Single-Channel Kinetic Analysis of Chimeric α7–5HT_3AReceptors

Abstract

The receptor chimera ␣7-5HT 3A 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 EC 50 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 5HT 3A receptor sequence,...

Number and Locations of Agonist Binding Sites Required to Activate Homomeric Cys-Loop Receptors

“…To recognize bursts and quantify their durations, a critical closed time ( crit ) was defined as the point of intersection between the second briefest and the succeeding components, and openings separated by closings briefer than this time constitute a burst (Rayes et al, 2005). For most of the recordings, crit ranged from 1 to 2 ms.…”

“…Mutant subunits were constructed using the QuikChange site-directed mutagenesis kit (Stratagene) and were confirmed by sequencing the entire cDNA insert. The high conductance form (HC) of the ␣7-5HT 3A chimeric receptor (Eiselé et al, 1993) was constructed as described previously (Rayes et al, 2005). In brief, three arginine residues responsible for the low conductance of the 5HT 3A receptor were mutated to glutamine (Q), aspartic acid (D), and alanine (A) (Kelley et al, 2003).…”

“…Solutions free of magnesium and with low calcium were used to minimize channel block by divalent cations (Rayes et al, 2005). Single-channel currents were recorded and low-pass filtered to 10 kHz using an Axopatch 200 B patch-clamp amplifier (Molecular Devices), digitized at 5 s intervals, and detected by the half-amplitude threshold criterion using the program TAC (Bruxton Corporation) (Bouzat et al, 2004;Rayes et al, 2005). Open-time histograms were fitted by the sum of exponential functions by maximum likelihood using the program TACFit (Bruxton Corporation).…”

“…To fill this gap, we studied a model homomeric Cys-loop receptor composed of the ligand binding domain from the ␣7 nicotinic receptor and pore and cytoplasmic domains from the 5-HT 3A receptor (Palma et al, 1996;Bouzat et al, 2004Bouzat et al, , 2008Rayes et al, 2005). To vary the number of agonist binding sites, we installed mutations that prevent agonist binding and, to report the presence of the mutant subunit, installed mutations that alter the single-channel conductance (Kelley et al, 2003).…”

Water-soluble LYNX1 Residues Important for Interaction with Muscle-type and/or Neuronal Nicotinic Receptors

“…It consists of two distinct parts: extracellular domain of chick ␣7 nAChR and the rest (transmembrane and cytoplasmic domains) of ␣1 human GlyR. This ␣7-GlyR chimera was selected for two main reasons: (i) at transient transfection in cell lines, it effectively forms recombinant pentameric channels producing whole-cell inward currents up to 2 nA on application of acetylcholine or nicotine; (ii) in contrast to native ␣7 nAChR (24,25) and the chimera composed of the ␣7 nAChR and subtype 3 of 5-hydroxytryptamine receptor (26,27), the ␣7-GlyR does not show desensitization (16). Moreover, activation kinetics of this chimeric receptor is very slow, which allows recording of responses reliably and independently of small variations in the speed of perfusion.…”