Figure 2. GLR-1(A/T) Forms a Nontoxic, Constitutively Open Channel that Can Be Expressed in the Command Interneurons(A) Alignment of the C-terminal region of TM III of ionotropic glutamate receptors. Shown are representative receptors of the non-NMDA and NMDA classes from C. elegans GLR-1 (1), rat GluR1 (2), rat GluR5 (3), Drosophila GluR1 (4), rat NR1 (5), rat Ka1 (6), rat delta2 (7), and mouse delta2 (8). The conserved alanine found in TM III is shown in red. This residue is mutated to a threonine (shown in green) in the mouse Lurcher strains Lc and LcJ. We have engineered an identical substitution into C. elegans GLR-1(A/T).(B and C) GFP expression in transgenic strains. Under control of the
glr-1 promoter, larval transgenic strains expressed chimeric proteins that contained GFP fused to the N terminus of wild-type GLR-1 (B) or GLR-1(A/T) (C). Images acquired with confocal microscopy show that transgenic strains expressed the GLR-1(A/T)::GFP fusion protein in the appropriate neurons and that these neurons show no evidence of gross morphological abnormalities.(D-F) GLR-1(A/T) forms a ligand-independent leaky channel in Xenopus oocytes. cRNA was prepared in vitro from cDNA clones encoding GLR-1, GLR-1(A/T), or GLR-1(A/T;Q/R) and was injected into Xenopus oocytes. After 3 days, transmembrane currents were recorded by standard two-electrode voltage-clamp techniques (Marcus-Sekura and Hitchcock 1987).(D) I-V relations obtained from oocytes bathed in standard oocyte ringer solution that contained 2 mM Ca2+ (OR2). Uninjected oocytes (dashed line, n = 7) were indistinguishable from oocytes injected with cRNA encoding GLR-1 (open squares, n = 12); both had a small leakage conductance, and the current reversed direction from inward to outward near 50 mV (reversal potential). In contrast, oocytes injected with cRNAs encoding either singly mutant (open circles, GLR-1[A/T], n = 11) or double mutant (open triangles, GLR-1[A/T;Q/R], n = 11) receptors were significantly leakier than control oocytes. In addition, the reversal potential has shifted right to a more depolarized level, indicating the presence of a membrane conductance with a reversal potential that is near 20 mV.(E) GLR-1(A/T) is primarily permeant to cations. Shown are I-V relations obtained from oocytes (D) that were switched to a salt solution in which Na+ (open symbols) was replaced by the large organic cation NMDG (closed symbols). GLR-1(A/T)-injected oocytes (circles) were leakier than GLR-1(A/T;Q/R)-injected oocytes (triangles), both before and after the switch to NMDG, which was accompanied by a leftward shift of the reversal potential to a more hyperpolarized level.(F) GLR-1(A/T;Q/R) is less permeant to the divalent cation Ca2+. Shown are I-V relations obtained from oocytes in (E) that were switched to a salt solution in which the NMDG containing 2 mM Ca2+ solution (closed symbols) was changed to an isoosmotic NMDG solution containing 50 mM Ca2+ (open symbols). GLR-1(A/T)-injected oocytes (circles) became significantly leakier after the switch to the high-Ca2+ NMDG solution. This conductance increase was accompanied by a rightward shift of the reversal potential to a more depolarized level, indicating that GLR-1(A/T) is permeant to Ca2+. In contrast, oocytes that expressed GLR-1(A/T; Q/R) had only a small change in conductance and reversal potential when changed to the solution containing 50 mM Ca2+.