Extracellular trypsin increases ASIC1a selectivity for monovalent versus divalent cations.
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.
Neaga
Emil
E
Department of Biophysics and Physiology, Faculty of Biology, University of Bucharest, Splaiul Independentei 91-95, Bucharest 76201, Romania.
Amuzescu
Bogdan
B
Dinu
Cristina
C
Macri
Beatrice
B
Pena
Florentina
F
Flonta
Maria-Luiza
ML
D062565:Acid Sensing Ion Channels; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D020013:Calcium Signaling / Q000187:drug effects / Q000502:physiology; D002413:Cations, Divalent / Q000378:metabolism*; D002414:Cations, Monovalent / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D002463:Cell Membrane Permeability / Q000187:drug effects / Q000502:physiology; D004305:Dose-Response Relationship, Drug; D045604:Extracellular Fluid / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration / Q000187:drug effects; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D008565:Membrane Proteins / Q000187:drug effects* / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000187:drug effects* / Q000378:metabolism; D009448:Neurochemistry / Q000379:methods; D009475:Neurons, Afferent / Q000187:drug effects* / Q000378:metabolism; D009480:Neuropharmacology / Q000379:methods*; D009482:Neurophysiology / Q000379:methods; D018408:Patch-Clamp Techniques / Q000379:methods; D015222:Sodium Channels / Q000187:drug effects* / Q000378:metabolism; D014357:Trypsin / Q000494:pharmacology*
C568266:ASIC1 protein, human; D062565:Acid Sensing Ion Channels; D002413:Cations, Divalent; D002414:Cations, Monovalent; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D015222:Sodium Channels; D014357:Trypsin; D002118:Calcium