A functional AMPA receptor-calcium channel complex in the postsynaptic membrane

Abstract

Ca(2+) channels play critical roles in the regulation of synaptic activity. In contrast to the well established function of voltage-activated Ca(2+) channels in the presynaptic membrane for neurotransmitter release, some studies are just beginning to elucidate the functions of the Ca(2+) channels in the postsynaptic membrane. In this study, we demonstrated the functional association of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors with the neuronal Ca(2+) channels. A series of biochemical studies showed the specific association of Ca(v)2.1 (alpha(1A)-class) and Ca(v)2.2 (alpha(1B)-class) with AMPA receptors in the postsynaptic membrane. Our electrophysiological and Ca(2+) imaging analyses of recombinant Ca(v)2.1 and AMPA receptors also showed functional coupling of the two channels. Considering the critical roles of postsynaptic intracellular concentration of Ca(2+) ([Ca(2+)](i)) increase and AMPA receptor trafficking for long-term potentiation (LTP) and long-term depression (LTD), the functional association of Ca(2+) channels with the AMPA receptors may provide new insights into the mechanism of synaptic plasticity.

Fig. 1.

Comparison of the Ca²⁺ channel complex partially purified from different tissues or through different methods. Sucrose gradient fractionation of Ca²⁺ channel complexes and subsequent immunoblotting for Ca²⁺ channel subunits demonstrate that the size of partially purified Ca²⁺ channel complexes differs depending on tissue and purification method. (A) Sucrose gradient fractionation of WGA-bound Ca²⁺ channel complex from rabbit brains. (B) Sucrose gradient fractionation of WGA-bound Ca²⁺ channel complex from rabbit skeletal muscles (SKM). (C) Sucrose gradient fractionation of heparin-bound Ca²⁺ channel complex from rabbit brains. The numbers at the top indicate the fraction of the sucrose gradient from top to bottom. Molecular mass standards (×10⁻³) are indicated on the left side of the panels. (D) Densitometry of Ca²⁺ channel subunits from Western blots of sucrose gradient fractions of WGA-bound Ca²⁺ channel complex. Fraction #, fraction number of sucrose gradient; Sucrose %, percentage of sucrose.

Fig. 2.

Association of AMPA receptors and PSD95 with neuronal Ca²⁺ channels. The cosedimentation and coimmunoprecipitation of AMPA receptors and PSD95 with Ca²⁺ channel subunits demonstrate specific association of these proteins in a complex. (A) Cosedimentation of AMPA receptor subunits and PSD95 with α₁2.2. (B) Densitometry of α₁2.2, AMPA receptors, and PSD95 from Western blots of sucrose gradient fractions. Fraction #, fraction number of sucrose gradient; Sucrose %, percentage of sucrose. (C) Immunoprecipitation of Ca²⁺ channel complexes using three different Ca²⁺ channel subunit antibodies and subsequent immunoblotting for GluR2/3 and PSD95. The first lane (Input) was loaded with the protein aliquots saved before immunoprecipitation. The antibodies used for immunoprecipitation are indicated at the top of each lane: Sheep 37 (anti-α₁2.1/2), VD2₁ (anti-β), and Rabbit 239 (anti-γ_2/3).

Fig. 3.

Subcellular fractionation of rabbit brains. (A) Pre- or postsynaptic marker proteins. (B and C) Ca²⁺ channel subunits. Subcellular fractionation of rabbit brains demonstrates the enrichment of Ca²⁺ channel subunits in the postsynaptic membrane as well as in the presynaptic membrane. The first (synap), second (presynap), and third (PSD) columns were loaded with synaptosomal proteins, presynaptic proteins, and PSD proteins, respectively.

Fig. 4.

Functional coupling between Ca_v2.1 and AMPA receptors. The activity of heterologously expressed Ca_v2.1 or AMPA receptors was analyzed by using HEK or HEK-BI 24-4 cells. The activities of Ca_v2.1 or AMPA receptors were significantly altered by the coexpression of the two receptors. (A) Superimposed plots of steady-state activation (G/G_max) curves of Ca_v2.1. (B) Superimposed plots of Ca_v2.1 activation kinetics (TP, time-to-peak). *, statistically significant difference in certain value between two groups. Electrodes were filled with internal solution containing 155 mM CsCl, 11 mM EGTA, 0.3 mM Li-GTP, 4 mM Mg-ATP, and 10 mM Hepes (pH 7.4 with CsOH). The recording chamber was filled with external solution containing 10 mM CaCl₂, 125 mM tetraethylammonium chloride (TEA-Cl), 10 mM Hepes, and 5 mM glucose. The external solution was adjusted to pH 7.3 with CsOH and to 297 mosmol/liter with sucrose. Test potentials were applied for 350 ms from a holding potential of −90 mV. (C) Current–voltage relationship of Ca_v2.1 coexpressed with AMPA receptors with (AMPA, bold line) or without AMPA treatment. The normal pipette solution contained 85 mM Cs-aspartate, 40 mM CsCl, 2 mM MgCl₂, 5 mM EGTA, 2 mM Na₂ATP, 5 mM Hepes, and 8 mM creatinine-phosphate (pH adjusted to 7.2 with CsOH). The external solution for recording of Ca²⁺ channel current contained 3 mM BaCl₂, 150 mM TEA-Cl, 10 mM Hepes, and 10 mM glucose (pH adjusted to 7.4 with TEA-OH). (D) Ca²⁺ response induced by AMPA (30 μM) and the AMPA receptor-specific modulator cyclothiazide (CTZ, 30 μM) HEK cells expressing GluR1 alone (HEK 293) or GluR1 plus Ca_v2.1 (HEK24-4) after 8 min exposure to high K⁺ (50 mM) solution. The “Ratio” was obtained by exciting fura-2 alternately at 340 and 380 nm.