Styrax blocks inward and outward current of Kir2.1 channel.
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ABSTRACT:
SUBMITTER: Ren S
PROVIDER: S-EPMC5279881 | biostudies-other | 2017 Jan
REPOSITORIES: biostudies-other
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Project description:Shaker-type K(+) channels in plants display distinct voltage-sensing properties despite sharing sequence and structural similarity. For example, an Arabidopsis K(+) channel (SKOR) and a tomato K(+) channel (LKT1) share high amino acid sequence similarity and identical domain structures; however, SKOR conducts outward K(+) current and is activated by positive membrane potentials (depolarization), whereas LKT1 conducts inward current and is activated by negative membrane potentials (hyperpolarization). The structural basis for the "opposite" voltage-sensing properties of SKOR and LKT1 remains unknown. Using a screening procedure combined with random mutagenesis, we identified in the SKOR channel single amino acid mutations that converted an outward-conducting channel into an inward-conducting channel. Further domain-swapping and random mutagenesis produced similar results, suggesting functional interactions between several regions of SKOR protein that lead to specific voltage-sensing properties. Dramatic changes in rectifying properties can be caused by single amino acid mutations, providing evidence that the inward and outward channels in the Shaker family from plants may derive from the same ancestor.
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Project description:Background and purposePentamidine is a drug used in treatment of protozoal infections. Pentamidine treatment may cause sudden cardiac death by provoking cardiac arrhythmias associated with QTc prolongation and U-wave alterations. This proarrhythmic effect was linked to inhibition of hERG trafficking, but not to acute block of ion channels contributing to the action potential. Because the U-wave has been linked to the cardiac inward rectifier current (I(K1)), we examined the action and mechanism of pentamidine-mediated I(K1) block.Experimental approachPatch clamp measurements of I(K1) were made on cultured adult canine ventricular cardiomyocytes, K(IR)2.1-HEK293 cells and K(IR)2.x inside-out patches. Pentamidine binding to cytoplasmic amino acid residues of K(IR)2.1 channels was studied by molecular modelling.Key resultsPentamidine application (24 h) decreased I(K1) in cultured canine cardiomyocytes and K(IR)2.1-HEK293 cells under whole cell clamp conditions. Pentamidine inhibited I(K1) in K(IR)2.1-HEK293 cells 10 min after application. When applied to the cytoplasmic side under inside-out patch clamp conditions, pentamidine block of I(K1) was acute (IC(50)= 0.17 microM). Molecular modelling predicted pentamidine-channel interactions in the cytoplasmic pore region of K(IR)2.1 at amino acids E224, D259 and E299. Mutation of these conserved residues to alanine reduced pentamidine block of I(K1). Block was independent of the presence of spermine. K(IR)2.2, and K(IR)2.3 based I(K1) was also sensitive to pentamidine blockade.Conclusions and implicationsPentamidine inhibits cardiac I(K1) by interacting with three negatively charged amino acids in the cytoplasmic pore region. Our findings may provide new insights for development of specific I(K1) blocking compounds.
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Project description:BackgroundThe ATP-sensitive K+ (K(ATP)) channel is found in a variety of tissues extending from the heart and vascular smooth muscles to the endocrine pancreas and brain. Common to all K(ATP) channels is the pore-forming subunit Kir6.x, a member of the family of small inwardly rectifying K+ channels, and the regulatory subunit sulfonylurea receptor (SURx). In insulin secreting β-cells in the endocrine part of the pancreas, where the channel is best studied, the K(ATP) channel consists of Kir6.2 and SUR1. Under physiological conditions, the K(ATP) channel current flow is outward at membrane potentials more positive than the K+ equilibrium potential around -80 mV. However, K(ATP) channel kinetics have been extensively investigated for inward currents and the single-channel kinetic model is based on this type of recording, whereas only a limited amount of work has focused on outward current kinetics.MethodsWe have estimated the kinetic properties of both native and cloned K(ATP) channels under varying ionic gradients and membrane potentials using the patch-clamp technique.ResultsAnalyses of outward currents in K(ATP) and cloned Kir6.2ΔC26 channels, alone or co-expressed with SUR1, show openings that are not grouped in bursts as seen for inward currents. Burst duration for inward current corresponds well to open time for outward current.ConclusionsOutward K(ATP) channel currents are not grouped in bursts regardless of membrane potential, and channel open time for outward currents corresponds to burst duration for inward currents.
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Project description:BackgroundAlthough octopamine has long been known to have major roles as both transmitter and modulator in arthropods, it has only recently been shown to be functionally important in molluscs, playing a role as a neurotransmitter in the feeding network of the snail Lymnaea stagnalis. The synaptic potentials cannot explain all the effects of octopamine-containing neurons on the feeding network, and here we test the hypothesis that octopamine is also a neuromodulator.ResultsThe excitability of the B1 and B4 motoneurons in the buccal ganglia to depolarising current clamp pulses is significantly (P < < 0.05) increased by (10 microM) octopamine, whereas the B2 motoneuron becomes significantly less excitable. The ionic currents evoked by voltage steps were recorded using 2-electrode voltage clamp. The outward current of B1, B2 and B4 motoneurons had two components, a transient IA current and a sustained IK delayed-rectifier current, but neither was modulated by octopamine in any of these three buccal neurons. The fast inward current was eliminated in sodium-free saline and so is likely to be carried by sodium ions. 10 microM octopamine enhanced this current by 33 and 45% in the B1 and B4 motoneurons respectively (P < < 0.05), but a small reduction was seen in the B2 neuron. A Hodgkin-Huxley style simulation of the B1 motoneuron confirms that a 33% increase in the fast inward current by octopamine increases the excitability markedly.ConclusionWe conclude that octopamine is also a neuromodulator in snails, changing the excitability of the buccal neurons. This is supported by the close relationship from the voltage clamp data, through the quantitative simulation, to the action potential threshold, changing the properties of neurons in a rhythmic network. The increase in inward sodium current provides an explanation for the polycyclic modulation of the feeding system by the octopamine-containing interneurons, making feeding easier to initiate and making the feeding bursts more intense.
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