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 PURPOSE: Pentamidine 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 APPROACH: Patch 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 RESULTS: Pentamidine 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 IMPLICATIONS: Pentamidine 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:Regulating the residence time of membrane proteins on the cell surface can modify their response to extracellular cues and allow for cellular adaptation in response to changing environmental conditions. The fate of membrane proteins that are internalized from the plasma membrane and arrive at the limiting membrane of the late endosome/multivesicular body (MVB) is dictated by whether they remain on the limiting membrane, bud into internal MVB vesicles, or bud outwardly from the membrane. The molecular details underlying the disposition of membrane proteins that transit this pathway and the mechanisms regulating these trafficking events are unclear. We established a cell-free system that reconstitutes budding of membrane protein cargo into internal MVB vesicles and onto vesicles that bud outwardly from the MVB membrane. Both budding reactions are cytosol-dependent and supported by Saccharomyces cerevisiae (yeast) cytosol. We observed that inward and outward budding from the MVB membrane are mechanistically distinct but may be linked, such that inhibition of inward budding triggers a re-routing of cargo from inward to outward budding vesicles, without affecting the number of vesicles that bud outwardly from MVBs.
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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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