Dieter Platzer

A C‐terminal peptide of the GIRK1 subunit directly blocks the G protein‐activated K+ channel (GIRK) expressed in Xenopus oocytes

1 In order to find out the functional roles of cytosolic regions of a G protein‐activated, inwardly rectifying potassium channel subunit we studied block of GIRK channels, expressed in Xenopus laevis oocytes, by synthetic peptides in isolated inside‐out membrane patches. 2 A peptide (DS6) derived from the very end of the C‐terminus of GIRK1 reversibly blocked GIRK activity with IC50 values of 7.9 ± 2.0 or 3.5 ± 0.5 μg ml−1 (corresponding to 3.7 ± 0.9 or 1.7 ± 0.2 μmol l−1) for GIRK1/GIRK5 or GIRK1/GIRK4 channels, respectively. 3 Dose dependency studies of GIRK activation by purified βγ subunits of the G protein (Gβγ) showed that DS6 block of GIRK channels is not the result of competition of the peptide with functional GIRK channels for the available Gβγ. 4 Burst duration of GIRK channels was reduced, whereas long closed times between bursts were markedly increased, accounting for the channel block observed. 5 Block by the DS6 peptide was slightly voltage dependent, being stronger at more negative potentials. 6 These data support the hypothesis that the distal part of the carboxy‐terminus of GIRK1 is a part of the intrinsic gate that keeps GIRK channels closed in the absence of Gβγ.

Single Channel Analysis of the Regulation of GIRK1/GIRK4 Channels by Protein Phosphorylation

G-Protein activated, inwardly rectifying potassium channels (GIRKs) are important effectors of G-protein beta/gamma-subunits, playing essential roles in the humoral regulation of cardiac activity and also in higher brain functions. G-protein activation of channels of the GIRK1/GIRK4 heterooligomeric composition is controlled via phosphorylation by cyclic AMP dependent protein kinase (PKA) and dephosphorylation by protein phosphatase 2A (PP(2)A). To study the molecular mechanism of this unprecedented example of G-protein effector regulation, single channel recordings were performed on isolated patches of plasma membranes of Xenopus laevis oocytes. Our study shows that: (i) The open probability (P(o)) of GIRK1/GIRK4 channels, stimulated by coexpressed m(2)-receptors, was significantly increased upon addition of the catalytic subunit of PKA to the cytosolic face of an isolated membrane patch. (ii) At moderate concentrations of recombinant G(beta1/gamma2), used to activate the channel, P(o) was significantly reduced in patches treated with PP(2)A, when compared to patches with PKA-cs. (iii) Several single channel gating parameters, including modal gating behavior, were significantly different between phosphorylated and dephosphorylated channels, indicating different gating behavior between the two forms of the protein. Most of these changes were, however, not responsible for the marked difference in P(o) at moderate G-protein concentrations. (iv) An increase of the frequency of openings (f(o)) and a reduction of dwell time duration of the channel in the long-lasting C(5) state was responsible for facilitation of GIRK1/GIRK4 channels by protein phosphorylation. Dephosphorylation by PP(2)A led to an increase of G(beta1/gamma2) concentration required for full activation of the channel and hence to a reduction of the apparent affinity of GIRK1/GIRK4 for G(beta1/gamma2). (v) Although possibly not directly the target of protein phosphorylation/dephosphorylation, the last 20 C-terminal amino acids of the GIRK1 subunit are required for the reduction of apparent affinity for the G-protein by PP(2)A, indicating that they constitute an essential part of the off-switch.

Quantification of Shock‐Induced Microscopic Virtual Electrodes Assessed by Subcellular Resolution Optical Potential Mapping in Guinea Pig Papillary Muscle

Introduction: The primary objective of this study was the quantitative description of shock‐induced, locally occurring virtual electrodes in natural cardiac tissue.

Simulation of excitation of single cardiomyocytes under field stimulation

In this work we try to explain the observed basic electrical behavior of isolated cardiac cells with as few assumptions as necessary. Membrane kinetics is assumed to be sufficiently characterized using the Beeler-Reuter equations. The model is used to complement experimental data, which stem from optical recordings simultaniously taken at multiple sites on a single cell during and after application of an external field stimulus.

Modeling the complex behavior of single cardiomyocytes during and after field stimulation

Recently available experimental data resulting from refined high resolution opti- cal potential measurements augment the modeling effort to describe single cell behavior during exter- nal field stimulation, in particular the process of cell excitation. Using a simplified cell model based on the Luo-Rudy description of membrane cur- rents, we compare the results of simulation stud- ies with experimental evidence. Besides striking similarity in many aspects, there are also note- able discrepancies, e.g. different dispersions of re- sulting maximum upstroke velocities, that form a challenge to cardiac cell and membrane modeling.

