Identification and characterization of the murine TRPM4 channel

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Abstract

The transient receptor potential (TRP) channels form a superfamily with six transmembrane structures, which is common in other types of voltage-dependent channels. The TRP-melastatin (TRPM) subfamily includes the putative tumor-suppressor melastatin, which was originally found as a down-regulated protein in melanoma tumor cell lines. Here, we report a novel TRP-related protein that is a murine orthologue of human TRPM4. The function of the novel murine TRPM4 was studied in HEK-293 cells using a fluorescent calcium indicator, fura-2. The removal and re-introduction of extracellular calcium triggered changes in the intracellular calcium only in cells expressing TRPM4a, which suggests that this novel channel plays a role in the calcium entry process. We also isolated a splice variant of TRPM4 that was proven to be non-functional. Both TRPM4 variants integrated into the plasma membrane. Furthermore, FRET analysis revealed that TRPM4a and TRPM4b localized close together, suggesting a multimerization of the two molecules.

Section snippets

Materials and methods

RNA isolation and reverse transcriptase-polymerase chain reaction analysis. Total RNA was isolated from the brain of a C57/BL6 mouse using an RNeasy Kit (Qiagen, Valencia, CA). The reverse transcription reaction was performed in a solution of 10 pmol oligo(dT) primer, 1 μg RNA, 1× first-strand cDNA buffer (Life Technologies, Rockville, MD), 10 mM dithiothreitol, 0.4 mM dNTPs, 40 U RNasin, and 200 U Superscript II in a volume of 25 μl, at 42 °C for 45 min.

For the reverse transcriptase-polymerase chain

Molecular cloning

Our cloning strategy was based on a detailed expressed sequence tag (EST) database search with all known TRPM members using the program BLASTN (NCBI; http://www.ncbi.nlm.nih.gov/blast/). One clone (Accession No.: XM-133450) was identified with the conserved transmembrane amino acid motif FLFLLITYVILTFVLLLN MLIALMGG (single amino acid code) and further analysis of the mouse DNA database identified several additional murine EST clones (BF582966, BQ918283, BF582749, and BG243789) in the mouse

Discussion

We discovered a novel murine TRPM and a variant form. Comparison of the amino acid sequences of all the known TRPM4s revealed that they are highly conserved. TRPM4 is widely expressed in both excitable and non-excitable cells. We also clarified the structure of the murine TRPM4 gene. Our intracellular calcium measurements suggested that the novel TRPM4a is a calcium-permeable cation channel, while TRPM4b, the new splice variant, has no such function. EGFP-fused TRPM4a and TRPM4b were localized

Acknowledgements

This research was partly sponsored by Grants-in-Aid from the Ministry of Education, Science and Culture, Japan, and from the Japan Foundation for Cardiovascular Research.

References (8)

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