Consequences of somatic mutations of GIRK1 detected in primary malign tumors on expression and function of G-protein activated, inwardly rectifying, K+ channels.

A search in the GDC Data Portal revealed 304 documented somatic mutations of the KCNJ3 gene in primary tumors (out of 10.202 cases). Most affected tumor types were carcinomas from uterus, skin and lung, while breast cancer exerted the lowest number of somatic mutations. We focused our research on 15 missense mutations within the region between TM1 and TM2, comprising the pore helix and ion selectivity signature. Expression was measured by confocal laser scan microscopy of eGFP tagged GIRK1 subunits, expressed with and without GIRK4 in oocytes of Xenopus laevis. GIRK ion currents were activated via coexpressed m2Rs and measured by the Two Electrode Voltage Clamp technique. Magnitude of the total GIRK current, as well as the fraction of current inducible by the agonist, were measured. Ion selectivity was gauged by assessment of the PNa+/PK+ ratio, calculated by the GIRK current reversal potential in extracellular media at different Na+ and K+ concentrations. None of the tested mutations was able to form functional GIRK1 homooligomeric ion channels. One of the mutations, G145A, which locates directly to the ion selectivity signature, exerted an increased PNa+/PK+ ratio. Generally, the missense mutations studied can be categorized into three groups: (i) normal/reduced expression accompanied by reduced/absent function (S132Y, F136L, E139K, G145A, R149Q, R149P, G178D, S185Y, Q186R), (ii) normal/increased expression as well as increased function (E140M, A142T, M184I) and (iii) miniscule expression but increased function relative to expression levels (I151N, G158S). We conclude, that gain of function mutations, identical or similar to categories (ii) and (iii), may potentially be involved in genesis and progression of malignancies in tissues that exert a high rate of occurrence of somatic mutations of KCNJ3.

Accuracy considerations for capacitance estimation by voltage steps in cardiomyocytes.

Electrophysiologists routinely use simple voltage steps to evaluate cell membrane capacitance derived from corresponding current responses. Frequently, the resting membrane voltage Vrest is employed as holding potential for the subsequent command voltage step and more or less accurate methods are utilised to analyse the transient current. Another choice as holding potential is the peak of the "quasi steady-state" current to voltage relationship, Vpeak. The aim of this study is the systematic evaluation of capacitance estimation accuracy from voltage step experiments depending on the choice of holding potential and analysis method. In this paper, a simulation approach is employed to analyse the current response of a model patch-clamp circuit. Four commonly accepted methods are implemented, utilizing different aspects of the transient current (charge, membrane time constant, and influence of the series resistance) in various combinations and with various degrees of refinement. This simulation study indicates an acceptable accuracy of the elaborated methods for capacitance estimation at holding potentials Vrest and Vpeak over a broad range of capacitance as well as series resistance values. Simple integration of the current transient provides sufficient accuracy at holding potentials, which effectively minimizes changes in resistive membrane current flow during command voltage steps (particularly around Vpeak). However, biphasic command protocols performed at Vpeak activate voltage dependent sodium channels, thereby possibly leading to the threshold voltage for an action potential. Compared to Vrest, all methods utilizing monophasic step protocols, gain additional accuracy, when applied at Vpeak as holding potential.

Postural hemodynamic parameters in older persons have a seasonal dependency : A pilot study.

AIMS: It is known that blood pressure regulation differs seasonally. It is unknown, however, how the cardiovascular system in patients with a stroke reacts to postural changes in different seasons. The aim was therefore to investigate how different temperatures in cold and warm seasons influence the reactions of haemodynamic mechanisms as well as heart rate variability during a sit-to-stand test in patients with stroke and a control group. METHODS: Hemodynamic responses were assessed in both groups during a sit-to-stand test (5 min sitting followed by 5 min standing) beat to beat within two different seasons. Systolic blood pressure (SBP), diastolic blood pressure (DBP), mean blood pressure (MBP), heart rate (HR), stroke index (SI), cardiac index (CI) and heart rate variability (HRV) were continuously monitored. RESULTS: During the sitting baseline period delta values of DBP (+15.1 [Standard error (SE) 3.75] mmHg, p < 0.05) and MBP (+14.35 [SE 4.18] mmHg, p < 0.05) were significantly higher in colder months compared to warmer months whereas SI (-3.86 [SE 1.43] ml/beat/m2, p < 0.05) and CI (-0.4 [SE 0.11] l/min/m2, p < 0.05) were lower in colder months compared to warmer months in non-stroke participants. In patients with stroke during sitting, baseline period delta values of DBP (+19.92 [SE 8.03] mmHg, p < 0.05) and MBP (+19.29 [SE 8.6] mmHg, p < 0.05) were significantly higher in colder months compared to warmer months but SI (-5.43 [SE 1.96] ml/beat/m2, p < 0.05) was significantly lower in colder months compared to warmer months. After standing, there was a significant decrease in SBP in warmer months (-16.84 [SE 4.38] mmHg, p < 0.05) and a decrease in DBP in warmer months (-7.8 [SE 2.3] mmHg, p < 0.05) and colder months (-6.73 [SE 1.5] mmHg, p < 0.05) in non-stroke participants and a decrease in MBP in warmer months (-12.5 [SE 2.8] mmHg, p < 0.05) and colder months (-8.93 [SE 1.8] mmHg, p < 0.05) in non-stroke participants and in warmer months (-14.54 [SE 4.1] mmHg, p < 0.05) in patients with stroke. CONCLUSION: Elderly with and without stroke respond to orthostatic stress with a greater drop in blood pressure in the warmer seasons.

