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Cell type influences the molecular mechanisms involved in hormonal regulation of ERG K+ channels

  • Ion Channels, Receptors and Transporters
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Abstract

While the thyrotropin-releasing hormone (TRH) effect of raising intracellular Ca2+ levels has been shown to rely on Gq/11 and PLC activation, the molecular mechanisms involved in the regulation of ERG K+ channels by TRH are still partially unknown. We have analysed the effects of βγ scavengers, Akt/PKB inactivation, and TRH receptor (TRH-R) overexpression on such regulation in native and heterologous expression cell systems. In native rat pituitary GH3 cells β-ARK/CT, Gαt, and phosducin significantly reduced TRH inhibition of rERG currents, whereas in HEK-H36/T1 cells permanently expressing TRH-R and hERG, neither of the βγ scavengers affected the TRH-induced shift in V 1/2. Use of specific siRNAs to knock Akt/PKB expression down abolished the TRH effect on HEK-H36/T1 cell hERG, but not on rERG from GH3 cells. Indeed, wortmannin or long insulin pretreatment also blocked TRH regulation of ERG currents in HEK-H36/T1 but not in GH3 cells. To determine whether these differences could be related to the amount of TRH-Rs in the cell, we studied the TRH concentration dependence of the Ca2+ and ERG responses in GH3 cells overexpressing the receptors. The data indicated that independent of the receptor number additional cellular factor(s) contribute differently to couple the TRH-R to hERG channel modulation in HEK-H36/T1 cells. We conclude that regulation of ERG currents by TRH and its receptor is transduced in GH3 and HEK-H36/T1 cell systems through common and different elements, and hence that the cell type influences the signalling pathways involved in the TRH-evoked responses.

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Acknowledgements

We thank Teresa González for the technical assistance and Dr. Kevin Dalton for proof reading the text. We also thank Dr. Angel Martínez-Nistal, head of the Scientific and Technical Services of the University of Oviedo, for the loan of the epifluorescence light source for our patch-clamp setup. This work was supported in part by Spanish Ministerio de Ciencia e Innovación (MICINN) Grant BFU2009-11262 and Spanish Ion Channel Initiative (SICI) Consolider-Ingenio Project CSD2008-00005. L.C. and J. F-T were supported by predoctoral fellowships from the Ministerio de Educación y Ciencia (MEC) and the Fondo de Investigación Científica y Técnica (FICYT) of Spain. A.M. holds a postdoctoral contract from SICI-Consolider 2008.

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  1. Departamento de Bioquímica y Biología Molecular, Edificio Santiago Gascón, Campus del Cristo, Universidad de Oviedo, 33006, Oviedo, Spain

    Luis Carretero, Francisco Barros, Pablo Miranda, Jorge Fernández-Trillo, Angeles Machín, Pilar de la Peña & Pedro Domínguez

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  1. Luis Carretero
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Correspondence to Pedro Domínguez.

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Suppl. Figure 1

Effect of the βγ scavengers βARK/CT and phosducin on Ca2+ liberation from GH3 cell intracellular stores in response to TRH. The time course of variations in [Ca2+] i levels is shown for Fura-2 loaded cells transfected with control plasmid, β-ARK/CT, or phosducin. Addition of 1 μM TRH is indicated by horizontal lines on top of the traces. Note the relatively high basal [Ca2+] i of the GH3 cells due to the spontaneous electrical activity and the oscillatory resting calcium levels of the individual cells. Continuous traces averaged point by point and their corresponding S.E.M. are shown. Data correspond to cells from the same microscope fields either showing fluorescence of the transfection marker (YFP+, left) or not (YFP−, right). Variations of [Ca2+] i levels in the cells showing some detectable peak Ca2+ increase after visual inspection are illustrated. In all cases, the number of averaged cells is in parenthesis. Averaged values from all data points before TRH addition and that of the initial maximum are indicated. The lower panel shows the averaged fold [Ca2+] i increase after TRH addition corresponding to the upper six panels. Analogous results were obtained in two additional experiments. (JPEG 127 kb)

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Carretero, L., Barros, F., Miranda, P. et al. Cell type influences the molecular mechanisms involved in hormonal regulation of ERG K+ channels. Pflugers Arch - Eur J Physiol 463, 685–702 (2012). https://doi.org/10.1007/s00424-012-1094-y

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