Single-Channel Kinetic Analysis of Chimeric 7–5HT3A Receptors

The receptor chimera 7–5HT3A has served as a prototype for understanding the pharmacology of 7 neuronal nicotinic receptors, yet its low single channel conductance has prevented studies of the activation kinetics of single receptor channels. In this study, we show that introducing mutations in the M3–M4 cytoplasmic linker of the chimera alters neither the apparent affinity for the agonist nor the EC50 but increases the amplitude of agonist-evoked single channel currents to enable kinetic analysis. Channel events appear as single brief openings flanked by long closings or as bursts of several openings in quick succession. Both the open and closed time distributions are described as the sum of multiple exponential components, but these do not change over a wide range of acetylcholine (ACh), nicotine, or choline concentrations. Bursts elicited by a saturating concentration of ACh contain brief and long openings and closings, and a cyclic scheme containing two open and two closed states is found to adequately describe the data. The analysis indicates that once fully occupied, the receptor opens rapidly and efficiently, and closes and reopens several times before it desensitizes. Channel closing and desensitization occur at similar rates and account for the invariant open and closed time distributions. The Cys-loop superfamily of neurotransmitter receptors includes receptors activated by acetylcholine (ACh), GABA, glycine, and 5-HT. Each of these neurotransmitters activates a corresponding pentameric receptor composed of identical subunits (i.e., a homopentamer). Because homopentameric receptors diverged least from the common ancestral receptor, they are expected to share fundamental mechanisms common to all members of the receptor superfamily and thus serve as prototypes. The homomeric 7 ACh receptor contributes to a wide range of neurophysiological processes, has been implicated in neurological diseases, and is a target for pharmacological agents (Gotti and Clementi, 2004). Nevertheless, little is known about the mechanism by which ACh activates 7 receptors, mainly because they are difficult to express in mammalian cells. The limited cell surface expression was traced to decreased palmitoylation of 7 (Drisdel et al., 2004); more recently, however, the chaperone protein ric-3 was found to promote cell-surface expression. As a result, ACh-evoked whole cell currents have been recorded from human embryonic kidney cells coexpressing 7 and ric-3 (Williams et al., 2005), but single channel currents have not been reported. Furthermore, although 7 receptors express in oocytes, few studies have reported single-channel currents, and analysis of single channel current kinetics has not been reported. In addition, several conductance states of channels were observed in both wild-type and mutant 7 AChRs expressed in oocytes (Palma et al., 1999; Fucile et al., 2000), complicating kinetic analysis. Chimeric receptors, composed of 7 sequence from the N terminus to the start of M1 followed by 5HT3A receptor sequence, form functional homopentameric receptors when expressed in mammalian cells (Eisele et al., 1993). For nearly a decade, the 7–5HT3 chimera has served as a prototype for investigating the pharmacology of homomeric 7 receptors, but its unitary conductance is too low to allow direct detection of single channel currents. However, by substituting neutral or negatively charged residues for three arginines in the cytoplasmic linker spanning the M3 and M4 domains (Kelley et al., 2003), we were able to resolve ACh-evoked single channel currents (Bouzat et al., 2004). We report here the kinetic analysis of currents through 7–5HT3 receptors and suggest that the 7–5HT3 chimera with increased conductance is a good model for studying the molecular pharmacology of homomeric Cys-loop receptors. This work was supported by grants from Consejo Nacional de Investigaciones Cientı́ficas y Técnicas, Universidad Nacional del Sur, Agencia Nacional de Promoción Cientı́fica y Tecnológica (to C.B.) and National Institutes of Health grant NS31744 (to S.M.S.). Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org. doi:10.1124/mol.105.015438. ABBREVIATIONS: ACh, acetylcholine; 5-HT3A, serotonin type 3A; BTX, -bungarotoxin; LC, low conductance; HC, high conductance. 0026-895X/05/6805-1475–1483$20.00 MOLECULAR PHARMACOLOGY Vol. 68, No. 5 Copyright © 2005 The American Society for Pharmacology and Experimental Therapeutics 15438/3061803 Mol Pharmacol 68:1475–1483, 2005 Printed in U.S.A. 1475 at A PE T Jornals on Sptem er 0, 2017 m oharm .aspeurnals.org D ow nladed from Materials and Methods Site-Directed Mutagenesis and Expression of 7–5HT3 Receptors. Mutant subunits were constructed using the QuikChange site-directed mutagenesis kit (Stratagene, Inc., La Jolla, CA). To increase unitary conductance for single-channel recordings, we mutated three arginine residues responsible for the low conductance of the serotonin type 3A receptor (5-HT3A) as follows: R432Q, R436D, and R440A (Kelley et al., 2003). Mutant subunits were confirmed by restriction enzyme analysis and sequencing. BOSC cells were transfected with 7–5HT3 subunit cDNAs using calcium phosphate precipitation, as described previously (Bouzat et al., 1994, 2000). A plasmid encoding green fluorescent protein (pGreen lantern) was also included to allow identification of transfected cells under fluorescence optics. Cells were used for single-channel measurements 1 or 2 days after transfection. Steady-State ACh Binding. Transfected cells were harvested by gentle agitation in phosphate-buffered saline, centrifuged at 1000g for 1 min, and re-suspended in potassium Ringer’s solution (140 mM KCl, 5.4 mM NaCl, 1.8 mM CaCl2, 1.7 mM MgCl2, 25 mM HEPES, 30 mg/liter bovine serum albumin, adjusted to pH 7.4 with 10 to 11 mM NaOH). Agonist binding was determined by competition of specified concentrations of ACh against the initial rate of I-bungarotoxin (BTX) binding as described previously (Sine et al., 1995). The total number of binding sites was determined by incubating cells with 25 nM I-BTX for 1 h and subtracting a blank determined in the presence of 10 M -BTX (Quiram and Sine, 1998). After computing fractional occupancy from the initial rates of toxin binding (Sine and Taylor, 1979), the following equation was fitted to the data: 1-fractional occupancy 1 [L]H/([L]H KH), where [L] is agonist concentration, K is the apparent dissociation constant, and nH is the Hill coefficient. Single-Channel and Macroscopic Patch-Clamp Recordings. Single-channel recordings were obtained in the cell-attached configuration (Hamill et al., 1981) at a membrane potential of 70 mV and at 20°C. The bath and pipette solutions contained 142 mM KCl, 5.4 mM NaCl, 0.2 mM CaCl2, and 10 mM HEPES, pH 7.4. Solutions free of magnesium and with low-calcium were used to minimize channel block by divalent cations. Patch pipettes were pulled from 7052 capillary tubes (Garner Glass, Claremont, CA) and coated with Sylgard (Dow Corning, Midland, MI). Pipette resistances ranged from 5 to 7 M . Agonists [ACh, ( )-nicotine, and choline (Sigma, St. Louis, MO)] were added to the pipette solution. Single-channel currents were recorded using an Axopatch 200 B patch-clamp amplifier (Molecular Devices, Sunnyvale, CA), digitized at 5s intervals with the PCI-6111E interface (National Instruments, Austin, TX), recorded to the computer hard disk using the program Acquire (Bruxton Corporation, Seattle, WA), and detected by the half-amplitude threshold criterion using the program TAC 4.0.10 (Bruxton Corporation) at a final bandwidth of 10 kHz (Gaussian digital filter). Openand closed-time histograms were plotted using a logarithmic abscissa and a square root ordinate (Sigworth and Sine, 1987) and fitted to the sum of exponential functions by maximum likelihood using the program TACFit (Bruxton Corporation, Seattle, WA). The Kd for fast blockade by the agonist was calculated according to the equation: Io/Ib 1 [B]/Kd, where Io is the current in the absence of blockade, Ib is the current at different concentrations of blocker (agonist), [B] is the blocker concentration, and Kd is the dissociation equilibrium constant for binding to the blocking site (Sine and Steinbach, 1984; Grosman and Auerbach,