Abstract
Sustained proton activation of native ASIC channels in primary sensory neurons or HEK293 cells leads to a reduction in the peak amplitude of transient inward currents and the progressive development of a persistent component, which hinders titration experiments in pharmacological studies. Here we report that extracellular trypsin applied for 5 min at 10-45 microg/ml and/or a short exposure to high Ca2+ (75 mM for less than 1 min) alleviate the persistent component, improving reproducibility of acid-elicited transients. Selectivity measurements performed in current clamp mode, in essentially bi-ionic conditions, prove that these two treatments decrease hASIC1a permeability for divalent but not for monovalent cations, producing a significant change in P(Na)/P(Ca) from 8.2+/-2.1 (mean+/-S.D.) to 26.0+/-7.8 (trypsin) or 24.5+/-11.1 (high Ca2+). The slope conductance of the unit inward Ca2+ transient was also lowered from 5.7 to 2.7 pS after trypsin.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Acid Sensing Ion Channels
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Calcium / metabolism
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Calcium / pharmacology
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Calcium Signaling / drug effects
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Calcium Signaling / physiology
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Cations, Divalent / metabolism*
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Cations, Monovalent / metabolism*
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Cell Line
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Cell Membrane / chemistry
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Cell Membrane / drug effects
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Cell Membrane / metabolism
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Cell Membrane Permeability / drug effects
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Cell Membrane Permeability / physiology
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Dose-Response Relationship, Drug
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Extracellular Fluid / drug effects
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Extracellular Fluid / metabolism
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Humans
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Hydrogen-Ion Concentration / drug effects
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Membrane Potentials / drug effects
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Membrane Potentials / physiology
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Membrane Proteins / drug effects*
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Membrane Proteins / metabolism
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Nerve Tissue Proteins / drug effects*
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Nerve Tissue Proteins / metabolism
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Neurochemistry / methods
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Neurons, Afferent / drug effects*
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Neurons, Afferent / metabolism
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Neuropharmacology / methods*
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Neurophysiology / methods
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Patch-Clamp Techniques / methods
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Sodium Channels / drug effects*
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Sodium Channels / metabolism
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Trypsin / pharmacology*
Substances
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ASIC1 protein, human
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Acid Sensing Ion Channels
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Cations, Divalent
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Cations, Monovalent
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Membrane Proteins
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Nerve Tissue Proteins
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Sodium Channels
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Trypsin
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Calcium