1 Department of Physiology, School of Basic Medical Science, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China 
2 Shanghai Institute of Brain Functional Genomics, the Key Laboratories of Ministry of Education and Science and Technology Commission of Shanghai Municipality, East China Normal University, Shanghai 200062, China

Abstract 

Objective 
To re-confirm and characterize the biophysical and pharmacological properties of endogenously expressed human acid-sensing ion channel 1a (hASIC1a) current in HEK293 cells with a modified perfusion Methods. 
Methods 
With cell floating method, which is separating the cultured cell from coverslip and putting the cell in front of perfusion tubing, whole cell patch clamp technique was used to record hASIC1a currents evoked by low pH external solution. 
Results 
Using cell floating method, the amplitude of hASIC1a currents activated by pH 5.0 in HEK293 cells is twice as large as that by the conventional method where the cells remain attached to coverslip. The time to reach peak at two different recording conditions is (21±5) ms and (270±25) ms, respectively. Inactivation time constants are (496±23) ms and (2284±120) ms, respectively. The cell floating method significantly increases the amiloride potency of block on hASIC1a [IC50 is (3.4±1.1) μmol/L and (2.4±0.9) μmol/L, respectively]. Both recording Methods have similar pH activation EC50 (6.6±0.6, 6.6±0.7, respectively). 
Conclusion 
ASICs channel activation requires fast exchange of extracellular solution with the different pH values. With cell floating method, the presence of hASIC1a current was re-confirmed and the biophysical and pharmacological properties of hASIC1a channel in HEK293 cells was precisely characterized. This method could be used to study all ASICs and other ligand-gated channels that require fast extracellular solution exchange.

Keywords

acid-sensing ion channel; patch-clamp recording; pH

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