Structural and electrostatic properties of the 5-HT3 receptor pore revealed by substituted cysteine accessibility mutagenesis.

5-HT(3) receptors are members of the Cys loop family of ligand-gated ion channels. We used the substituted cysteine accessibility method to identify amino acid residues in the channel forming domain, M2 that face the water-accessible surface and to locate their position in the ion conduction pathway. Cysteine was substituted for each residue, one at a time, in the M2 segment (Asp(274)-Asp(298)). 5-Hydroxytryptamine EC(50) values for functional mutants did not vary from wild type (1.4 +/- 0.2 microm) by more than 10-fold, and five mutants were nonfunctional. Covalent modification of the mutant receptors with sulfydryl reagents revealed 11 residues to be water-accessible, with a pattern consistent with an alpha-helix except at Leu(285) and Leu(293). The data suggest that charge selectivity begins at a more cytoplasmic level than Val(291). Modification at some positions (Val(291), Leu(293), Ile(294), Leu(287), and Ser(280)) resulted in channels that were locked open. Reaction rates with accessible cysteines were voltage-dependent at some residues, suggesting that access occurs via the ion channel. Overall the data observed are similar but not identical to that reported for other members of the family and confirms the high degree of structural and functional homology between receptors in the Cys loop receptor family.

Intrinsic metabolic depression in cells isolated from the hepatopancreas of estivating snails.

Many animals across the phylogenetic scale are routinely capable of depressing their metabolic rate to 5-15% of that at rest, remaining in this state sometimes for years. However, despite its widespread occurrence, the biochemical processes associated with metabolic depression remain obscure. We demonstrate here the development of an isolated cell model for the study of metabolic depression. The isolated cells from the hepatopancreas (digestive gland) of the land snail (Helix aspersa) are oxygen conformers; i.e., their rate of respiration depends on pO(2). Cells isolated from estivating snails show a stable metabolic depression to 30% of control (despite the long and invasive process of cell isolation) when metabolic rate at the physiological pH and pO(2) of the hemolymph of estivating snails is compared with metabolic rate at the physiological pH and pO(2) of the hemolymph of control snails. When the extrinsic effects of pH and pO(2) are excluded, the intrinsic metabolic depression of the cells from estivating snails is still to below 50% of control snails. The in vitro effect of pO(2) on metabolic rate is independent of pH and state (awake or estivating), but the effects of pH and state significantly interact. This suggests that pH and state change affect metabolic depression by similar mechanisms but that the metabolic depression by hypoxia involves a separate mechanism.