GIRK1 triggers multiple cancer-related pathways in the benign mammary epithelial cell line MCF10A.

Excessive expression of subunit 1 of GIRK1 in ER+ breast tumors is associated with reduced survival times and increased lymph node metastasis in patients. To investigate possible tumor-initiating properties, benign MCF10A and malign MCF7 mammary epithelial cells were engineered to overexpress GIRK1 neoplasia associated vital parameters and resting potentials were measured and compared to controls. The presence of GIRK1 resulted in resting potentials negative to the controls. Upon GIRK1 overexpression, several cellular pathways were regulated towards pro-tumorigenic action as revealed by comparison of transcriptomes of MCF10AGIRK1 with the control (MCF10AeGFP). According to transcriptome analysis, cellular migration was promoted while wound healing and extracellular matrix interactions were impaired. Vital parameters in MCF7 cells were affected akin the benign MCF10A lines, but to a lesser extent. Thus, GIRK1 regulated cellular pathways in mammary epithelial cells are likely to contribute to the development and progression of breast cancer.

A single point mutation in the TRPC3 lipid-recognition window generates supersensitivity to benzimidazole channel activators.

Mutation of a single residue within the recently identified lipid (diacylglycerol) recognition window of TRPC3 (G652A) was found to abolish channel activation via endogenous lipid mediators while retaining sensitivity to the non-lipid activator GSK1702934A (abb. GSK). The mechanism of this change in chemical sensing by TRPC3 was analysed by whole-cell and single channel electrophysiology as well as Ca2+ imaging. Currents initiated by GSK or the structural (benzimidazole) analog BI-2 were significantly larger in cells expressing the G652A mutant as compared to wild type (WT) channels. Whole cell patch-clamp experiments revealed that enhanced sensitivity to benzimidazoles was not due to augmented potency but reflected enhanced efficacy of benzimidazoles. Single channel analysis demonstrated that neither unitary conductance nor I-V characteristics were altered by the G652A mutation, precluding altered pore architecture as the basis of enhanced efficacy. These experiments uncovered a distinct gating pattern of BI-2-activated G652A mutant channels, featuring a unique, long-lived open state. Moreover, G652A mutant channels lacked PLC/diacylglycerol mediated cross-desensitization for GSK activation as typically observed for TRPC3. Lack of desensitization in G652A channels enabled large GSK/BI-2-induced Ca2+ signals in conditions that fully desensitized TRPC3 WT channels. We demonstrate that the lipid-recognition window of TRPC3 determines both sensitivity to lipid mediators and chemical gating by benzimidazoles. TRPC3 mutations within this lipid interaction site are suggested as a basis for chemogenetic targeting of TRPC3-signaling.

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

Transient 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.

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 KCNJ3using 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.

Live-cell imaging of ER-PM contact architecture by a novel TIRFM approach reveals extension of junctions in response to store-operated Ca2+-entry.

Nanometer-spaced appositions between endoplasmic reticulum and plasma membrane (ER-PM junctions) stabilized by membrane-joining protein complexes are critically involved in cellular Ca2+-handling and lipid trafficking. ER-PM junctional architecture and plasticity associated with inter-membrane communication are as yet barely understood. Here, we introduce a method to precisely characterize ER-PM junction morphology and dynamics with high temporal resolution and minimal disturbance of junctional intermembrane communication. We show that expression of soluble cytosolic fluorophores in combination with TIRFM enables to delineate ER and PM distance in the range of 10-150 nm. Live-cell imaging of sub-plasmalemmal structures in RBL-2H3 mast cells by this method, designated as fluorescence density mapping (FDM), revealed profound dynamics of ER-PM contact sites in response to store-depletion. We report the existence of a Ca2+-dependent process that expands the junctional ER to enlarge its contact surface with the PM, thereby promoting and stabilizing STIM1-Orai1 competent ER-PM junctions.

