Calcium modulation of 5-HT3 receptor binding and function.
Calcium modulates the 5-HT3 receptor response by reducing peak current amplitude and increasing rates of activation, deactivation and desensitisation, but the binding site(s) and mechanism(s) of this modulation are unknown. Here we study residues that may be involved in calcium binding in two partially overlapping regions of the extracellular domain (E213-E215-E218 and D204-E218-V219). The modulatory effects of calcium were assessed by radioligand binding and whole-cell patch-clamp. Comparisons of [3H]granisetron binding showed an increase in Kd in 10mM calcium that was abolished by the substitutions E213Q, E215Q, D204N and V219L. E218Q mutant receptors displayed no specific binding or function, and immunofluorescence showed that they did not reach the cell surface. E213Q increased inherent rates of desensitisation, but the relative effects of calcium on these rates, and on the reduction in current amplitude, were similar to wild type receptors. Current responses and calcium-mediated effects at E215Q mutant receptors were indistinguishable from wild type. D204N and V219L mutants were non-functional. A calcium impermeable mutant (E277A/S297R) revealed no changes in peak amplitude or kinetics with increased calcium. Our results are consistent with residues D204, E218 and V219 participating in receptor assembly, structure and/or trafficking to the plasma membrane, and we speculate that this might rely upon the stabilising effect of bound calcium. E213, E215, D204 and V219 may contribute to a calcium binding site that is responsible for the calcium-mediated effects on ligand binding. However, the major site for calcium-dependent modulation of the 5-HT3 current is located within the ion channel or cell interior.
Thompson
Andrew J
AJ
Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1QW, UK.
Lummis
Sarah C R
SC
D002118:Calcium / Q000378:metabolism / Q000494:pharmacology*; D002461:Cell Line, Transformed; D004305:Dose-Response Relationship, Drug; D017829:Granisetron / Q000494:pharmacology; D006801:Humans; D008564:Membrane Potentials / Q000187:drug effects / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D018408:Patch-Clamp Techniques; D011485:Protein Binding / Q000187:drug effects; D044406:Receptors, Serotonin, 5-HT3 / Q000235:genetics / Q000502:physiology*; D012701:Serotonin / Q000494:pharmacology; D012702:Serotonin Antagonists / Q000494:pharmacology; D014162:Transfection / Q000379:methods; D014316:Tritium / Q000494:pharmacology
D002118:Calcium; D017829:Granisetron; D044406:Receptors, Serotonin, 5-HT3; D012701:Serotonin; D012702:Serotonin Antagonists; D014316:Tritium