TRPM4b channel suppresses store-operated Ca2+ entry by a novel protein–protein interaction with the TRPC3 channel

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Abstract

We identified human TRPC3 protein by yeast two-hybrid screening of a human brain cDNA library with human TRPM4b as a bait. Immunoprecipitation and confocal microscopic analyses confirmed the protein–protein interaction between TRPM4b and TRPC3, and these two TRPs were found to be highly colocalized at the plasma membrane of HEK293T cells. Overexpression of TRPM4b suppressed TRPC3-mediated whole cell currents by more than 90% compared to those in TRPC3-expressed HEK293T cells. Furthermore, HEK293T cells stably overexpressing red fluorescent protein (RFP)–TRPM4b exhibited an almost complete abolition of UTP-induced store-operated Ca2+ entry, which is known to take place via endogenous TRPC channels in HEK293T cells. This study is believed to provide the first clear evidence that TRPM4b interacts physically with TRPC3, a member of a different TRP subfamily, and regulates negatively the channel activity, in turn suppressing store-operated Ca2+ entry through the TRPC3 channel.

Section snippets

Materials and methods

Yeast two-hybrid screening. Human TRPM4b cDNA was cloned into the NdeI and SalI sites of pGBKT7 (Clontech) to serve as a bait vector (BD/TRPM4b). A Pretransformed Human Fetal Brain Matchmaker cDNA Library (Clontech) was used as the prey library. Human TRPC3 was also fused in-frame with the GAL4-DNA activating domain of the pGADT7 AD vector (AD/TRPC3). Both BD/TRPM4b and AD/TRPC3 were cotransformed into the yeast strain AH109 to reconfirm the protein–protein interaction between TRPM4b and TRPC3.

Protein–protein interaction between TRPM4 b and TRPC3

We used a yeast two-hybrid system to identify proteins that bind to human TRPM4b. Positive clones were isolated independently from a human fetal brain cDNA library. Sequence analysis showed that one of the positive clones encodes a human TRPC3 channel. Using the full-length cDNA of TRPM4b and TRPC3, we reconfirmed the protein–protein interaction between these two channels by yeast two-hybrid-based assay. As shown in Fig. 1A, the growth of transformed yeast in media lacking His is completely

Discussion

In this study, we screened human TRPC3 as a novel binding partner of human TRPM4b, and confirmed that overexpressed TRPM4b and TRPC3 are highly colocalized in the plasma membrane of HEK293T cells. We also determined that TRPM4b suppressed TRPC3-mediated currents and SOC in cells stably expressing TRPM4b.

TRPM4 has been established as a non-selective, Ca2+-impermeable cation channel that is activated by intracellular Ca2+[1], [2], [3], [4]. To the best of our knowledge, we have shown clearly for

Acknowledgments

This work was supported by Grants from the Korea Science and Engineering Foundation (R13-2005-012-02002-0, R13-2005-012-01002-0), the Korean Research Foundation (KRF-2006-005-J04204) and in part by a Grant from Gyeongsang National University (RPP-2006-056).

References (24)

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1

Address: Department of Microbiology, Research Institute of Life Science, Gyeongsang National University, Jinju 660-701, Republic of Korea.

2

Address: School of Engineering, Information and Communications University, Daejeon 305-732, Republic of Korea.

3

Address: Sensory Research Group, National Creative Research Initiatives, College of Pharmacy, Seoul National University, Seoul 151-742, Republic of Korea.

4

Contributed equally to this work.

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