events and intrinsic channel gating more important at the steady state.
Sands
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SB
Department of Physiology and Biophysics, College of Medicine, University of California, Irvine 92717.
Barish
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ME
D000818:Animals; D015640:Ion Channel Gating / Q000502:physiology; D007473:Ion Channels / Q000502:physiology*; D008274:Magnesium / Q000378:metabolism; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D016716:PC12 Cells; D010276:Parasympatholytics / Q000494:pharmacology; D051381:Rats; D011978:Receptors, Nicotinic / Q000502:physiology*; D013997:Time Factors
D007473:Ion Channels; D010276:Parasympatholytics; D011978:Receptors, Nicotinic; D008274:Magnesium