Fractional calcium currents through recombinant GluR channels of the NMDA, AMPA and kainate receptor subtypes.
1. Simultaneous fluorescence and whole-cell current measurements using the calcium indicator dye fura-2 were made in HEK 293 cells expressing recombinant glutamate receptor (GluR) channels, and fractional Ca2+ currents (the proportion of whole-cell current carried by Ca2+; Pf) were determined. 2. Cells expressing N-methyl-D-aspartate receptor (NMDAR) channels showed glutamate activated Ca2+ inflow in the voltage range -60 to 40 mV in normal extracellular solution. Ca2+ inflow decreased in a voltage-dependent manner at membrane potentials more negative than -30 mV due to Mg2+ block. Voltage dependence of block at negative potentials was stronger in cells expressing the NR1-NR2A as compared with cells expressing NR1-NR2C subunits. 3. Fractional Ca2+ currents through NMDARs were independent of extracellular Mg2+ and varied between 8.2% (NR1-NR2C subunits) and 11% (NR1-NR2A subunits) in normal extracellular solution (1.8 mM Ca2+) at -60 mV membrane potential. Pf values increased with increasing [Ca2+]o in the range of 0.5-10mM [Ca2+]o in a saturating fashion. 4. In cells expressing alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptor (AMPAR) subunits which were unedited at the Q/R site of the putative transmembrane segment TM2 (Q-form), or in cells coexpressing unedited and edited subunits (R-form), the glutamate-evoked Ca2+ inflow increased from 20 to -80 mV in an almost linear way. 5. Fractional Ca2+ currents through AMPAR channels depended on subunit composition. Pf values of Q-form homomeric channels at -60 mV and 1.8 mM [Ca2+]o were between 3.2 and 3.9%. They were slightly voltage dependent and increased with [Ca2+]o in the range 1.8-10mM. Pf values in cells co-expressing Q- and R-form subunits were almost one order of magnitude smaller (0.54%). 6. Relative concentrations of Q-form and R-form GluR-B subunit-specific cDNAs used for cell transfection determined the expression of functionally different heteromeric AMPARS. Pf decreased with increasing relative concentration of R-form encoding CDNAs from 3.4 to 1.4%, demonstrating that editing of the Q/R site of GluR-B subunits decreases Ca2+ inflow through heteromeric AMPARs. 7. Cells expressing the GluR-6 subunit of the kainate receptor (KAR) family were characterized by Pf values which depended on the editing in the TM1 and TM2 segments. Pf values were largest for the Q-form (1.55-2.0%) and lowest for R-form channels (< 0.2%), suggesting that Q/R site editing also decreases Ca2+ inflow through KAR channels. Cells co-expressing both subunit forms showed an intermediate value (0.58%).(ABSTRACT TRUNCATED AT 400 WORDS)
Burnashev
N
N
Max-Planck-Institut für medizinische Forschung, Abteilung Zellphysiologie, Heidelberg, Germany.
Zhou
Z
Z
Neher
E
E
Sakmann
B
B
D002120:Calcium Channel Agonists / Q000494:pharmacology; D015220:Calcium Channels / Q000378:metabolism*; D002460:Cell Line; D004594:Electrophysiology; D016257:Fura-2; D006801:Humans; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D012333:RNA, Messenger / Q000096:biosynthesis; D018091:Receptors, AMPA / Q000378:metabolism*; D018092:Kainic Acid Receptors / Q000378:metabolism*; D016194:Receptors, N-Methyl-D-Aspartate / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism
D002120:Calcium Channel Agonists; D015220:Calcium Channels; D016257:Fura-2; D008274:Magnesium; D012333:RNA, Messenger; D018091:Receptors, AMPA; D018092:Kainic Acid Receptors; D016194:Receptors, N-Methyl-D-Aspartate; D011994:Recombinant Proteins