An optimized recording method to characterize biophysical and pharmacological properties of acid-sensing ion channel.
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) micromol/L and (2.4+/- 0.9) micromol/L, respectively]. Both recording methods have similar pH activation EC50 (6.6+/-0.6, 6.6+/-0.7, respectively).


CONCLUSIONS
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 were precisely characterized. This method could be used to study all ASICs and other ligand-gated channels that require fast extracellular solution exchange.
Li
Ai
A
Department of Physiology, School of Basic Medical Science, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.
Si
Wen
W
Hu
Xin-Wu
XW
Liu
Chang-Jin
CJ
Cao
Xiao-Hua
XH
D062565:Acid Sensing Ion Channels; D000584:Amiloride / Q000494:pharmacology; D001703:Biophysics / Q000295:instrumentation / Q000379:methods; D018929:Cell Culture Techniques / Q000295:instrumentation / Q000379:methods; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry / Q000187:drug effects* / Q000378:metabolism*; D003470:Culture Media / Q000737:chemistry / Q000494:pharmacology; D045604:Extracellular Fluid / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration / Q000187:drug effects; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000187:drug effects* / Q000378:metabolism*; D009480:Neuropharmacology / Q000295:instrumentation / Q000379:methods; D018408:Patch-Clamp Techniques / Q000295:instrumentation / Q000379:methods; D010477:Perfusion / Q000295:instrumentation / Q000379:methods; D026941:Sodium Channel Blockers / Q000494:pharmacology; D015222:Sodium Channels / Q000737:chemistry / Q000187:drug effects* / Q000378:metabolism*; D013997:Time Factors
D062565:Acid Sensing Ion Channels; D003470:Culture Media; D009419:Nerve Tissue Proteins; D026941:Sodium Channel Blockers; D015222:Sodium Channels; D000584:Amiloride