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Participation of HERG channel cytoplasmic structures on regulation by the G protein-coupled TRH receptor

  • Ion Channels, Receptors and Transporters
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Abstract

Human ether-a-go-go-related gene (HERG) channels heterologously expressed in Xenopus oocytes are regulated by the activation of G protein-coupled hormone receptors that, like the thyrotropin-releasing hormone (TRH) receptor, activate phospholipase C. Previous work with serially deleted HERG mutants suggested that residues 326–345 located in the proximal domain of the channels amino terminus might be required for the hormonal modulation of HERG activation. Generation of new channel mutants deleted in this region further point to the amino acid sequence between residues 326 and 332 as a possible determinant of the TRH effects, but individual or combined single-point mutations in this sequence demonstrate that maintenance of its consensus sites for phosphorylation and/or interaction with regulatory components is not important for the modulatory response(s). The TRH-induced effects also remained unaltered when a basic amino acid cluster located between residues 362 and 366 is eliminated. Additionally, no effect of TRH was observed in channels carrying single-point mutations at the beginning of the intracellular loop linking transmembrane domains S4 and S5. Our results indicate that a correct structural arrangement of the amino terminal domains is essential for the hormone-induced modifications of HERG activation. They also suggest that the hormonal regulatory action is transmitted to the transmembrane channel core through interactions between the cytoplasmic domains and the initial portion of the S4–S5 linker.

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Acknowledgements

This work was supported by grants SAF2003-00329 from the Ministerio de Ciencia y Tecnología, BFU2006-10936 from the Ministerio de Educación y Ciencia of Spain (both partially cofinanced by FEDER European Funds) and IB05-002 from the Principado de Asturias (Spain). C.A.R. and D.G.M. are predoctoral fellows from the Spanish Ministerio de Ciencia y Tecnología (refs. BES-2004-3872 and AP2000-4363). P.M. holds a predoctoral fellowship from FICYT of Asturias (ref. BP03-108).

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Authors and Affiliations

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

    Carlos Alonso-Ron, Francisco Barros, Diego G. Manso, David Gómez-Varela, Pablo Miranda, Luis Carretero, Pedro Domínguez & Pilar de la Peña

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  1. Carlos Alonso-Ron
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  2. Francisco Barros
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Correspondence to Francisco Barros.

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Supplemental Fig. 1

Deactivation properties of different constructs and effect of structural modifications in the amino terminus and the S4-S5 loop on TRH-induced acceleration of HERG deactivation. (A) Summary of fast deactivation rates at −100 mV. We considered for analysis only the time constants of the fast deactivation current that corresponds to the major component of current at negative voltages and that shows minimal contamination with residual oocyte chloride currents triggered by the hormone treatment. The rates of deactivation were determined from negative-amplitude biexponential fits to the decaying phase of tail currents at −100 mV as detailed in ref [1] of the paper. (B) Comparison of TRH effects on deactivation kinetics in channels carrying different structural alterations in the amino-terminus. Bars in the histogram represent TRH-induced reductions in the magnitude of fast deactivation time constant at −100 mV as compared to that observed in wild-type channels, that amounted 22.6 ± 3.7% (n = 24) in this set of experiments. In this case, a 50% reduction corresponds to a doubled speed of closing. (C) Comparison of TRH effects on deactivation kinetics in channels carrying single-point mutations in the S4–S5 loop (residues 540–546) and the 326–331 sequence of the proximal domain (GIF 96 kb)

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Alonso-Ron, C., Barros, F., Manso, D.G. et al. Participation of HERG channel cytoplasmic structures on regulation by the G protein-coupled TRH receptor. Pflugers Arch - Eur J Physiol 457, 1237–1252 (2009). https://doi.org/10.1007/s00424-008-0599-x

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