Abstract
We investigated the regulation of T-type channels by Rho-associated kinase (ROCK). Activation of ROCK via the endogenous ligand lysophosphatidic acid (LPA) reversibly inhibited the peak current amplitudes of rat Cav3.1 and Cav3.3 channels without affecting the voltage dependence of activation or inactivation, whereas Cav3.2 currents showed depolarizing shifts in these parameters. LPA-induced inhibition of Cav3.1 was dependent on intracellular GTP, and was antagonized by treatment with ROCK and RhoA inhibitors, LPA receptor antagonists or GDPßS. Site-directed mutagenesis of the Cav3.1 α1 subunit revealed that the ROCK-mediated effects involve two distinct phosphorylation consensus sites in the domain II-III linker. ROCK activation by LPA reduced native T-type currents in Y79 retinoblastoma and in lateral habenular neurons, and upregulated native Cav3.2 current in dorsal root ganglion neurons. Our data suggest that ROCK is an important regulator of T-type calcium channels, with potentially far-reaching implications for multiple cell functions modulated by LPA.
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Acknowledgements
We thank T. Snutch and J. McRory for providing wild-type Cav3 cDNAs, and M. Molineux for early recording tests in vitro. This work was supported by grants from the Canadian Institutes for Health Research. R.W.T. and G.W.Z. are Scientists of the Alberta Heritage Foundation for Medical Research (AHFMR). G.W.Z. is a Canada Research Chair. D.V. holds postdoctoral fellowships from the AHFMR and the Heart and Stroke Foundation of Canada.
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Supplementary information
Supplementary Fig. 1
Expression of endogenous LPA receptors in tsA-201 cells as assayed by RT-PCR analysis (PDF 40 kb)
Supplementary Fig. 2
Analysis of Rho kinase phosphorylation sites on the Cav3.1 channel. (PDF 35 kb)
Supplementary Fig. 3
Average peak current amplitudes obtained from Cav1.2, Cav2.1 and Cav2.2 calcium channels under control conditions, and after 15-min incubation with LPA with or without pretreatment by Fasudil. (PDF 50 kb)
Supplementary Table 1
Lack of direct effects of inhibitors on half-activation potentials and peak current amplitude of rat Cav3.1 T-type calcium channels. (PDF 52 kb)
Supplementary Table 2
The effects of LPA (10 μM) and Fasudil (10 μM) on paramters of tonic spike discharge in LHb neurons in vitro (PDF 56 kb)
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Iftinca, M., Hamid, J., Chen, L. et al. Regulation of T-type calcium channels by Rho-associated kinase. Nat Neurosci 10, 854–860 (2007). https://doi.org/10.1038/nn1921
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DOI: https://doi.org/10.1038/nn1921
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