Biochemical and Biophysical Research Communications
TRPM4b channel suppresses store-operated Ca2+ entry by a novel protein–protein interaction with 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).
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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.