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. 2000 Oct 1;20(19):7238-45.
doi: 10.1523/JNEUROSCI.20-19-07238.2000.

Homer proteins regulate coupling of group I metabotropic glutamate receptors to N-type calcium and M-type potassium channels

P J Kammermeier  1 , B Xiao, J C Tu, P F Worley, S R Ikeda
Affiliations

Homer proteins regulate coupling of group I metabotropic glutamate receptors to N-type calcium and M-type potassium channels

P J Kammermeier et al. J Neurosci. .

Abstract

Group I metabotropic glutamate receptors (mGluR1 and 5) couple to intracellular calcium pools by a family of proteins, termed Homer, that cross-link the receptor to inositol trisphosphate receptors. mGluRs also couple to membrane ion channels via G-proteins. The role of Homer proteins in channel modulation was investigated by expressing mGluRs and various forms of Homer in rat superior cervical ganglion (SCG) sympathetic neurons by intranuclear cDNA injection. Expression of cross-linking-capable forms of Homer (Homer 1b, 1c, 2, and 3, termed long forms) occluded group I mGluR-mediated N-type calcium and M-type potassium current modulation. This effect was specific for group I mGluRs. mGluR2 (group II)-mediated inhibition of N-channels was unaltered. Long forms of Homer decreased modulation of N- and M-type currents but did not selectively block distinct G-protein pathways. Short forms of Homer, which cannot self-multimerize (Homer 1a and a Homer 2 C-terminal deletion), did not alter mGluR-ion channel coupling. When coexpressed with long forms of Homer, short forms restored the mGluR1a-mediated calcium current modulation in an apparent dose-dependent manner. Homer 2b induced cell surface clusters of mGluR5 in SCG neurons. Conversely, a uniform distribution was observed when mGluR5 was expressed alone or with Homer short forms. These studies indicate that long and short forms of Homer compete for binding to mGluRs and regulate their coupling to ion channels. In vivo, the immediate early Homer 1a is anticipated to enhance ion channel modulation and to disrupt coupling to releasable intracellular calcium pools. Thus, Homer may regulate the magnitude and predominate signaling output of group I mGluRs.

