Ca2+ permeability of mouse and chick nicotinic acetylcholine receptors expressed in transiently transfected human cells.
1. Combinations of cDNAs encoding mouse and chick nicotinic acetylcholine receptor (nAChR) subunits were transiently transfected into human BOSC 23 cells, and the expressed receptors were studied by simultaneously recording transmembrane currents and fluorescence transients using the whole-cell patch-clamp technique, and confocal microscopy with the Ca2+ indicator dye fluo-3. 2. The fractional Ca2+ current, Pf, of nAChRs was evaluated as the normalized ratio of nicotine-evoked fluorescence transient over total charge entering the cell (F/Q ratio). Mouse fetal muscle nAChR channels had a Pf, alphabetagammadelta value of 2.1 %. The substitution of the gamma subunit with the epsilon subunit resulted in a 2-fold increase in Pf (4.2 %). The difference in Ca2+ permeability was confirmed by determination of Ca2+/Cs+ permeability ratios. 3. Among the chick neuronal nAChRs tested, Pf,alpha3beta4 was 4.6 %, while Pf, alpha4beta4 and Pf,alpha4beta2 were 3.0 % and 2.9 %, respectively. 4. The amplitude of the current elicited by the activation of alpha3beta4 nAChRs increased as the external Ca2+ concentration was raised from 2 to 110 mM, whereas currents flowing through all other nAChRs tested were reduced to various extents. 5. Our findings indicate that the adult-type muscle nAChR (alphabetaepsilondelta) is more permeable to Ca2+ than the fetal-type (alphabetagammadelta), while ganglionic-like alpha3beta4 nAChR is more permeable to Ca2+ than the examined alpha4-containing nAChRs. The functional significance is discussed.
Ragozzino
D
D
Istituto Pasteur-Fondazione Cenci-Bolognetti and Dipartimento di Medicina Sperimentale e Patologia, Università di Roma, Italy. Ragozzino@crs.ifo.it
Barabino
B
B
Fucile
S
S
Eusebi
F
F
D000814:Aniline Compounds; D000818:Animals; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D015220:Calcium Channels / Q000187:drug effects / Q000502:physiology*; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000502:physiology; D002463:Cell Membrane Permeability; D002645:Chickens; D005333:Fetus; D005456:Fluorescent Dyes; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D051379:Mice; D008956:Models, Chemical; D018482:Muscle, Skeletal / Q000502:physiology; D009474:Neurons / Q000378:metabolism*; D009538:Nicotine / Q000494:pharmacology*; D018408:Patch-Clamp Techniques; D055503:Protein Multimerization; D011978:Receptors, Nicotinic / Q000096:biosynthesis / Q000502:physiology*; D011994:Recombinant Proteins / Q000096:biosynthesis; D014162:Transfection; D014966:Xanthenes
D000814:Aniline Compounds; D015220:Calcium Channels; D005456:Fluorescent Dyes; D046911:Macromolecular Substances; D011978:Receptors, Nicotinic; D011994:Recombinant Proteins; D014966:Xanthenes; C059715:Fluo-3; D009538:Nicotine; D002118:Calcium