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J Neurosci Methods. 2005 Jun 15;144(2):241-8. Epub 2004 Dec 19.

Extracellular trypsin increases ASIC1a selectivity for monovalent versus divalent cations.

Author information

1
Department of Biophysics and Physiology, Faculty of Biology, University of Bucharest, Splaiul Independentei 91-95, Bucharest 76201, Romania.

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.

PMID:
15910984
DOI:
10.1016/j.jneumeth.2004.11.012
[Indexed for MEDLINE]

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