A key role for STIM1 in store operated calcium channel activation in airway smooth muscle

BACKGROUND: Control of cytosolic calcium plays a key role in airway myocyte function. Changes in intracellular Ca(2+ )stores can modulate contractile responses, modulate proliferation and regulate synthetic activity. Influx of Ca(2+ )in non excitable smooth muscle is believed to be predominantly through store operated channels (SOC) or receptor operated channels (ROC). Whereas agonists can activate both SOC and ROC in a range of smooth muscle types, the specific trigger for SOC activation is depletion of the sarcoplasmic reticulum Ca(2+ )stores. The mechanism underlying SOC activation following depletion of intracellular Ca(2+ )stores in smooth muscle has not been identified. METHODS: To investigate the roles of the STIM homologues in SOC activation in airway myocytes, specific siRNA sequences were utilised to target and selectively suppress both STIM1 and STIM2. Quantitative real time PCR was employed to assess the efficiency and the specificity of the siRNA mediated knockdown of mRNA. Activation of SOC was investigated by both whole cell patch clamp electrophysiology and a fluorescence based calcium assay. RESULTS: Transfection of 20 nM siRNA specific for STIM1 or 2 resulted in robust decreases (>70%) of the relevant mRNA. siRNA targeted at STIM1 resulted in a reduction of SOC associated Ca(2+ )influx in response to store depletion by cyclopiazonic acid (60%) or histamine but not bradykinin. siRNA to STIM2 had no effect on these responses. In addition STIM1 suppression resulted in a more or less complete abrogation of SOC associated inward currents assessed by whole cell patch clamp. CONCLUSION: Here we show that STIM1 acts as a key signal for SOC activation following intracellular Ca(2+ )store depletion or following agonist stimulation with histamine in human airway myocytes. These are the first data demonstrating a role for STIM1 in a physiologically relevant, non-transformed endogenous expression cell model

Automated whole-cell patch-clamp electrophysiology of neurons in vivo

Whole-cell patch-clamp electrophysiology of neurons is a gold-standard technique for high-fidelity analysis of the biophysical mechanisms of neural computation and pathology, but it requires great skill to perform. We have developed a robot that automatically performs patch clamping in vivo, algorithmically detecting cells by analyzing the temporal sequence of electrode impedance changes. We demonstrate good yield, throughput and quality of automated intracellular recording in mouse cortex and hippocampus.National Institutes of Health (U.S.) (NIH EUREKA Award program (1R01NS075421))National Institutes of Health (U.S.) ((NIH) Director′s New Innovator Award (DP2OD002002)National Science Foundation (U.S.) ((NSF) CAREER award (CBET 1053233))New York Stem Cell Foundation (Robertson Neuroscience Award)Dr. Gerald Burnett and Marjorie BurnettNational Science Foundation (U.S.) (grant CISE 1110947)National Science Foundation (U.S.) (grant EHR 0965945)American Heart Association (10GRNT4430029

A Fungal Metabolite Asperparaline A Strongly and Selectively Blocks Insect Nicotinic Acetylcholine Receptors: The First Report on the Mode of Action

Asperparalines produced by Aspergillus japonicus JV-23 induce paralysis in silkworm (Bombyx mori) larvae, but the target underlying insect toxicity remains unknown. In the present study, we have investigated the actions of asperparaline A on ligand-gated ion channels expressed in cultured larval brain neurons of the silkworm using patch-clamp electrophysiology. Bath-application of asperparaline A (10 µM) had no effect on the membrane current, but when delivered for 1 min prior to co-application with 10 µM acetylcholine (ACh), it blocked completely the ACh-induced current that was sensitive to mecamylamine, a nicotinic acetylcholine receptor (nAChR)-selective antaogonist. In contrast, 10 µM asperparaline A was ineffective on the γ-aminobutyric acid- and L-glutamate-induced responses of the Bombyx larval neurons. The fungal alkaloid showed no-use dependency in blocking the ACh-induced response with distinct affinity for the peak and slowly-desensitizing current amplitudes of the response to 10 µM ACh in terms of IC50 values of 20.2 and 39.6 nM, respectively. Asperparaline A (100 nM) reduced the maximum neuron response to ACh with a minimal shift in EC50, suggesting that the alkaloid is non-competitive with ACh. In contrast to showing marked blocking action on the insect nAChRs, it exhibited only a weak blocking action on chicken α3β4, α4β2 and α7 nAChRs expressed in Xenopus laevis oocytes, suggesting a high selectivity for insect over certain vertebrate nAChRs

Dynamic clamp with StdpC software

Dynamic clamp is a powerful method that allows the introduction of artificial electrical components into target cells to simulate ionic conductances and synaptic inputs. This method is based on a fast cycle of measuring the membrane potential of a cell, calculating the current of a desired simulated component using an appropriate model and injecting this current into the cell. Here we present a dynamic clamp protocol using free, fully integrated, open-source software (StdpC, for spike timing-dependent plasticity clamp). Use of this protocol does not require specialist hardware, costly commercial software, experience in real-time operating systems or a strong programming background. The software enables the configuration and operation of a wide range of complex and fully automated dynamic clamp experiments through an intuitive and powerful interface with a minimal initial lead time of a few hours. After initial configuration, experimental results can be generated within minutes of establishing cell recording

