Ion permeation and conduction in a human recombinant 5‐HT₃ receptor subunit (h5‐HT_3A)

Abstract

A human recombinant homo‐oligomeric 5‐HT3 receptor (h5‐HT3A) expressed in a human embryonic kidney cell line (HEK 293) was characterized using the whole‐cell recording configuration of the patch clamp technique. 5‐HT evoked transient inward currents (EC50= 3.4 μM; Hill coefficient = 1.8) that were blocked by the 5‐HT3 receptor antagonist ondansetron (IC50= 103 pM) and by the non‐selective agents metoclopramide (IC50= 69 nM), cocaine (IC50= 459 nM) and (+)‐tubocurarine (IC50= 2.8 μM). 5‐HT‐induced currents rectified inwardly and reversed in sign (E5‐HT) at a potential of −2.2 mV. N‐Methyl‐D‐glucamine was finitely permeant. Permeability ratios PNa/PCs and PNMDG/PCs were 0.90 and 0.083, respectively. Permeability towards divalent cations was assessed from measurements of E5‐HT in media where Ca2+ and Mg2+ replaced Na+. PCa/PCs and PMg/PCs were calculated to be 1.00 and 0.61, respectively. Single channel chord conductance (γ) estimated from fluctuation analysis of macroscopic currents increased with membrane hyperpolarization from 243 fS at −40 mV to 742 fS at −100 mV. Reducing [Ca2+]o from 2 to 0.1 mM caused an increase in the whole‐cell current evoked by 5‐HT. A concomitant reduction in [Mg2+]o produced further potentiation. Fluctuation analysis indicates that a voltage‐independent augmentation of γ contributes to this phenomenon. The data indicate that homo‐oligomeric receptors composed of h5‐HT3A subunits form inwardly rectifying cation‐selective ion channels of low conductance that are permeable to Ca2+ and Mg2+.

An Interaction Involving an Arginine Residue in the Cytoplasmic Domain of the 5-HT3A Receptor Contributes to Receptor Desensitization Mechanism

“…Noise Analysis-Single channel conductances were determined using nonstationary variance analysis (20). Whole cell patch-clamp recording was performed at room temperature using an Axopatch 1D or 200B (Axon Instruments) amplifier.…”

The structural basis of function in Cys-loop receptors

“…Electrophysiological data support the suggestion that the charged groups line portals within the sides of the ICD, and influence the ion flux between the cytoplasm and the inner vestibule at the base of the pore (Miyazawa et al 1999 ;Unwin, 2000). As the widest region of the portals resembles the diameter of a hydrated permeant ion, they provide an explanation for the homomeric 5-HT 3 channel having a much smaller unitary conductance than most nAChRs, despite their similar ionic permeabilities and very similar M2 sequences (Brown et al 1998 ;Lambert et al 1989 ;Malone et al 1991 ;Mochizuki et al 2000 ;Yakel et al 1990 ;Yang, 1990). Additional studies have shown that the conductance of the channel can be dynamically altered by sulphydryl modifying reagents (Deeb et al 2007) and the permeability of divalent cations is also altered by mutations in this region (Livesey et al 2008).…”

“…The effects of these ions vary according to the receptor type and subunit composition. For example, in the a7 nAChR, these cations potentiate responses, while 5-HT 3 R responses are typically reduced (Brown et al 1998;Hu & Lovinger, 2005 ;Niemeyer & Lummis, 2001 ;Thompson & Lummis, 2008a). Ionbinding sites in many Cys-loop receptors have been identified in the channel (Bertrand et al 1993 ;Eddins et al 2002aEddins et al , 2002bGill et al 1995 ;Hu & Lovinger, 2005 ;Livesey et al 2008 ;Niemeyer & Lummis, 2001 ;Noam et al 2008 ;Quirk et al 2004 ;Thompson & Lummis, 2008a ;Van Hooft & Wadman, 2003), but there are also binding sites in other regions of these proteins.…”

“…Five symmetrically placed M2 helices from each of the five subunits create a hydrophobic region that is 3 Å at its narrowest and less than 3Á5 Å over a distance of approximately 8 Å in the closed state, and has been referred to as a hydrophobic girdle (Miyazawa et al 2003). Ion permeabilities suggest that the diameter of the open channel is between 7Á4 Å and 8Á4 Å for cation channels and between 5Á2 Å and 6Á2 Å for anion channels (Brown et al 1998 ;Cohen et al 1992 ;Fatima-Shad & Barry, 1993 ;Rundstrom et al 1994 ;Wang & Imoto, 1992). Originally, the channel gate was predicted to be close to the cytoplasmic end of M2 (Wilson & Karlin, 1998 ;Wilson et al 2000).…”

“…In the 5-HT 3 R, A-subunits can form functional homomeric channels with a conductance <1 pS, but when combined with B-subunits receptors display a much larger conductance (9-17 pS; Brown et al 1998;Davies et al 1999 ;Derkach et al 1989 ;Hussy et al 1994). By replacing parts of the A-subunit sequence with homologous regions from the B-subunit, Kelley et al (2003) identified three amino acids that govern the differences between the low conductance of the homomeric receptor and the higher conductance of the heteromeric receptor, and which align with a polar stripe of residues identified by Finer-Moore & Stroud (1984).…”