The blocking efficacy of 4,9-anhydro-TTX (4,9-ah-TTX) and TTX on several isoforms of voltage-dependent sodium channels, expressed in Xenopus laevis oocytes, was tested (Na(v1.2), Na(v1.3), Na(v1.4), Na(v1.5), Na(v1.6), Na(v1.7), and Na(v1.8)). Generally, TTX was 40-231 times more effective, when compared with 4,9-ah-TTX, on a given isoform. An exception was Na(v1.6), where 4,9-ah-TTX in nanomole per liter concentrations sufficed to result in substantial block, indicating that 4,9-ah-TTX acts specifically at this peculiar isoform. The IC(50) values for TTX/4,9-ah-TTX were as follows (in nmol/l): 7.8 +/- 1.3/1,260 +/- 121 (Na(v1.2)), 2.8 +/- 2.3/341 +/- 36 (Na(v1.3)), 4.5 +/- 1.0/988 +/- 62 (Na(v1.4)), 1,970 +/- 565/78,500 +/- 11,600 (Na(v1.5)), 3.8 +/- 1.5/7.8 +/- 2.3 (Na(v1.6)), 5.5 +/- 1.4/1,270 +/- 251 (Na(v1.7)), and 1,330 +/- 459/>30,000 (Na(v1.8)). Analysis of approximal half-maximal doses of both compounds revealed minor effects on voltage-dependent activation only, whereas steady-state inactivation was shifted to more negative potentials by both TTX and 4,9-ah-TTX in the case of the Na(v1.6) subunit, but not in the case of other TTX-sensitive ones. TTX shifted steady-state inactivation also to more negative potentials in case of the TTX-insensitive Na(v1.5) subunit, where it also exerted profound effects on the time course of recovery from inactivation. Isoform-specific interaction of toxins with ion channels is frequently observed in the case of proteinaceous toxins. Although the sensitivity of Na(v1.1) to 4,9-ah-TTX is not known, here we report evidence on a highly isoform-specific TTX analog that may well turn out to be an invaluable tool in research for the identification of Na(v1.6)-mediated function, but also for therapeutic intervention.
Besides being activated by G‐protein β/γ subunits, G‐protein activated potassium channels (GIRKs) are regulated by cAMP‐dependent protein kinase. Back‐phosphorylation experiments have revealed that the GIRK1 subunit is phosphorylated in vivo upon protein kinase A activation in Xenopus oocytes, whereas phosphorylation was eliminated when protein kinase A was blocked. In vitro phosphorylation experiments using truncated versions of GIRK1 revealed that the structural determinant is located within the distant, unique cytosolic C‐terminus of GIRK1. Serine 385, serine 401 and threonine 407 were identified to be responsible for the incorporation of radioactive 32P into the protein. Furthermore, the functional effects of cAMP injections into oocytes on currents produced by GIRK1 homooligomers were significantly reduced when these three amino acids were mutated. The data obtained in the present study provide information about the structural determinants that are responsible for protein kinase A phosphorylation and the regulation of GIRK channels. Structured digital abstract http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-7260296, http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-7260317, http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-7260333, http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-7260347, http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-7260361, http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-7260270: PKA‐cs (uniprotkb:http://www.ebi.uniprot.org/entry/P00517) phosphorylates (http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0217) Girk1 (uniprotkb:http://www.ebi.uniprot.org/entry/P63251) by protein kinase assay (http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0424)
G-protein activated inwardly rectifying K+ channels (GIRKs) of the heterotetrameric GIRK1/GIRK4 composition mediate IK + ACh in atrium and are regulated by cAMP dependent protein kinase (PKA). Phosphorylation of GIRK1/GIRK4 complexes promotes the activation of the channel by the G-protein Gβγ-dimer (“heterologous facilitation”). Previously we reported that 3 serines/threonines (S/Ts) within the GIRK1 subunit are phosphorylated by the catalytic subunit of PKA (PKA-cs) in-vitro and are responsible for the acute functional effects exerted by PKA on the homooligomeric GIRK1F137S (GIRK1⁎) channel. Here we report that homooligomeric GIRK4WT and GIRK4S143T (GIRK4⁎) channels are clearly regulated by PKA phosphorylation. Heterooligomeric channels of the GIRK1S385CS401CT407C/GIRK4WT composition, where the GIRK1 subunit is devoid of PKA mediated phosphorylation, exhibited reduced but still significant acute effects (reduction during agonist application was ≈ 49% compared to GIRK1WT/GIRK4WT). Site directed mutagenesis of truncated cytosolic regions of GIRK4 revealed four serines/threonines (S/Ts) that were heavily phosphorylated by PKA-cs in vitro. Two of them were found to be responsible for the acute effects exerted by PKA in vivo, since the effect of cAMP injection was reduced by ≈ 99% in homooligomeric GIRK4⁎T199CS412C channels. Coexpression of GIRK1WT/GIRK4T199CS412C reduced the acute effect by ≈ 65%. Only channels of the GIRK1S385CS401CT407C/GIRK4T199CS412C composition were practically devoid of PKA mediated effects (reduction by ≈ 97%), indicating that both subunits contribute to the heterologous facilitation of IK + ACh.
Four isoforms of GIRK channels (GIRK1-4) have been described in humans. In addition, several splice variants of more or less unknown function have been identified from several tissues and species. In our study, we investigated the structure and function of a new variant of GIRK1 that has been isolated from rat brain. Because of wide similarities with a previously described variant, we also named it GIRK1d. This variant lacks a region corresponding to exon 2 of full-length GIRK1, leading to a truncated GIRK1 that lacks the main part of the C-terminus. To study GIRK1d we used the Xenopus laevis expression system, the two-electrode voltage clamp method, and confocal laser scan microscopy. We found that our GIRK1d variant preferentially binds GIRK2 or GIRK4 over GIRK1. Furthermore, it largely reduces conductances mediated by GIRK1/2 or GIRK1/4 hetero-multimeric channels when coexpressed and nearly totally abolishes currents when replacing GIRK1 in hetero-multimeric channels.
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