Rectification of acetylcholine-elicited currents in PC12 pheochromocytoma cells.

Abstract

The current-voltage (I-V) relationship for acetylcholine-elicited currents in the rat pheochromocytoma cell line PC12 is nonlinear. Two voltage-dependent processes that could account for the whole-cell current rectification were examined, receptor channel gating and single receptor channel permeation. We found that both factors are involved in the rectification of the whole-cell currents. The voltage dependence of channel gating determines the shape of the I-V curve at negative potentials. The single-channel I-V relationship is inwardly rectifying and largely responsible for the characteristic shape ofthe whole-cell I-V curve at positive potentials. Johnson, Washington University School of Medicine). Cultures were fed three times a week and used 5-10 days after plating.Physiological Recordings. Recordings were obtained using the List EPC-7 patch clamp. Records from whole-cell and outside-out patches (12) were acquired using the PCLAMP programs (Axon Instruments, Foster City, CA) and a 386 Zenith personal computer. Data were analyzed using BINFITS (C. Lingle, Washington University School of Medicine). Pipettes were pulled from KG33 borosilicate capillaries by using a P-80/PC micropipette puller (Sutter Instrument, San Rafael, CA) and coated with Sylgard 182 (Dow). After fire-polishing, pipette resistances were 2-5 MQ when the pipettes were filled with sodium isethionate intracellular solution (see below). For whole-cell recordings, the EPC-7 circuitry was used to cancel the capacity charging transients and to compensate for the series resistance, which ranged from 5 to 30 MQ. Series resistances were 70-80% compensated. Whole-cell and single-channel records were filtered at 2 kHz with an eight-pole low-pass Bessel filter. All experiments were conducted at room temperature (18-19'C) unless specified otherwise.Whole-cell I-V relationships were determined from peak currents elicited by ACh application to cells held at various potentials between -80 and +80 mV.Voltage-jump records were obtained by bracketing a series of jumps in the presence of ACh with jumps done in the absence of agonist. Voltage-jump records were leaksubtracted averages of 12-18 jumps. Current relaxations were fit with a single exponential or the sum of two exponential components by using the BINFITS program. Best fits were determined by visual inspection.Single-channel I-V relationships were determined using voltage ramps and voltage jumps. An AI2020 event detector (Axon Instruments, Foster City, CA) was used to trigger the computer to initiate the voltage ramp or jump when a single Abbreviations: ACh, acetylcholine; SCG, superior cervical ganglion;I-V, current-voltage. 4794The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Inward rectification of acetylcholine‐elicited currents in rat phaeochromocytoma cells.

“…In contrast to muscle-type receptors where the weak inward rectification can be explained by the voltage dependence of the apparent channel closing rate (Dionne & Stevens, 1975), the mechanisms underlying the inward rectification of ACh-elicited currents in ganglion cells have not been fully elucidated. The voltage dependence of gating for ganglionic ACh receptors in excised patches appears to be relatively weak (Ifune & Steinbach, 1990b;Mathie, Colquhoun & Cull-Candy, 1990) and is not steep enough to be the sole explanation for the rectification of whole-cell ACh elicited currents. In excised patches, the single channel current-voltage relationship displays inward rectification in the presence of intracellular Mg2+ (Neuhaus & Cachelin, 1990;Ifune & Steinbach, 1990bMathie et al 1990).…”

“…The voltage dependence of gating for ganglionic ACh receptors in excised patches appears to be relatively weak (Ifune & Steinbach, 1990b;Mathie, Colquhoun & Cull-Candy, 1990) and is not steep enough to be the sole explanation for the rectification of whole-cell ACh elicited currents. In excised patches, the single channel current-voltage relationship displays inward rectification in the presence of intracellular Mg2+ (Neuhaus & Cachelin, 1990;Ifune & Steinbach, 1990bMathie et al 1990). However, many investigators have shown that removal of divalent cations from the intracellular solution does not have a dramatic effect on the current-voltage relationship of the macroscopic ACh-elicited current (Hirano et al 1987;Mathie et al 1987;Yawo, 1989;Neuhaus & Cachelin, 1990).…”

Inhibitory effects of capsaicin on acetylcholine‐evoked responses in rat phaeochromocytoma cells

“…Inhibition by capsaicin of ACh-activated current ACh (30 to 300 jiM) elicited a transient inward current in PC12 cells as was previously described (Ifune & Steinbach, 1990). Capsaicin (1 and 3 tM) suppressed this ACh-elicited current (Figure 3).…”

“…Ifune & Steinbach (1990) in PC12 cells. Capsaicin did not affect the voltage-dependence of the ACh-activated current in four out of four cells tested: the current activated in the presence of capsaicin also exhibited inward rectification and the inward component in the negative potential range was uniformly suppressed (Figure 5a).…”

Nicotinic Acetylcholine Receptors

“…This suggested that rectification is caused either by channel block by intracellular components (that are not present in artificial intracellular solutions) and/or by voltagedependence of the channel kinetics (Mathie et al, 1990). We now know that both are important: the major role is played by channel block by micromolar concentration of the intracellular polyamine, spermine (Haghighi & Cooper, 1998), with a minor contribution by intracellular magnesium ions (Mathie et al, 1990;Ifune & Steinbach, 1990;Ifune & Steinbach, 1992) and by the voltage dependence of the channel P open . While this work was carried out on native neuronal nicotinic receptors of autonomic ganglia (and on recombinant α4β2 and α3β4 channels), it is likely that similar mechanisms underlie the rectification of other neuronal nicotinic receptors (Alkondon et al, 1994).…”