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Ca2+ influx induces various physiological events such as contraction, exocytosis, gene expression, and cell growth and death. Transient receptor potential (TRP) and store-operated Ca2+ (SOC) channels are the representative of non-voltage-gated Ca2+ entry channels, and serve as major Ca2+ entry routes for non-excitable cells. Many reports indicate that Ca2+ influx through these channels is causally associated with cell cycle progression or growth, but the mechanism underlying it seems to vary depending on the cell type. To question how the proliferative potential and cell cycle progression of adult stem cells are controlled is an attractive theme for regenerative medicine and bioengineering, but the implication of non-voltage-gated Ca2+ entry channels therein is poorly elucidated. In this study, we investigated the roles of TRP and STIM/Orai channels on cell cycle progression of bone marrow stromal cells (BMSCs) from adult rat which include mesenchymal stem cells. Cultured BMSCs were synchronized in G1, S, G2 or M phases, and the mRNA levels of respective TRPC and STIM/Orai subtypes were assessed by the quantitative real-time PCR. In the S phase, the expression levels of TRPC1, STIM and Orai were significantly enhanced. On the other hand, that of TRPC6 was greatly reduced in the S phase and significantly enhanced in the G1 phase. In experiments measuring the intracellular Ca2+ concentration with Fura 2, SOC-mediated Ca2+ influx, which was evoked by thapsigargin or cyclopiazonic acid, was inhibited by 1µM Gd3+. The Ca2+ influx was significantly enhanced in the S phase. Gd3+ 1µM), SKF96365 or pyrazole compound (Pyr2) suppressed cell proliferation. To seek a role of TRPC6 channels for cell cycle progression, we evaluated the resting membrane potential (RMP) of cells using a voltage-sensitive dye DiBAC4(3) or by the current-clamp mode of patch clamp technique. siRNA knockdown of TRPC6 expression but not of TRPC1 produced a significant negative shift of RMP. RMP in the S phase was deeper than in the other cell cycle stages. From these observations, we speculate that TRPC6 channels may work as a modulator of RMP thereby controlling the driving force for Ca2+ influx in the S phase. Cell cycle analysis by the flow cytometry revealed that siRNA knockdown of TRPC1, TRPC6 or STIM/Orai expression differentially affected the distribution of cell cycle stages. The fraction of cells residing in the G0/G1 phase was increased by siRNA of STIM/Orai but decreased by siRNA of TRPC1 or TRPC6. siRNA of TRPC6 significantly increased the fraction of cells in the G2/M phase. To elucidate these observations more explicitly, the correlation between cell cycle-regulating molecules such as cyclins and the functions of TRPC1/C6 and STIM/Orai will need to be investigated. [ABSTRACT FROM AUTHOR]

TRPM4 is a molecular correlate for Ca2+-activated monovalent cation selective channels ubiquitously expressed in both excitable and non-excitable tissues. Recent investigations have revealed that increased expression of this channel protein may be the main causes for certain types of familial atrio-ventricular blocks and other arrhythmogenic propensity associated with cardiac remodeling. However, the apparent Ca2+ sensitivity of this channel evaluated in heterologous systems is almost out of physiological range (typically >10μM), so that it remains still puzzling how exactly it could contribute to arrhythmogenicity. In this study, we re-evaluated the Ca2+ sensitivity of TRPM4 channels in both expression systems and immortalized atrial cardiomyocyte cell line HL-1, and explored their roles in inducing arrhythmic changes by means of mathematical model simulations. In cell-free conditions, the Ca2+ sensitivity of human TRPM4 channels expressed in HEK293 cells rapidly declined requiring more than several-hundred μM Ca2+ for half maximal activation (Kd) near the resting membrane potential. However, a much higher Ca2+ sensitivity was observed when cells were maintained as intact as possible and intracellular Ca2+ concentration was changed via ionomycin-mediated Ca2+ influx (Kd: ~ 0.5μM). A similar estimate of Kd was obtained in experiments evaluating the relationship between the integral of voltage-dependent Ca2+ influx and the magnitude of tail cationic current in HEK cells coexpressing TRPM4 and voltage-dependent Ca2+ channels (VDCC) subunits (α1C/β2/α2δ). Furthermore, repetitive activation of VDCC at high frequencies induced the development of sustained TRPM4 current at -80mV. 4-5 day treatment of HL-1 myocytes with angiotensin II resulted in enhanced expression of TRPM4 channel protein and current density, and induced spontaneous action potentials (APs) with prolongation of AP duration, which could be abolished by a relatively selective concentration (10μM) of a TRPM4 channel blocker, 9-phenanthrol. In silico simulation of cardiac action potentials (APs) based on 2000 Luo-Rudy or Nygren models which incorporated the Ca2+-and voltage-dependent kinetics of TRPM4 channel reproduced the late phase-3 AP prolongation and moderate basal depolarization when increasing the expression level of TRPM4 by several fold. These results suggest that the intact Ca2+ sensitivity of TRPM4 channel would be sufficiently high to induce arrhythmic changes, when the channel expression or function is up-regulated. [ABSTRACT FROM AUTHOR]