I(f) blocking potency of ivabradine is preserved under elevated endotoxin levels in human atrial myocytes.

Lower heart rate is associated with better survival in patients with multiple organ dysfunction syndrome (MODS), a disease mostly caused by sepsis. The benefits of heart rate reduction by ivabradine during MODS are currently being investigated in the MODIfY clinical trial. Ivabradine is a selective inhibitor of the pacemaker current If and since If is impaired by lipopolysaccharide (LPS, endotoxin), a trigger of sepsis, we aimed to explore If blocking potency of ivabradine under elevated endotoxin levels in human atrial cardiomyocytes. Treatment of myocytes with S-LPS (containing the lipid A moiety, a core oligosaccharide and an O-polysaccharide chain) but not R595 (an O-chain lacking LPS-form) caused If inhibition under acute and chronic septic conditions. The specific interaction of S-LPS but not R595 to pacemaker channels HCN2 and HCN4 proves the necessity of O-chain for S-LPS-HCN interaction. The efficacy of ivabradine to block If was reduced under septic conditions, an observation that correlated with lower intracellular ivabradine concentrations in S-LPS- but not R595-treated cardiomyocytes. Computational analysis using a sinoatrial pacemaker cell model revealed that despite a reduction of If under septic conditions, ivabradine further decelerated pacemaking activity. This novel finding, i.e. If inhibition by ivabradine under elevated endotoxin levels in vitro, may provide a molecular understanding for the efficacy of this drug on heart rate reduction under septic conditions in vivo, e.g. the MODIfY clinical trial.

Intracellular dyssynchrony of diastolic cytosolic [Ca²âº] decay in ventricular cardiomyocytes in cardiac remodeling and human heart failure.

RATIONALE: Synchronized release of Ca²âº into the cytosol during each cardiac cycle determines cardiomyocyte contraction. OBJECTIVE: We investigated synchrony of cytosolic [Ca²âº] decay during diastole and the impact of cardiac remodeling. METHODS AND RESULTS: Local cytosolic [Ca²âº] transients (1-µm intervals) were recorded in murine, porcine, and human ventricular single cardiomyocytes. We identified intracellular regions of slow (slowCaR) and fast (fastCaR) [Ca²âº] decay based on the local time constants of decay (TAUlocal). The SD of TAUlocal as a measure of dyssynchrony was not related to the amplitude or the timing of local Ca²âº release. Stimulation of sarcoplasmic reticulum Ca²âº ATPase with forskolin or istaroxime accelerated and its inhibition with cyclopiazonic acid slowed TAUlocal significantly more in slowCaR, thus altering the relationship between SD of TAUlocal and global [Ca²âº] decay (TAUglobal). Na⁺/Ca²âº exchanger inhibitor SEA0400 prolonged TAUlocal similarly in slowCaR and fastCaR. FastCaR were associated with increased mitochondrial density and were more sensitive to the mitochondrial Ca²âº uniporter blocker Ru360. Variation in TAUlocal was higher in pig and human cardiomyocytes and higher with increased stimulation frequency (2 Hz). TAUlocal correlated with local sarcomere relengthening. In mice with myocardial hypertrophy after transverse aortic constriction, in pigs with chronic myocardial ischemia, and in end-stage human heart failure, variation in TAUlocal was increased and related to cardiomyocyte hypertrophy and increased mitochondrial density. CONCLUSIONS: In cardiomyocytes, cytosolic [Ca²âº] decay is regulated locally and related to local sarcomere relengthening. Dyssynchronous intracellular [Ca²âº] decay in cardiac remodeling and end-stage heart failure suggests a novel mechanism of cellular contractile dysfunction.

A novel homology model of TRPC3 reveals allosteric coupling between gate and selectivity filter.

Utilizing a novel molecular model of TRPC3, based on the voltage-gated sodium channel from Arcobacter butzleri (Na(V)AB) as template, we performed structure-guided mutagenesis experiments to identify amino acid residues involved in divalent permeation and gating. Substituted cysteine accessibility screening within the predicted selectivity filter uncovered amino acids 629-631 as the narrowest part of the permeation pathway with an estimated pore diameter of < 5.8Å. E630 was found to govern not only divalent permeability but also sensitivity of the channel to block by ruthenium red. Mutations in a hydrophobic cluster at the cytosolic termini of transmembrane segment 6, corresponding to the S6 bundle crossing structure in Na(V)AB, distorted channel gating. Removal of a large hydrophobic residue (I667A or I667E) generated channels with approximately 60% constitutive activity, suggesting I667 as part of the dynamic structure occluding the permeation path. Destabilization of the gate was associated with reduced Ca2+ permeability, altered cysteine cross-linking in the selectivity filter and promoted channel block by ruthenium red. Collectively, we present a structural model of the TRPC3 permeation pathway and localize the channel's selectivity filter and the occluding gate. Moreover, we provide evidence for allosteric coupling between the gate and the selectivity filter in TRPC3.