Entropic Tension in Crowded Membranes

Unlike their model membrane counterparts, biological membranes are richly decorated with a heterogeneous assembly of membrane proteins. These proteins are so tightly packed that their excluded area interactions can alter the free energy landscape controlling the conformational transitions suffered by such proteins. For membrane channels, this effect can alter the critical membrane tension at which they undergo a transition from a closed to an open state, and therefore influence protein function \emph{in vivo}. Despite their obvious importance, crowding phenomena in membranes are much less well studied than in the cytoplasm. Using statistical mechanics results for hard disk liquids, we show that crowding induces an entropic tension in the membrane, which influences transitions that alter the projected area and circumference of a membrane protein. As a specific case study in this effect, we consider the impact of crowding on the gating properties of bacterial mechanosensitive membrane channels, which are thought to confer osmoprotection when these cells are subjected to osmotic shock. We find that crowding can alter the gating energies by more than $2\;k_BT$ in physiological conditions, a substantial fraction of the total gating energies in some cases. Given the ubiquity of membrane crowding, the nonspecific nature of excluded volume interactions, and the fact that the function of many membrane proteins involve significant conformational changes, this specific case study highlights a general aspect in the function of membrane proteins.Comment: 20 pages (inclduing supporting information), 4 figures, to appear in PLoS Comp. Bio

Functional consequences of Kir2.1/Kir2.2 subunit heteromerization

Kir2 subunits form channels that underlie classical strongly inwardly rectifying potassium currents. While homomeric Kir2 channels display a number of distinct and physiologically important properties, the functional properties of heteromeric Kir2 assemblies, as well as the stoichiometries and the arrangements of Kir2 subunits in native channels, remain largely unknown. Therefore, we have implemented a concatemeric approach, whereby all four cloned Kir2 subunits were linked in tandem, in order to study the effects of Kir2.1 and Kir2.2 heteromerization on properties of the resulting channels. Kir2.2 subunits contributed stronger to single-channel conductance than Kir2.1 subunits, and channels containing two or more Kir2.2 subunits displayed conductances indistinguishable from that of a Kir2.2 homomeric channel. In contrast, single-channel kinetics was a more discriminating property. The open times were significantly shorter in Kir2.2 channels compared with Kir2.1 channels and decreased nearly proportionally to the number of Kir2.2 subunits in the heteromeric channel. Similarly, the sensitivity to block by barium also depended on the proportions of Kir2.1 to Kir2.2 subunits. Overall, the results showed that Kir2.1 and Kir2.2 subunits exert neither a dominant nor an anomalous effect on any of the properties of heteromeric channels. The data highlight opportunities and challenges of using differential properties of Kir2 channels in deciphering the subunit composition of native inwardly rectifying potassium currents

Reverse mode Na+/Ca2+ exchange mediated by STIM1 contributes to Ca2+ influx in airway smooth muscle following agonist stimulation

<p>Abstract</p> <p>Background</p> <p>Agonist stimulation of airway smooth muscle (ASM) results in IP₃mediated Ca²⁺release from the sarcoplasmic reticulum followed by the activation of store operated and receptor operated non-selective cation channels. Activation of these non-selective channels also results in a Na⁺influx. This localised increase in Na⁺levels can potentially switch the Na⁺/Ca²⁺exchanger into reverse mode and so result in a further influx of Ca²⁺. The aim of this study was to characterise the expression and physiological function of the Na⁺/Ca²⁺exchanger in cultured human bronchial smooth muscle cells and determine its contribution to agonist induced Ca²⁺influx into these cells.</p> <p>Methods</p> <p>The expression profile of NCX (which encodes the Na⁺/Ca²⁺exchanger) homologues in cultured human bronchial smooth muscle cells was determined by reverse transcriptase PCR. The functional activity of reverse mode NCX was investigated using a combination of whole cell patch clamp, intracellular Ca²⁺measurements and porcine airway contractile analyses. KB-R7943 (an antagonist for reverse mode NCX) and target specific siRNA were utilised as tools to inhibit NCX function.</p> <p>Results</p> <p>NCX1 protein was detected in cultured human bronchial smooth muscle cells (HBSMC) cells and NCX1.3 was the only mRNA transcript variant detected. A combination of intracellular Na⁺loading and addition of extracellular Ca²⁺induced an outwardly rectifying current which was augmented following stimulation with histamine. This outwardly rectifying current was inhibited by 10 μM KB-R7943 (an antagonist of reverse mode NCX1) and was reduced in cells incubated with siRNA against NCX1. Interestingly, this outwardly rectifying current was also inhibited following knockdown of STIM1, suggesting for the first time a link between store operated cation entry and NCX1 activation. In addition, 10 μM KB-R7943 inhibited agonist induced changes in cytosolic Ca²⁺and induced relaxation of porcine peripheral airways.</p> <p>Conclusions</p> <p>Taken together, these data demonstrate a potentially important role for NCX1 in control of Ca²⁺homeostasis and link store depletion via STIM1 directly with NCX activation.</p