KCNJ3 is a new independent prognostic marker for estrogen receptor positive breast cancer patients

Numerous studies showed abnormal expression of ion channels in different cancer types. Amongst these, the potassium channel gene KCNJ3 (encoding for GIRK1 proteins) has been reported to be upregulated in tumors of patients with breast cancer and to correlate with positive lymph node status. We aimed to study KCNJ3 levels in different breast cancer subtypes using gene expression data from the TCGA, to validate our findings using RNA in situ hybridization in a validation cohort (GEO ID GSE17705), and to study the prognostic value of KCNJ3 using survival analysis. In a total of > 1000 breast cancer patients of two independent data sets we showed a) that KCNJ3 expression is upregulated in tumor tissue compared to corresponding normal tissue (p < 0.001), b) that KCNJ3 expression is associated with estrogen receptor (ER) positive tumors (p < 0.001), but that KCNJ3 expression is variable within this group, and c) that ER positive patients with high KCNJ3 levels have worse overall (p < 0.05) and disease free survival probabilities (p < 0.01), whereby KCNJ3 is an independent prognostic factor (p <0.05). In conclusion, our data suggest that patients with ER positive breast cancer might be stratified into high risk and low risk groups based on the KCNJ3 levels in the tumor.

Model-augmented investigations on field-stimulated cardiomyocytes

An experimental and theoretical approach was used to reveal and explain particular complex behavior of cardiac cells under electric field stimuli. The use of straightforward modeling forms a strong alliance with the optical recording technique to adequately explain the underlying mechanisms. Optical recordings reveal behavior that can not be explained by membrane kinetic descriptions established under physiological conditions and lead to an augmented ionic current model for the cardiac ventricular cells under investigation.

Modeling geometrical aspects in cardiac stimulation and propagation experiments

A two-dimensional model is used to complement the analysis of experimental results recorded from cardiac preparations with high resolution mapping systems at a microscopic size scale. This includes field stimulation experiments of isolated cardiomyocytes observed with a fast optical mapping system and propagation measurements on multicellular preparations recorded with a recently developed high density microelectrode array.

Electrical Inhomogeneities of Guinea Pig Atrial Tissue in High Resolution Optical Potential Mapping

Inhomogeneities in the tissue are a determining factor for cardiac function and can be either structural or functional. Using the voltage sensitive fluorescent dye di-4-ANEPPS on incubation stained guinea pig preparations allows to map the tissues response to externally applied electric shocks. Previous work on ventricular muscle preparations revealed a membrane potential morphology that could be relevant during defibrillation [1]. Using a two pulse stimulus protocol we are able to map the excitation response to a short triggering field pulse and to consequently map the membrane potential morphology resulting from a second field pulse, which is applied in the depolarized state and is attributed to the interaction of the electric field with the tissue. This approach is now carried over to atrial preparations. A custom developed object-positioning and image-stitching method allows for wide view analysis of the atrial tissue structure. Objective-magnifications of 10x, 20x, 40x and 63x enable a multi-scale based analysis with resolutions up to 15 micrometers. This is augmented with information from transmitted light microscopy so that it is possible to correlate the membrane potential patterns with the visible structure of the atrial tissue, providing further insight into atrial excitation and conduction.[1] Windisch,H. et al. (2007) Quantification of shock-induced microscopic virtual electrodes assessed by subcellular resolution optical potential mapping in guinea pig papillary muscle. J Cardiovasc Electrophysiol; 18(10):1086-1094

Structure Related Modification of the Shock Induced Excitation in Guinea Pig Papillary Muscle

Here, we present our recent findings from mapping experiments in field stimulated guinea pig papillary muscle. We monitored the developing local excitation during applied shocks (2.5-10 V/cm, 5 or 10 ms) with very high spatial and temporal resolution. Time maps, based on the occurrence of the maximal upstroke velocities, on exceeding 50% of the signal amplitudes, and on exceeding a presumed excitation threshold of -60 mV were constructed. The local, micro-structure related modulation of the excitation process was gained by subtracting a first-order fit (representing the general tendencies) of these time maps from the original ones. The resulting local time maps show the small, locally appearing temporal deviations related to local tissue discontinuities. In general, structure related modulations were found during the whole excitation phase, even during complex signal developments. In regions with positive shock induced polarizations, with increasing shock strength, the local temporal deviations were diminished; in negatively polarized regions, increased, respectively.