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-e were made in HEK 293 cells expressin g recombinant glutamate receptor (GluR) channels, and fractional Ca2currents (the proportion of whole-cell current carried by Ca2+; P-f) w ere determined. 2. Cells expressing N-methyl-D-aspartate receptor (NMD AR) channels showed glutamate-activated Ca2+ inflow in the voltage ran ge -60 to 40 mV in normal extracellular solution. Ca2+ inflow decrease d in a voltage-dependent manner at membrane potentials more negative t han -30 mV due to Mg2+ block. Voltage dependence of block at negative potentials was stronger in cells expressing the NR1-NR2A as compared w ith cells expressing NR1-NR2C subunits. 3. Fractional Ca2+ currents th rough NMDARs were independent of extracellular Mg2+ and varied between 8.2% (NR1-NR2C subunits) and 11% (NR1-NR2A subunits) in normal extrac ellular solution (1.8 mM Ca2+) at -60 mV membrane potential. P-f value s increased with increasing [Ca2+](o) in the range of 0.5-10 mM [Ca2+] (o) in a saturating fashion. 4. In cells expressing pha-amino-3-hydrox y-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 al most linear way. 5. Fractional Ca2+ currents through AMPAR channels de pended on subunit composition. P-f values of Q-form homomeric channels at -60 mV and 1.8 mM [Ca2+](o) were between 3.2 and 3.9%. They were s lightly voltage dependent and increased with [Ca2+](o) in the range 1. 8-10 mM. P-f values in cells co-expressing Q- and R-form subunits were almost one order of magnitude smaller (0.54%). 6. Relative concentrat ions of Q-form and R-form GluR-B subunit-specific cDNAs used for cell transfection determined the expression of functionally different heter omeric AMPARs. P-f 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 heterom eric AMPARs. 7. Cells expressing the GluR-B subunit of the kainate rec eptor (KAR) family were characterized by P-f values which depended on the editing in the TM1 and TM2 segments. P-f values were largest for t he Q-form (1.55-2.0%) and lowest for R-form channels (< 0.2%), suggest ing that Q/R site editing also decreases Ca2+ inflow through KAR chann els. Cells co-expressing both subunit forms showed an intermediate val ue (0.58%). 8. Fractional Ca2+ currents measured with normal [Ca2+](o) were different from P-f values predicted with constant field assumpti ons from reversal potentials measured in bi-ionic (Ca2+-Cs+) condition s with high [Ca2+](o). 9. The results indicate that the size of P(f)s mediated by recombinant GluR channels in normal extracellular solution varies over a 50-fold range between less than 0.2 and 11%, depending on the combination of GluR channel subunits expressed. Assembly of dif ferent subunit combinations, relative abundance of subunit specific mR NAs and editing of mRNA are major mechanisms which control this wide r ange of Ca2+ inflow through different versions of GluR channels under physiological conditions.