Single-channel properties of α3ß4, α3ß4α5 and α3ß4ß2 nicotinic acetylcholine receptors in mice lacking specific nicotinic acetylcholine receptor subunits.

Previous attempts to measure the functional properties of recombinant nicotinic acetylcholine receptors (nAChRs) composed of known receptor subunits have yielded conflicting results. The use of knockout mice that lack α5, ß2, α5ß2 or α5ß2α7 nAChR subunits enabled us to measure the single-channel properties of distinct α3ß4, α3ß4α5 and α3ß4ß2 receptors in superior cervical ganglion (SCG) neurons. Using this approach, we found that α3ß4 receptors had a principal conductance level of 32.6 ± 0.8 pS (mean ± SEM) and both higher and lower secondary conductance levels. α3ß4α5 receptors had the same conductance as α3ß4 receptors, but differed from α3ß4 receptors by having an increased channel open time and increased burst duration. By contrast, α3ß4ß2 receptors differed from α3ß4 and α3ß4α5 receptors by having a significantly smaller conductance level (13.6 ± 0.5 pS). After dissecting the single-channel properties of these receptors using our knockout models, we then identified these properties - and hence the receptors themselves - in wild-type SCG neurons. This study is the first to identify the single-channel properties of distinct neuronal nicotinic receptors in their native environment.

A diffusion-adjusted regional blood flow model to predict solute kinetics during haemodialysis.

BACKGROUND: Sequestration of creatinine, in both erythrocytes and other cells, has complicated the widespread application of creatinine kinetics in haemodialysis. The goal of this study was to determine whether creatinine kinetics could be described using a regional blood flow (RBF) model that also incorporated diffusion between intra- and extracellular fluids. METHODS: Transport between intra- and extracellular spaces was modelled by diffusion using a specific rate constant k(s) for creatinine equilibration in whole blood (0.022 min(-1)) determined in a separate study. This k(s) was applied to all body spaces and to creatinine removal from blood coursing through the dialyzer. Erythrocyte and plasma creatinine and urea concentrations during haemodialysis measured and reported by others were used to test the model. RESULTS: The model accurately predicted the reported time course of creatinine in plasma and erythrocytes as well as the time course of urea in plasma when using the much higher k(s) for urea (158 min(-1)). However, it did not explain an increased erythrocyte to plasma urea gradient found at the end of haemodialysis. CONCLUSION: The results suggest that a diffusion-adjusted regional blood flow (DA-RBF) model can be used to explain compartmentalization of creatinine or urea throughout the body during haemodialysis, although possible additional compartmentalization of urea in erythrocytes, and perhaps in the tissues, still needs to be accounted for. This new model should be applicable to modelling of other non-protein-bound candidate uraemic toxins, also.

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.

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. METHODS AND RESULTS: Multiscale optical potential mapping using 10x, 20x, and 40x magnifying objectives, achieving resolutions of 0.13, 0.065, and 0.033 mm, was performed when applying uniform shocks (+/-10 V/cm, 5 ms) during diastole and action potential plateau. A procedure was developed to identify local potential deviations as depolarizing or hyperpolarizing peaks and to quantify their occurrence and characteristic amplitudes, lateral extents, and dynamics. At shock onset, peaks of either polarity developed significantly faster (tau = 0.92 +/- 0.65 ms, N = 64) than the average bulk polarization (tau = 2.25 +/- 0.96 ms, P < 0.001) and appeared locally fixed, changing their polarity at shock reversal. The mean peak magnitude (21.2 +/- 12 mV) and the amplitude distribution were essentially independent from the magnification. The peak density continuously increased with decreasing peak extent (taken at 70% of the amplitude), reaching a maximum of approximately 3 peaks/mm2 in the range of approximately 30-65 microm. There was no correlation between peak amplitude and size throughout. Potentially exciting peaks were found with a density of 0.04-0.2 peaks/mm2 corresponding to estimated 1-5 peaks/mm3. CONCLUSIONS: Our results suggest that microscopic inhomogeneities form a substantial substrate for far-field excitation in natural cardiac tissue. Here, we effectively bridged the gap between the extensively studied myocyte cultures and larger heart preparations.

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.