Homer 1a uncouples metabotropic glutamate receptor 5 from postsynaptic effectors

Abstract

Metabotropic glutamate receptors (mGluRs) and Homer proteins play critical roles in neuronal functions including plasticity, nociception, epilepsy, and drug addiction. Furthermore, Homer proteins regulate mGluR1/5 function by acting as adapters and facilitating coupling to effectors such as the inositol triphosphate receptor. However, although Homer proteins and their interaction with mGluRs have been the subject of intense study, direct measurements of Homer-induced changes in postsynaptic mGluR-effector coupling have not been reported. This question was addressed here by examining glutamatergic excitatory postsynaptic currents (EPSCs) in rat autaptic hippocampal cultures. In most neurons, the group I mGluR agonist (S)-3,5-dihydroxyphenylglycine strongly inhibited the EPSC acutely. This modulation occurred postsynaptically, was mediated primarily by mGluR5, and was inositol triphosphate receptor-dependent. Expression of the dominant negative, immediate early form of Homer, Homer 1a, strongly reduced EPSC modulation, but the W24A mutant of Homer 1a, which cannot bind mGluRs, had no effect. (S)-3,5-dihydroxyphenylglycine-mediated intracellular calcium responses in the processes of Homer 1a-expressing neurons were reduced compared with those in Homer 1a W24A-expressing cells. However, neither the distribution of mGluR5 nor the modulation of somatic calcium channels was altered by Homer 1a expression. These data demonstrate that Homer 1a can reduce mGluR5 coupling to postsynaptic effectors without relying on large changes in the subcellular distribution of the receptor. Thus, alteration of mGluR signaling by changes in Homer protein expression may represent a viable mechanism for fine-tuning synaptic strength in neurons.

Fig. 1.

EPSCs in hippocampal autapses. (A) Phase contrast image of a hippocampal neuron grown in autaptic culture for 7 days. (B) Sample EPSC trace illustrating the voltage protocol used to evoke an action potential volley from a holding potential of −80 mV (above), the action current, and the EPSC (as indicated). (C) Sensitivity of the EPSC to 10 μM kynurenic acid. Sample currents before (Con), during (KYN), and after (Wash) application of KYN are shown. (D) Average + SEM inhibition by KYN and 20 μM CNQX (6-cyano-7-nitroquinoxaline-2,3-dione). The number of cells in each group is indicated in parentheses.

Fig. 2.

The hippocampal autaptic EPSC is modulated by mGluR5. (A) Sensitivity of the EPSC to 50 μM DHPG. Sample currents before (Control), during (50 μM DHPG), and after (Wash) application of DHPG are shown. (B) Dose–response relationship for DHPG inhibition of the EPSC. The estimated half-maximal effective concentration (EC₅₀) was 3.9 μM.

Fig. 3.

Modulation of the EPSC by DHPG occurs postsynaptically. (A) Sample paired-pulse current traces illustrating the paired-pulse ratio in uninhibited (Con) and DHPG-inhibited currents. (B) Average + SEM P2/P1 ratio before (open bar) and during (filled bar) EPSC inhibition by the indicated drug.

Fig. 4.

Expression of Homer 1a uncouples mGluR5 from EPSC modulation. (A) Scatter plot illustrating the maximal inhibition by DHPG of the EPSC in each uninfected (Con; filled circles) and Homer 1a-expressing (H1a; open circles; neurons expressing either Homer 1a IRES GFP and GFP IRES Homer 1 were used) autaptic neuron. The mean (heavy black line) and median (gray line) inhibition for each group are also shown. The Inset shows DHPG inhibition in a sample Homer 1a-expressing neuron. (B) Epifluorescence image showing GFP expression in one GFP IRES Homer 1a-expressing neuron. (C) Scatter plot illustrating the maximal inhibition by DHPG of the EPSC in each uninfected (Con; filled squares) and Homer 1a W24A-expressing (W24A; open squares) autaptic neuron. The mean (black lines) and median (gray lines) inhibition for each group are also shown. (D) Epifluorescence image showing GFP expression in one Homer 1a W24A IRES GFP-expressing neuron.

Fig. 5.

Modulation of the EPSC by DHPG requires the IP₃R but not PKC. (A) Sample current traces showing DHPG modulation of the autaptic EPSC in control cells (Left) and cells recorded in the presence of 100 μg/ml intracellular low-molecular-weight heparin (Right). (B) Scatter plot illustrating the maximal EPSC inhibition in each cell by 50 μM DHPG in control cells (open circles), cells recorded with intracellular heparin (filled circles), or 3 μM intracellular bisindolylmaleimide (filled squares). The mean (black lines) and median (gray lines) inhibition for each group are also shown.

Fig. 6.

Homer 1a reduces mGluR5-mediated rise in intracellular calcium. (A) Homer 1a expression reduces the mGluR5-induced intracellular calcium signal in the processes of hippocampal neurons. Ratio of Fura 2 fluorescence (340:380 nm excitation) time course (average ± SEM) for hippocampal neurons expressing either Homer 1a W24A (gray) or Homer 1a (black). Data were baseline-subtracted from the average of all points 10 sec prior to DHPG application. The data include measurements from 61 points on the processes of six infected neurons and 59 points on the processes of five infected neurons in Homer 1a W24A and Homer 1a-expressing cells, respectively. (B) Peak calcium responses minus baseline in response to 50 μM DHPG and 10 μM oxo-M, as indicated in dSRed-transfected neurons (filled bars) and neurons transfected with dSRed plus Homer 1a (open bars). Calcium measurements were obtained as in A.

Fig. 7.

Expression of Homer 1a in hippocampal neurons does not detectably alter the distribution of mGluR5. (A) GFP-fluorescence image of a GFP-infected hippocampal neuron (Left) and AF555 fluorescence indicating mGluR5 expression (Right) from the same field. (B) GFP-fluorescence image of a Homer 1a IRES GFP-infected hippocampal neuron (Left) and AF555 fluorescence indicating mGluR5 expression (Right) from the same field. Both images illustrate mGluR5 expression in the cell body and processes of these representative cells. (C) Control fluorescence images illustrating the lack of signal in the GFP channel in an uninfected, mGluR5-positive cell (Left) and the lack of AF555 fluorescence in a GFP-transfected, mGluR5-negative neuron (Right), confirming the lack of bleed-over across the two channels.

Fig. 8.

Expression of Homer 1a does not alter DHPG-mediated modulation of currents through somatic calcium channels. (A) Sample current traces from the indicated voltage protocol (Inset) illustrating DHPG modulation of the barium currents from somatic outside-out patches in uninfected (Upper) and Homer 1a-expressing (Lower) hippocampal neurons. (B) Average + SEM inhibition of the somatic, outside-out patch barium current in uninfected and Homer 1a-expressing neurons, as indicated. Inhibitions were calculated from peak currents during a voltage ramp from −80 to +80 mV (see Methods). The number of cells in each group is indicated in parentheses.