Selective Association of TRPC Channel Subunits in Rat Brain Synaptosomes

TLDR

TRPC genes encode ubiquitous Ca²⁺‑permeable channels activated by GPCR‑PLC signaling and are thought to form tetramers whose subunit composition is unknown. The study aimed to determine the subunit arrangement of TRPC channels by heterologously expressing channel pairs in Sf9 cells and immunoprecipitating them with subtype‑specific antibodies. The authors performed co‑immunoprecipitation of TRPC pairs in Sf9 cells, examined their interaction with the PDZ‑containing protein INAD, and validated subunit associations in rat brain synaptosomes. TRPC1,4,5 and TRPC3,6,7 form distinct tetrameric complexes that do not cross‑associate, as demonstrated by reciprocal co‑immunoprecipitation, INAD binding assays, and synaptosomal analyses, providing the first direct evidence of selective subunit assembly in nerve terminals.

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.