Programme Contents

MA-stretch residues are critical for ion conduction of 5-HT_3A receptors

5-Hydroxytryptamine type 3 (5-HT₃) receptors are members of the Cys-loop ligand-gated ion channel receptor superfamily. Members of this family, that include nicotinic acetylcholine (nACh), glycine and GABA_A receptors, convert the chemical messages conveyed by neurotransmitters into electrical impulses through the selective conduction of ions. Electron microscopic images of the Torpedo marmorata nACh receptor suggest that residues within a helical stretch (the MA-stretch) of the M3-M4 cytoplasmic loop line intracellular portals that are an obligate pathway for ion flux (Unwin, 2005). It has recently been demonstrated that three arginine residues (R432, R436, R440) within the human 5-HT_3A MA-stretch are critical determinants of single channel conductance (γ) (Kelley et al., 2003). The collective replacement of these residues by their 5-HT_3B counterparts (producing the 5-HT_3AQDA receptor) increased γ 29-fold, with R436D exerting the greatest influence.

In the current study, the influence of all 5-HT_3A receptor MA-stretch residues (positions 426 to 442) on γ was investigated. A combination of alanine and arginine scanning and the substituted cysteine accessibility method (SCAM) was employed. Site-directed mutagenesis was used to introduce alanine, arginine and cysteine residues along the MA-stretch, one at a time. Mutations were introduced into a 5-HT_3AQDA receptor (Kelley et al., 2003) to allow detection of single channel events evoked by 5-HT in outside-out membrane patches. All mutant subunits produced functional receptors when expressed in tsA-201 cells, with the exception of the 5-HT_3AQDA(W442R) and 5-HT_3AQDA(W442C) subunits.

The introduction of alanine residues along the MA-stretch typically resulted in a decrease in γ compared to 5-HT_3AQDA receptors. In particular, with the exception of the R426A mutation, removal of a charged residue resulted in a significant change in γ (p < 0.01). The greatest change in γ was a ∼2-fold decrease observed with the D436A mutation. Alanine mutant receptors were treated as controls for the remaining experiments.

The mutation of MA-stretch residues to the positively charged arginine residue typically resulted in a decrease in γ compared to alanine controls. This decrease was significant with the introduction of arginine at positions 427 and 431 to 440 (p < 0.05, n = 3-7). The greatest reduction in γ was observed with arginine present at the 436 and 440 positions. In contrast, γ was not reduced when arginine occupied positions 427, 428 and 431. Unexpectedly, the L429R mutation significantly increased γ (p < 0.01, n = 12).

Typically, the introduction of cysteine residues had little effect on receptor γ compared to control alanine mutants. Of the cysteine mutant subunits analyzed, addition of the positively charged methanethiosulfonate (MTS) reagent, MTSEA (200 μM), to the electrode solution reduced receptor γ compared to controls. The changes in γ mimicked the effects observed with the introduction of the positively charged arginine residues at all but three positions, suggesting successful reaction of MTSEA with introduced cysteine residues. This decrease in γ was significant at receptors containing the E434C to D436C and V438C to A440C mutations (p < 0.001, n = 4-5), the greatest change being seen with the modification of cysteine residues at positions 435, 436 and 440.

This work establishes that a substantial portion of the 5-HT_3A MA-stretch influences ion conduction. In particular, residues at the 436 and 440 positions are the major critical determinants of γ.

Kelley SP, Dunlop JI, Kirkness EF, Lambert JJ & Peters JA. (2003) Nature, 424: 321-4.

Unwin N. (2005) The Journal of Molecular Biology, 346: 967-89.