Wolfgang Schreibmayer

Tolperisone: A typical representative of a class of centrally acting muscle relaxants with less sedative side effects

Tolperisone, a piperidine derivative, is assigned to the group of centrally acting muscle relaxants and has been in clinical use now for decades. The review summarizes the known pharmacokinetics, pharmacodynamics, toxicology and side effects in humans and the clinical use of tolperisone. A future perspective for further exploration of this drug is given.

Cloning and characterisation of GIRK1 variants resulting from alternative RNA editing of the KCNJ3 gene transcript in a human breast cancer cell line

The aim of this study was to investigate the impact of increased mRNA levels encoding GIRK1 in breast tumours on GIRK protein expression. mRNA levels encoding hGIRK1 and hGIRK4 in the MCF7, MCF10A and MDA‐MB‐453 breast cancer cell lines were assessed and the corresponding proteins detected using Western blots. cDNAs encoding for four hGIRK1 splice variants (hGIRK1a, 1c, 1d and 1e) were cloned from the MCF7 cell line. Subcellular localisation of fluorescence labelled hGIRK1a–e and hGIRK4 and of endogenous GIRK1 and GIRK4 subunits was monitored in the MCF7 cell line. All hGIRK1 splice variants and hGIRK4 were predominantly located within the endoplasmic reticulum. Heterologous expression in Xenopus laevis oocytes and two electrode voltage clamp experiments together with confocal microscopy were performed. Only the hGIRK1a subunit was able to form functional GIRK channels in connection with hGIRK4. The other splice variants are expressed, but exert a dominant negative effect on heterooligomeric channel function. Hence, alternative splicing of the KCNJ3 gene transcript in the MCF7 cell line leads to a family of mRNAs, encoding truncated versions of the hGIRK1 protein. The very high abundance of mRNAs encoding GIRK1 together with the presence of GIRK1 protein suggests a pathophysiological role in breast cancer. J. Cell. Biochem. 110: 598–608, 2010.

No blocking effects of the pentapeptide QYNAD on Na+ channel subtypes expressed in Xenopus oocytes or action potential conduction in isolated rat sural nerve

Reversible block of Na(+) channels by endogenous pentapeptide QYNAD has been reported to account for the fast relapses and remissions seen in autoimmune demyelinating disorders. Here it is shown that, in contrast to previous reports, synthetic QYNAD (10-100 microM) applied to Na(+) channels (Na(v)1.6 and 1.8) expressed in Xenopus oocytes was unable to block the peak current or inhibit channel kinetics. Furthermore, QYNAD (100 microM) applied to five isolated rat sural nerve in vitro did not demonstrate any change in the amplitude of compound nerve action potential or latency. The reason for the ineffectiveness of QYNAD has not been elucidated; it was apparently not related to a problem in the synthesis of the pentapeptide. Our experiments raise significant concerns about the suggestion that QYNAD peptide is a Na(+) channel blocker or modulator. However, in a protein library search the amino acid sequence of QYNAD was found to be related to ankyrin-G, which plays a role in Na(+) channel clustering in the node of Ranvier.

Four and a Half LIM Protein 1C (FHL1C): A Binding Partner for Voltage-Gated Potassium Channel Kv1.5

Four-and-a-half LIM domain protein 1 isoform A (FHL1A) is predominantly expressed in skeletal and cardiac muscle. Mutations in the FHL1 gene are causative for several types of hereditary myopathies including X-linked myopathy with postural muscle atrophy (XMPMA). We here studied myoblasts from XMPMA patients. We found that functional FHL1A protein is completely absent in patient myoblasts. In parallel, expression of FHL1C is either unaffected or increased. Furthermore, a decreased proliferation rate of XMPMA myoblasts compared to controls was observed but an increased number of XMPMA myoblasts was found in the G0/G1 phase. Furthermore, low expression of Kv1.5, a voltage-gated potassium channel known to alter myoblast proliferation during the G1 phase and to control repolarization of action potential, was detected. In order to substantiate a possible relation between Kv1.5 and FHL1C, a pull-down assay was performed. A physical and direct interaction of both proteins was observed in vitro. In addition, confocal microscopy revealed substantial colocalization of FHL1C and Kv1.5 within atrial cells, supporting a possible interaction between both proteins in vivo. Two-electrode voltage clamp experiments demonstrated that coexpression of Kv1.5 with FHL1C in Xenopus laevis oocytes markedly reduced K+ currents when compared to oocytes expressing Kv1.5 only. We here present the first evidence on a biological relevance of FHL1C.

