Canonical transient receptor potential 4 (TRPC4) channels are calcium-permeable, nonselective cation channels that are widely distributed in mammalian cells. It is generally speculated that TRPC4 channels are activated by Gq/11-PLC pathway or directly activated by Gi/o proteins. Although many mechanistic studies regarding TRPC4 have dealt with heterotrimeric G proteins, here, we first report the functional relationship between TRPC4 and small GTPase, Rasd1. Rasd1 selectively activated TRPC4 channels, and it was the only Ras protein among Ras protein family that can activate TRPC4 channels. For this to occur, it was found that certain population of functional Gαi1 and Gαi3 proteins are essential. Meanwhile, dexamethasone, a synthetic glucocorticoid and anti-inflammatory drug was known to increase messenger RNA (mRNA) level of Rasd1 in pancreatic β-cells. We have found that dexamethasone triggers TRPC4-like cationic current in INS-1 cells via increasing protein expression level of Rasd1. This relationship among dexamethasone, Rasd1, and TRPC4 could suggest a new therapeutic agent for hospitalized diabetes mellitus (DM) patients with prolonged dexamethasone prescription.
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We thank Dr. Won Kyung Ho for providing INS-1 cell line. We thank Dr. M. Schaefer for providing the mouse TRPC4 cDNA, Dr. Y. Mori for providing the mouse TRPC6, and Dr. Shuji Kaneko for providing the human TRPC5-GFP and acknowledge the Missouri S&T cDNA Resource Center (http://www.cdna.org) for providing the M2 receptor cDNAs. We thank Dr. Yong-Sung Juhnn for the human Gαi1 G202T, Gαi2 G203T, and Gαi3 G202T; Dr. Seong-Woo Jeong for all of the Gβγs; and Dr. Won Do Heo for the small G protein. We thank Elsevier for revising the manuscript (http://www.elsevier.com). This study was supported by a grant from the Korean Health Technology R&D Project of the Ministry of Health and Welfare of the Republic of Korea (HI13C0104).
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Specificity of mouse TRPC4β to Rasd1. (A) All panels indicate I-V relationship of currents measured from HEK293 cells expressing mouse TRPC4β-ECFP channel and constitutively active Rasd1 proteins, Rasd1G81A and Rasd1A178V. Enhanced Cyan Fluorescent Protein (pECFP) was expressed as a negative control for Rasd1 proteins since they are tagged with pECFP at their C-terminus (see materials and methods). All Rasd1 proteins were able to activate TRPC4 channels. (B) Summarized current density measured above. Rasd1G81V and Rasd1A178V induced significant TRPC4-like current increase. The comparison between pECFP and Rasd1G81A or Rasd1A178V was carried out with Student’s t-test. Statistical significance was denoted by an asterisk (*) at P < 0.05. (PDF 57 kb)
Specificity of Rasd1 to TRPC4β channels among other TRP channels. The effect of Rasd1S33V on other TRPC channels, TRPC4α, TRPC5 or TRPC6. Rasd1S33V could not activate mouse TRPC4α or mouse TRPC6. It rather decreased the activity of human TRPC5 channels. The comparison between TRPC channels and TRPC channels + Rasd1S33V was carried out with Student’s t-test. Statistical significance was denoted by an asterisk (*) at P < 0.05. n.s., not significant. (PDF 18 kb)
Time course of M2R-activated TRPC4β channels co-expressed with Rasd1 proteins. All panels indicate time course of currents measured from HEK293 cells expressing mouse TRPC4β channels, human muscarinic acetylcholine receptor type 2 (M2R) and indicated Ras family proteins, Rasd1S33V or Rasd1G31V. 100 μM of extracellular carbachol was applied in order to stimulate muscarinic receptors. (A) Under carbachol stimulation, TRPC4β channel was strongly activated with fast activation. (B) Under carbachol stimulation, TRPC4β channel showed minute activation, hence small inward current when Rasd1S33V was co-expressed. (C) The carbachol-M2R-mediated TRPC4β channel activation was fully recovered when Rasd1G31V, a dominant negative form of Rasd1, was co-expressed instead of Rasd1S33V. Black arrow heads (NT) and red arrow heads (Cs + CCh) indicate the time points where corresponding I-V curves were obtained. (PDF 192 kb)
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Wie, J., Kim, J., Ha, K. et al. Dexamethasone activates transient receptor potential canonical 4 (TRPC4) channels via Rasd1 small GTPase pathway. Pflugers Arch - Eur J Physiol 467, 2081–2091 (2015). https://doi.org/10.1007/s00424-014-1666-0