Molecular basis of the facilitation of the heterooligomeric GIRK1/GIRK4 complex by cAMP dependent protein kinase.

G-protein activated inwardly rectifying K(+) channels (GIRKs) of the heterotetrameric GIRK1/GIRK4 composition mediate I(K+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 Gßγ-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 GIRK1(F137S) (GIRK1(⁎)) channel. Here we report that homooligomeric GIRK4(WT) and GIRK4(S143T) (GIRK4(⁎)) channels are clearly regulated by PKA phosphorylation. Heterooligomeric channels of the GIRK1(S385CS401CT407C)/GIRK4(WT) 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 GIRK1(WT)/GIRK4(WT)). 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 GIRK1(WT)/GIRK4(T199CS412C) reduced the acute effect by ≈65%. Only channels of the GIRK1(S385CS401CT407C)/GIRK4(T199CS412C) composition were practically devoid of PKA mediated effects (reduction by ≈97%), indicating that both subunits contribute to the heterologous facilitation of I(K+ACh).

Identification of the structural determinant responsible for the phosphorylation of G-protein activated potassium channel 1 by cAMP-dependent protein kinase.

Besides being activated by G-protein beta/gamma 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 (32)P 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.

The GIRK1 brain variant GIRK1d and its functional impact on heteromultimeric GIRK channels.

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.

The TTX metabolite 4,9-anhydro-TTX is a highly specific blocker of the Na(v1.6) voltage-dependent sodium channel.

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.

A comparative study of the action of tolperisone on seven different voltage dependent sodium channel isoforms.

The specific, acute interaction of tolperisone, an agent used as a muscle relaxant and for the treatment of chronic pain conditions, with the Na(v1.2), Na(v1.3), Na(v1.4), Na(v1.5), Na(v1.6), Na(v1.7), and Na(v1.8) isoforms of voltage dependent sodium channels was investigated and compared to that of lidocaine. Voltage dependent sodium channels were expressed in the Xenopus laevis oocyte expression system and sodium currents were recorded with the two electrode voltage clamp technique. Cumulative dose response relations revealed marked differences in IC(50) values between the two drugs on identical isoforms, as well as between isoforms. A detailed kinetic analysis uncovered that tolperisone as well as lidocaine exhibited their blocking action not only via state dependent association/dissociation with voltage dependent sodium channels, but a considerable fraction of inhibition is tonic, i.e. permanent and basic in nature. Voltage dependent activation was affected to a minor extent only. A shift in steady-state inactivation to more negative potentials could be observed for most drug/isoform combinations. The contribution of this shift to overall block was, however, small at drug concentrations resulting in considerable overall block. Recovery from inactivation was affected notably by both drugs. Lidocaine application led to a pronounced prolongation of the time constant of the fast recovery process for the Na(v1.3), Na(v1.5), and Na(v1.7) isoforms, indicating common structural properties in the local anesthetic receptor site of these three proteins. Interestingly, this characteristic drug action was not observed for tolperisone.