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 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.

An optically controlled probe identifies lipid-gating fenestrations within the TRPC3 channel

AbstractTransient receptor potential canonical (TRPC) channels TRPC3, TRPC6 and TRPC7 are able to sense the lipid messenger diacylglycerol (DAG). The DAG-sensing and lipid-gating processes in these ion channels are still unknown. To gain insights into the lipid-sensing principle, we generated a DAG photoswitch, OptoDArG, that enabled efficient control of TRPC3 by light. A structure-guided mutagenesis screen of the TRPC3 pore domain unveiled a single glycine residue behind the selectivity filter (G652) that is exposed to lipid through a subunit-joining fenestration. Exchange of G652 with larger residues altered the ability of TRPC3 to discriminate between different DAG molecules. Light-controlled activation–deactivation cycling of TRPC3 channels by an OptoDArG-mediated optical ‘lipid clamp’ identified pore domain fenestrations as pivotal elements of the channel´s lipid-sensing machinery. We provide evidence for a novel concept of lipid sensing by TRPC channels based on a lateral fenestration in the pore domain that accommodates lipid mediators to control gating.A photoswitchable diacylglycerol enabled a screen that found critical TRPC3 lipid-sensing residues and identified a lateral fenestration in the pore domain that allows lipids to protrude toward the permeation pathway to control channel gating.

Single-channel properties of α3β4, α3β4α5 and α3β4β2 nicotinic acetylcholine receptors in mice lacking specific nicotinic acetylcholine receptor subunits

•  Under normal conditions, individual nerve cells express a cohort of several nicotinic acetylcholine receptor (nAChR) subtypes with unique functional properties. Therefore, single‐ channel recordings of such neurons reflect the mixed properties of the various expressed receptors. •  Previous attempts to recapitulate the properties of naturally occurring receptors using recombinant receptors of known subunit composition in heterologous expression systems have been largely unsuccessful, as the properties of these receptors vary widely among expression systems. •  We measured the properties of specific nAChRs in superior cervical ganglion neurons cultured from mice with targeted deletions of select nAChR subunit genes. Mice lacking both the α5 and the β2 subunits express α3β4 receptors, whereas single‐knockout (KO) mice lacking either α5 or β2 express α3β4β2 and α3β4α5 (plus α3β4) hetero‐oligomeric receptors, respectively. This approach allows one to investigate these receptors at the single‐channel level in their native environment. The single‐channel properties of nACh receptors in superior cervical ganglion (SCG) neurons from α5β2α7 triple‐KO mice were similar to the properties of receptors measured in α5β2 double‐KO mice. •  The principal conductance level of α3β4 receptors was 32.6 ± 0.8 pS (mean ± SEM), and these receptors also displayed both higher and lower secondary conductance levels. The conductance levels of α3β4α5 receptors were identical to α3β4 receptors, but the α3β4α5 receptors had longer open times and burst duration. α3β4β2 receptors had a lower conductance level and longer open times than α3β4 receptors. Interestingly, all three receptor types could be identified faithfully in wild‐type C57Bl/6J SCG neurons.

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 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.

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.

