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 μg/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 PNa/PCa 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.
Extracellular trypsin increases ASIC1a selectivity for monovalent versus divalent cations
Radu, Beatrice Mihaela;
2005-01-01
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 μg/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 PNa/PCa 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.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.