Selective Association of TRPC Channel Subunits in Rat Brain Synaptosomes

Monu Goel,William G Sinkins,William P Schilling

doi:10.1074/jbc.m207882200

Abstract

TRPC genes encode a ubiquitous family of ion channel proteins responsible for Ca(2+) influx following stimulation of G-protein-coupled membrane receptors linked to phospholipase C. These channels may be localized to large multimeric signaling complexes via association with PDZ-containing scaffolding proteins. Based on sequence homology, the TRPC channel family can be divided into two major subgroups: TRPC1, -C4, and -C5 and TRPC3, -C6, and -C7. Although TRPC channels are thought to be tetramers, the actual subunit composition remains unknown. To determine subunit arrangement, individual TRPC channel pairs were heterologously expressed in Sf9 insect cells and immunoprecipitated using affinity-purified rabbit polyclonal antibodies specific for each channel subtype. Reciprocal co-immunoprecipitations showed that TRPC1, -C4, and -C5 co-associate and that TRPC3, -C6, and -C7 co-associate but that cross-association between the two major subgroups does not occur. Additionally, the interaction between each TRPC channel and the PDZ-containing protein, INAD (protein responsible for the inactivation-no-after-potential Drosophila mutant), was examined. TRPC1, -C4, and -C5 co-immunoprecipitated with INAD, whereas TRPC3, -C6, and -C7 did not. To define channel subunit interactions in vivo, immunoprecipitations were performed from isolated rat brain synaptosomal preparations. The results revealed that TRPC1, -C4, and -C5 co-associate and that TRPC3, -C6, and -C7 co-associate in both cortex and cerebellum but that cross-association between the two major subgroups does not occur. These results demonstrate that TRPC channels are present in nerve terminals and provide the first direct evidence for selective assembly of channel subunits in vivo.

Highlights

TRP1 genes, originally identified as critical components of phototransduction in Drosophila, encode a ubiquitous and heterogeneous family of ion channel proteins that appear to play a fundamental role in cell signaling, cell growth, and cell death
The results revealed that TRPC1, -C4, and -C5 co-associate and that TRPC3, -C6, and -C7 co-associate in both cortex and cerebellum but that cross-association between the two major subgroups does not occur
We characterized rabbit polyclonal antibodies generated against amino acid sequences specific for each TRPC channel protein expressed in Sf9 insect cells using recombinant baculovirus

Summary

Introduction

TRP1 genes, originally identified as critical components of phototransduction in Drosophila, encode a ubiquitous and heterogeneous family of ion channel proteins that appear to play a fundamental role in cell signaling, cell growth, and cell death. Selective localization to specific domains of the membrane appears to play an important role in signal transduction [26] In this regard, studies in Drosophila photoreceptor cells have shown that TRP channels are held in a signaling complex (i.e. a signalplex) by a scaffolding protein called INAD [1]. The immunoprecipitation experiments revealed that TRPC1, -C4, and -C5 co-associate and that TRPC3, -C6, and -C7 co-associate in both cortex and cerebellum but that cross-association between the two major subgroups does not occur These results demonstrate that TRPC channels are present in nerve terminals, but they provide the first direct evidence for selective assembly of channel subunits in vivo. Selective subunit assembly and selective interaction with PDZ-containing proteins may underlie differences in the activation mechanism for the various TRPC channels and help to explain conflicting results with regard to their putative role as store-operated channels

Methods

Results

Conclusion