Ion permeation and conduction in a human recombinant 5-HT3 receptor subunit (h5-HT3A).
1. 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. 2. 5-HT evoked transient inward currents (EC50 = 3.4 microM; 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 microM). 3. 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. 4. 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. 5. Single channel chord conductance (gamma) estimated from fluctuation analysis of macroscopic currents increased with membrane hyperpolarization from 243 fS at -40 mV to 742 fS at -100 mV. 6. 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 gamma contributes to this phenomenon. 7. 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+.
Brown
A M
AM
Department of Pharmacology and Neuroscience, Ninewells Hospital and Medical School, The University of Dundee, Dundee DD1 9SY, UK.
Hope
A G
AG
Lambert
J J
JJ
Peters
J A
JA
D002118:Calcium / Q000378:metabolism; D002413:Cations, Divalent / Q000378:metabolism; D002460:Cell Line; D002463:Cell Membrane Permeability; D003042:Cocaine / Q000494:pharmacology; D004553:Electric Conductivity; D005071:Evoked Potentials / Q000187:drug effects*; D006801:Humans; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D008536:Meglumine / Q000493:pharmacokinetics; D008787:Metoclopramide / Q000494:pharmacology; D008956:Models, Chemical; D017294:Ondansetron / Q000494:pharmacology; D011985:Receptors, Serotonin / Q000096:biosynthesis / Q000502:physiology*; D044406:Receptors, Serotonin, 5-HT3; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D012701:Serotonin / Q000494:pharmacology*; D012702:Serotonin Antagonists / Q000494:pharmacology*; D012964:Sodium / Q000378:metabolism; D014162:Transfection; D014403:Tubocurarine
D002413:Cations, Divalent; D011985:Receptors, Serotonin; D044406:Receptors, Serotonin, 5-HT3; D011994:Recombinant Proteins; D012702:Serotonin Antagonists; D012701:Serotonin; D017294:Ondansetron; D008536:Meglumine; D012964:Sodium; D008274:Magnesium; D003042:Cocaine; D008787:Metoclopramide; D002118:Calcium; D014403:Tubocurarine