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

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.

FRET with multiply labeled HERG K(+) channels as a reporter of the in vivo coarse architecture of the cytoplasmic domains.

The intracellular N-terminus of human ether-a-go-go-related gene (HERG) potassium channels constitutes a key determinant of activation and deactivation characteristics and is necessary for hormone-induced modifications of gating properties. However, the general organization of the long amino and carboxy HERG terminals remains unknown. In this study we performed fluorescence resonance energy transfer (FRET) microscopy with a library of fluorescent HERG fusion proteins obtained combining site-directed and transposon-based random insertion of GFP variants into multiple sites of HERG. Determinations of FRET efficiencies with functional HERG channels labeled in different combinations localize the fluorophores, introduced in the amino and carboxy ends, in two quadratic planes of 7.8 and 8.6 nm lateral size, showing a vertical separation of nearly 8 nm without major angular torsion between the planes. Similar analysis using labels at positions 345 and 905 of the amino and carboxy terminals, located them slightly above the planes delimited by the amino and carboxy end labels, respectively. Our data also indicate an almost vertical arrangement of the fluorophores introduced in the NH(2) and COOH ends and at position 905, but a near 45 degrees angular rotation between the planes delimited by these labels and the 345-located fluorophores. Systematic triangulation using interfluorophore distances coming from multiply labeled channels provides an initial constraint on the overall in vivo arrangement of the HERG cytoplasmic domains, suggesting that the C-linker/CNBD region of HERG hangs centrally below the transmembrane core, with the initial portion of the amino terminus around its top and side surfaces directed towards the gating machinery.

Specificity of TRH receptor coupling to G-proteins for regulation of ERG K+ channels in GH3 rat anterior pituitary cells.

The identity of the G-protein coupling thyrotropin-releasing hormone (TRH) receptors to rat ether-à-go-go related gene (r-ERG) K+ channel modulation was studied in situ using perforated-patch clamped adenohypophysial GH(3) cells and dominant-negative variants (Galpha-QL/DN) of G-protein alpha subunits. Expression of dominant-negative Galpha(q/11) that minimizes the TRH-induced Ca2+ signal had no effect on r-ERG current inhibition elicited by the hormone. In contrast, the introduction of dominant-negative variants of Galpha13 and the small G-protein Rho caused a significant loss of the inhibitory effect of TRH on r-ERG. A strong reduction of this TRH effect was also obtained in cells expressing either dominant-negative Galpha(s) or transducin alpha subunits, an agent known to sequester free G-protein betagamma dimers. As a further indication of specificity of the dominant-negative effects, only the dominant-negative variants of Galpha13 and Rho (but not Galpha(s)-QL/DN or Galpha(t)) were able to reduce the TRH-induced shifts of human ERG (HERG) activation voltage dependence in HEK293 cells permanently expressing HERG channels and TRH receptors. Our results demonstrate that whereas the TRH receptor uses a G(q/11) protein for transducing the Ca2+ signal during the initial response to TRH, this G-protein is not involved in the TRH-induced inhibition of endogenous r-ERG currents in pituitary cells. They also identify G(s) (or a G(s)-like protein) and G13 as important contributors to the hormonal effect in these cells and suggest that betagamma dimers released from these proteins may participate in modulation of ERG currents triggered by TRH.

Relevance of the proximal domain in the amino-terminus of HERG channels for regulation by a phospholipase C-coupled hormone receptor.

We used Xenopus oocytes co-expressing thyrotropin-releasing hormone (TRH) receptors and human ether-a-go-go-related gene (HERG) K+ channel variants carrying different amino-terminal modifications to check the relevance of the proximal domain for hormonal regulation of the channel. Deletion of the whole proximal domain (Delta 138-373) eliminates TRH-induced modifications in activation and deactivation parameters. TRH effects on activation are also suppressed with channels lacking the second half of the proximal domain or only residues 326-373. However, normal responses to TRH are obtained with Delta 346-373 channels. Thus, whereas residues 326-345 are required for the hormonal modulation of HERG activation, different proximal domain sequences contribute to set HERG gating characteristics and its regulation by TRH.