Involvement of TRPM7 in cell growth as a spontaneously activated Ca2+ entry pathway in human retinoblastoma cells.

We investigated the possible involvement of the melastatin family protein TRPM7 in Ca(2+)-mediated proliferative control of human retinoblastoma (RB) cells. The growth of RB cell was facilitated by elevating the extracellular Ca(2+) concentration with a parallel increase in the magnitude of spontaneous Ca(2+) influx. Under nystatin-perforated voltage-clamp, RB cells exhibited an outward-rectifying, spontaneous cation current (I(spont)) having Ca(2+)/Mg(2+)-inhibited but -permeating properties. Various cation channel blockers inhibiting I(spont) (Gd(3+), La(3+), LOE908, 2-APB) suppressed the spontaneous Ca(2+) influx and decelerated the growth of RB cells with similar efficacies. Excision of the RB cell membrane (inside-out) into MgATP-free solution induced a 70pS single channel activity, which was effectively inhibited by millimolar concentrations of Mg(2+) or MgATP. RT-PCR and immunocytochemical experiments revealed the expression of TRPM7 mRNA and protein in RB cells, and heterologous expression of TRPM7 in HEK293 cells reproduced the key features of I(spont). In contrast, elimination of this protein from RB cells by siRNA silencing markedly reduced I(spont) density and the magnitude of spontaneous Ca(2+) influx, which was paralleled by decreased TRPM7 immunoreactivity, decelerated cell proliferation, and retarded G(1)/S cell cycle progression. These results suggest a significant regulatory role of TRPM7 for RB cell proliferation as a spontaneously activated Ca(2+) influx pathway.

Transient receptor potential protein as a novel non-voltage-gated Ca2+ entry channel involved in diverse pathophysiological functions.

In both excitable and non-excitable cells, many chemical and physical stimuli elicit continuous Ca2+ influx through yet poorly understood pathways distinct from voltage-gated Ca2+ channels, leading to activation and modulation of various cellular functions. The molecular entities of these pathways have long been enigmatic, but important clues have been obtained from recent investigations on the Drosophila transient receptor potential (TRP) protein and its mammalian homologues. TRP proteins function as non-voltage-gated Ca2+ channels that are constitutively active or gated by a multitude of stimuli including light, pheromones, lipids, temperature, acid, osmolarity, and oxidative stress; and thus they may play divergent roles in cell pathophysiology. This short paper briefly overviews the current knowledge about these channels with a main focus on their possible linkage with in vivo function.

Contribution of nifedipine-insensitive voltage-dependent Ca2+ channel to diameter regulation in rabbit mesenteric artery.

We investigated a possible role of nifedipine-insensitive high voltage-activated (NI-HVA) Ca2+ channels in arterial diameter regulation in the semi-terminal branches of rabbit mesenteric artery (RMA). From these branches, NI-HVA Ca2+ currents showing almost identical properties to those previously identified in a similar region of guinea-pig [Circulation Research 1999;85:596-605] were recorded with whole-cell patch clamp recording. With video-microscopic measurement, the diameter of RMA segments perfused intraluminally at a constant rate (2-6 mL/h; 269 +/- 9 micro m, n = 27) decreased by 50-60% by raising the external K+ concentration ([K+]o) to 75 mM, a substantial part of which remained after addition of 1-10 micro M nifedipine (44 +/- 5% of initial diameter, n = 27). This nifedipine-insensitive diameter decrease (NI-DD) appeared to consist of initial transient and subsequent tonic phases (this separation was, however, not always clear), was resistant to tetrodotoxin, and was completely abolished in Ca2+-free or 100 micro M Cd2+-containing bath solutions. The magnitude of NI-DD increased depending on [K+]o with a threshold concentration of 20-40 mM. Raising the external Ca2+ concentration dose-dependently increased the magnitude of NI-DD, the extent being more prominent in the late tonic phase. Combined application of caffeine (10 mM) with ryanodine (3 micro M) produced a large transient NI-DD, which strongly attenuated the NI-DD evoked by a subsequent elevation in [K+]o. Using the fura-2 spectrofluorimetric Ca2+ imaging technique, a nifedipine-insensitive [Ca2+]i increase showing similar [K+]o-dependence and Cd2+ sensitivity to NI-DD was observed. These properties of NI-DD accord with those of NI-HVA Ca2+ channels, thus suggesting their contribution to small arterial diameter regulation in RMA.