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Figures

Fig. 1.
Fig. 1.
SCG neurons natively express Homer 2 and Homer 3 but not Homer 1. SCG extracts were examined for expression of Homer isoforms. Antibodies were directed against Homer proteins derived from each gene. The anti-Homer1 antibody (left) recognized Homer 1b and Homer 1c. Anti-Homer2 (center) recognized Homer 2b, and anti-Homer3 (right) recognized Homer 3. Five, 10, and 15 μl of SCG extract were loaded into lanes 1, 2, and 3 of each group, respectively. The immunoblots were probed with Homer 1-, 2-, and 3-specific antibody.
Fig. 2.
Fig. 2.
Homer 2b expression induces clustering of mGluR5-myc. Left, Surface expression pattern of three cells (A–C represent different focusing plains) expressing mGluR5-myc and Homer 2b. Right, Surface expression pattern of three cells (A–C) expressing mGluR5-myc alone.
Fig. 3.
Fig. 3.
The long but not the short forms of Homer reduce calcium current inhibition through group I mGluRs. A, Sample currents (top) and time course (bottom) illustrating inhibition by 100 μmglutamate in a cell expressing mGluR1a. Cells were held at −80 mV and stepped to a +10 mV test pulse for 25 msec and then to a 50 msec +80 mV conditioning pulse, followed by a brief step back to the holding potential and another test pulse to +10 mV. In the time course,filled circles represent current measurements 10 msec into the first test pulse, the prepulse; open circlesrepresent measurements from the postpulse. Calibration: 0.2 nA, 20 msec. B, Sample currents and time course for a cell expressing mGluR1a and Homer 1a. Calibration: 0.2 nA, 20 msec.C, Currents and time course for a cell expressing mGluR1a and Homer 2b. Calibration: 0.5 nA, 20 msec. D, Summary of data from mGluR1a (open bars) and mGluR5a (hatched bars) cells. Error bars represent mean + SEM. The number of cells is indicated in parentheses to theright of each bar. *Statistically significant differences versus controls (ANOVA, p = 0.05).glu, Glutamate; con, control.
Fig. 4.
Fig. 4.
Homer does not alter modulation through mGluR2.A–C, Sample currents for inhibition by 100 μm glutamate in cells expressing mGluR2 (A), mGluR2 plus Homer 1a (B), and mGluR2 plus Homer 3 (C). Calibration: A, 1 nA;B, C, 0.7 nA; A–C, 20 msec.D, Summary of all mGluR2 data. No group produced a level of inhibition that differed from control (p= 0.05, ANOVA). glu, Glutamate; con, control.
Fig. 5.
Fig. 5.
The effect of Homer 2b can be partially reversed by coexpression of other forms of Homer. A, Sample currents illustrating inhibition by 100 μm glutamate in a cell expressing mGluR1a and Homer 2b (same cell as in Fig.1C). Calibration: 0.5 nA, 20 msec. B, Sample currents for a cell expressing mGluR1a, Homer 2b, and Homer 1a. Calibration: 0.5 nA, 20 msec. C, Sample currents for a cell expressing mGluR1a, Homer 2b, and H2N. Calibration: 0.7 nA, 20 msec. D, Data summary for control cells (mGluR1a only, from Fig. 3D), Homer 2b cells (cells expressing mGluR1a plus Homer 2b), and those coexpressing Homer 1a injected at 0.1 μg/μl [+2b, 1a (low)], 0.2 μg/μl [+2b, 1a (hi)], Homer 1b (+2b, 1b), and H2N (+2b, H2N). Data are mean + SEM. *Significant divergence from Homer 2b only cells (ANOVA, p = 0.05). In addition, inhibition in the +2b 1a (hi)group was significantly larger than in the +2b 1a (low)group. glu, Glutamate; con, control.
Fig. 6.
Fig. 6.
Long forms of Homer can occlude M-current modulation. A–C, Sample current traces and time courses from M-current inhibition by glutamate in cells expressing mGluR1a (A), mGluR1a plus Homer 1a (B), and mGluR1a plus Homer 2b (C). Cells were held at −30 mV and stepped to −60 mV for 500 msec every 10 sec. M-current in each time course was taken as the difference in current at the start and end of the step to −60 mV as M-type channels deactivated. Calibration:A, 0.2 nA; B, C, 0.1 nA;A–C, 20 msec. D, Summary of M-current modulation data. *Statistical significance (p = 0.05) compared with control (mGluR1a alone). glu, Glutamate; con, control.
Fig. 7.
Fig. 7.
Homer does not alter the voltage dependence of calcium current inhibition by glutamate. A, Postpulse versus prepulse inhibition plot for glutamate inhibition in cells expressing mGluR1a (filled circles) and mGluR1a plus Homer 2b (open circles). In this plot, slope is inversely proportional to voltage dependence. Slopes for mGluR1a and mGluR1a plus Homer 2b were 0.52 ± 0.09 and 0.60 ± 0.13 (slope ± 95% confidence interval). These values were not significantly different. B, Similar plot as inA, but for mGluR1a cells and cells expressing mGluR1a, Homer 2b, and Homer 1a. The slopes were 0.52 ± 0.09 and 0.53 ± 0.09, respectively. Solid lines represent linear regressions to each data set.
Fig. 8.
Fig. 8.
Schematic representation of the proposed mechanism of Homer function. A, When Homer 1a associates with group I mGluRs (or without any Homer proteins bound), receptors are diffusely distributed in the membrane. In this state, voltage-gated ion channels on the cell membrane are efficiently modulated, but the calcium signal via mGluRs and the IP3 receptor is weak.B, In the presence of long forms of Homer, group I mGluRs cluster together, presumably because of the interaction of the coiled coil, C-terminal tails of Homer. This interaction may also bring the complex in association with the IP3 receptor (schematic not shown). Under this condition, the intracellular calcium signal is strong, but modulation of voltage-gated ion channels is weak.
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