Abstract
TRPC5 forms non-selective cation channels. Here we studied the role of internal Ca(2+) in the activation of murine TRPC5 heterologously expressed in human embryonic kidney cells. Cell dialysis with various Ca(2+) concentrations (Ca(2+)(i)) revealed a dose-dependent activation of TRPC5 channels by internal Ca(2+) with EC(50) of 635.1 and 358.2 nm at negative and positive membrane potentials, respectively. Stepwise increases of Ca(2+)(i) induced by photolysis of caged Ca(2+) showed that the Ca(2+) activation of TRPC5 channels follows a rapid exponential time course with a time constant of 8.6 +/- 0.2 ms at Ca(2+)(i) below 10 microM, suggesting that the action of internal Ca(2+) is a primary mechanism in the activation of TRPC5 channels. A second slow activation phase with a time to peak of 1.4 +/- 0.1 s was also observed at Ca(2+)(i) above 10 microM. In support of a Ca(2+)-activation mechanism, the thapsigargin-induced release of Ca(2+) from internal stores activated TRPC5 channels transiently, and the subsequent Ca(2+) entry produced a sustained TRPC5 activation, which in turn supported a long-lasting membrane depolarization. By co-expressing STIM1 plus ORAI1 or the alpha(1)C and beta(2) subunits of L-type Ca(2+) channels, we found that Ca(2+) entry through either calcium-release-activated-calcium or voltage-dependent Ca(2+) channels is sufficient for TRPC5 channel activation. The Ca(2+) entry activated TRPC5 channels under buffering of internal Ca(2+) with EGTA but not with BAPTA. Our data support the hypothesis that TRPC5 forms Ca(2+)-activated cation channels that are functionally coupled to Ca(2+)-selective ion channels through local Ca(2+) increases beneath the plasma membrane.
Highlights
Taking advantage of the fact that the opening of a non-selective cation channels depolarizes the cell membrane, we use membrane potential imaging to study the activation of TRPC5 in non-voltage-clamped cells and found that TRPC5 channels are transiently activated by the Ca2ϩ release from internal stores, whereas Ca2ϩ entry supports a sustained activation of TRPC5 channels
In co-expression experiments with calcium-release-activated-calcium (CRAC) and L-type voltage-dependent Ca2ϩ channels, we found that the local rise of internal Ca2ϩ induced by the opening of these Ca2ϩ-selective ion channels is sufficient to activate TRPC5 channels, suggesting that TRPC5 channels represent Ca2ϩ-activated channels functionally coupled to Ca2ϩ-selective channels
Similar changes in the inward currents have been attributed to a switch between voltage-dependent and voltage-independent gating modes of TRPC5 channels activated via receptor stimulation [38]
Summary
Transfection, and Cell Culture—The murine TRPC5, STIM1, and ORAI1 were cloned from brain and subcloned in a bicistronic expression vector, which contains the cDNA of the green fluorescence protein as expression marker [21, 31]. Photolysis of Caged Ca2ϩ and Measurements of Internal Ca2ϩ—Cells were clamped at Ϫ70 mV using an EPC-9 amplifier (HEKA), and ion currents were continuously sampled at 10 kHz. The pipette solution contained 120 mM CsCl, 10 mM NaCl, 10 mM Hepes, 0.4 mM MgCl2, 5.6 mM nitrophenyl-EGTA, 0.2 mM FURA-2, 0.3 mM Furaptra, 2.185 mM CaCl2, pH 7.3 (CsOH). The external Ca2ϩ concentration (Ca2ϩo) was varied between 1.25 and 5 mM by adding the appropriated amount of CaCl2 to a solution that contained 140 mM NaCl, 5.4 mM KCl, 2 mM MgCl2, 10 mM Hepes, 5 mM glucose, pH 7.2 (NaOH). For the simultaneous imaging of membrane potential and internal Ca2ϩ, the cells were first loaded for 40 min at room temperature with 10 M FURA-2 AM (Invitrogen) dissolved in HBSS. Pooled data is given as the mean Ϯ S.E
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