Coexpression and characterization of the human large-conductance Ca(2+)-activated K (+) channel alpha + beta1 subunits in HEK293 cells.
Large-conductance Ca(2+)-activated K(+) channel is formed by a tetramer of the pore-forming alpha-subunit and distinct accessory beta-subunits (beta1-beta4) which contribute to BK(Ca) channel molecular diversity. Accumulative evidences indicate that not only alpha-subunit alone but also the alpha + beta subunit complex and/or beta-subunit might play an important role in modulating various physiological functions in most mammalian cells. To evaluate the detailed pharmacological and biophysical properties of alpha + beta1 subunit complex or beta1-subunit in BK(Ca) channel, we established an expression system that reliably coexpress hSloalpha + beta1 subunit complex in HEK293 cells. The coexpression of hSloalpha + beta1 subunit complex was evaluated by western blotting and immunolocalization, and then the single-channel kinetics and pharmacological properties of expressed hSloalpha + beta1 subunit complex were investigated by cell-attached and outside-out patches, respectively. The results in this study showed that the expressed hSloalpha + beta1 subunit complex demonstrated to be fully functional for its typical single-channel traces, Ca(2+)-sensitivity, voltage-dependency, high conductance (151 +/- 7 pS), and its pharmacological activation and inhibition.
Department of Aerospace Physiology, Fourth Military Medical University, 17#, Changle West Road, Xi'an, 710032, China.
Jun
J
Li
Chang-Lei
CL
Deng
Fang
F
Gao
Jiu-Hua
JH
Cheng
Zhi-Bin
ZB
Yu
Li
L
Liu
Man-Jiang
MJ
Xie
D001562:Benzimidazoles / Q000494:pharmacology; D015153:Blotting, Western; D002118:Calcium / Q000494:pharmacology; D002460:Cell Line; D048430:Cell Shape / Q000187:drug effects; D006801:Humans; D015640:Ion Channel Gating / Q000187:drug effects; D051037:Large-Conductance Calcium-Activated Potassium Channel alpha Subunits / Q000378:metabolism*; D051038:Large-Conductance Calcium-Activated Potassium Channel beta Subunits / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D010455:Peptides / Q000494:pharmacology; D010957:Plasmids / Q000235:genetics; D014162:Transfection
D001562:Benzimidazoles; D051037:Large-Conductance Calcium-Activated Potassium Channel alpha Subunits; D051038:Large-Conductance Calcium-Activated Potassium Channel beta Subunits; D010455:Peptides; C086491:NS 1619; C064719:iberiotoxin; D002118:Calcium