Emerging role(s) of G‐protein α‐subunits in the gating of GIRKs

In the early days following the discovery of heterotrimeric G-Proteins, the GTP associated Gα subunits were regarded as the active principle in signalling to G-protein effectors. Gβ/γ subunits were seen as inactive, being required as ‘docking-stations’ for α-subunits when signalling was over. The first direct Gβ/γ effector to be discovered was a G-protein activated potassium channel from atrium (GIRK; Logothetis et al. 1987). Subsequently, certain isoforms of phospholipase Cβ (PLCβ) and other G-protein effectors were also found to be triggered directly by the Gβ/γ dimer. Finally the view that Gβ/γ and the Gα subunits are in fact equal partners with respect to signalling became generally accepted (for review see Birnbaumer, 2007). Moreover, with the uncovering of G-protein effector isoforms, it became clear that some effector isoforms are regulated by Gαs, others by Gβ/γs, in a differential and eventually divergent way, sometimes both entities acting cooperatively on the same effector isoform (see e.g. Tang & Gilman, 1991). With respect to GIRKs, Gβ/γ dimers of pertussis toxin (PTX) sensitive G-proteins were long believed to act as the exclusive mediators of G-protein coupled receptor (GPCR) activation. Only sparse evidence for a role of G-protein α-subunits existed: in 1995, direct binding of Gβ/γ to both the carboxy- and the amino-terminal cytosolic parts (C-T and N-T, respectively) of the GIRK1 subunit was observed (Huang et al. 1995). Interestingly, also the inactive G-protein trimer, including the Gα subunit, was found to directly interact with the GIRK1 N-T in the same study. First functional effects of G-protein α-subunits on GIRK channel activation by Gβ/γ were described in 1996 (Schreibmayer et al. 1996). Despite these early findings the exact role(s) of G-protein α-subunits as mediators of GIRK gating remained obscure. In a seminal series of papers, starting in 2002 and coming to a preliminary end with Rubinstein et al. (2009) in a recent issue of The Journal of Physiology, scientists around Prof. Dascal from Tel Aviv University have established the GIRK1 subunit as target for the inactive, GDP bound, form of Gα. The entire story started with the seminal observation, that the basal, agonist-independent K+ current through GIRK1/GIRK2 heterooligomeric channels grows disproportionally with the level of expression in Xenopus oocytes. Thus, at high levels of expression of GIRK1/GIRK2 the basal current is abnormally high, indicating a limiting endogenous compound. Surprisingly, Gαi3 was identified as being this limiting compound, since Gαi3 overexpression sufficed to restore low basal current. Moreover, Gαi3 was found to directly interact with and bind to the GIRK1 N-T, confirming the previous data of Huang et al. (1995) and Peleg et al. (2002). In two subsequent papers (Ivanina et al. 2003, 2004), the binding sites for both Gβ/γ and Gαi1/Gαi3 on the GIRK1 and GIRK2 C-T and N-T were mapped with biochemical methods. In addition to confirming and extending data of several other groups, a low-affinity Gβ/γ-binding site was detected in the unique distal C-T of GIRK1. When compared to Gαi1, Gαi3 binding to GIRK1 and to GIRK2 was stronger. Accordingly, Gαi3 better supported activation by Gβ/γ when compared to Gαi1 in excised membrane patches as well as in whole oocytes. The correlation between the specificity of the two Gαs and their binding strength to GIRK subunits indicated that direct binding of Gαi plays a role in channel regulation. Subsequently, Rishal et al. (2005) discovered, that the high basal activity upon overexpression of GIRK1/GIRK2 was the result of co-trafficking of the Gβ/γ subunit with the GIRK1/GIRK2 heterooligomer, which ‘hauls’ Gβ/γ to the plasma membrane. Co-trafficking of Gα subunits was also observed, but to a lesser extent. Preassociation of Gβ/γ with GIRKs with signalling complexes was independently confirmed by others using BRET and FRET (Rebois et al. 2006; Riven et al. 2006). The knowledge on the regulation of GIRK channels by G-protein α-subunits was meanwhile extended independently by another group, when Koike-Tani et al. (2005) observed direct inhibition of GIRK1/GIRK2 heterooligomeric channels by Gαq. Rubinstein et al. (2007) refined and extended the experimental paradigms and further distinguished the regulation by Gαi3 from the effect of a simple Gβ/γ scavenger. Notably, Rubinstein et al. (2009) show that the regulation of GIRK1/GIRK2 by Gαi3 is not a peculiar property of the Xenopus laevis oocyte expression system, but also occurs in mammalian (HEK 293) cells. Furthermore, by using constitutively active and inactive mutants of Gαi3, it was demonstrated that Gαi3GDP, but not Gαi3GTP is required to regulate basal activity. Moreover, this regulation is isoform specific with respect to GIRK subunits: only GIRK1 binds to the Gαi3βγ heterotrimer, while GIRK2 does not. Accordingly, functional effects are conferred exclusively by the GIRK1 subunit. The responsible structural determinant is located in the unique proximal C-T of GIRK1. Rubinstein et al. propose a model where GIRK1/2 has two sites, an ‘activation’ site for Gβ/γ which is occupied by Gβ/γ when Gα is GTP-bound, and an ‘anchoring’ site where the ineffective Gαβγ heterotrimer is bound to GIRK, preventing Gβ/γ from activating the channel. New perspectives come forth and reveal important questions to be answered in the future. Does this regulation via GIRK associated Gα subunits participate in the specificity of G-protein isoform mediated signalling? Are also GIRK3 and GIRK4 subunits affected? Does this regulation of basal current by Gαi3GDP occur in native cells? In atrial cells, for example, the agonist independent, basal activity of IK+ACh is extremely low, to an extent that has never been observed in heterologous systems (see e.g. Schreibmayer et al. 1996). It will be interesting to see whether regulation by an endogenous Gα subunit is responsible.