Stoichiometry of expressed KCNQ2/KCNQ3 potassium channels and subunit composition of native ganglionic M channels deduced from block by tetraethylammonium.
KCNQ2 and KCNQ3 potassium-channel subunits can form both homomeric and heteromeric channels; the latter are thought to constitute native ganglionic M channels. We have tried to deduce the stoichiometric contributions of KCNQ2 and KCNQ3 subunits to currents generated by the coexpression of KCNQ2 and KCNQ3 cDNA plasmids in Chinese hamster ovary (CHO) cells, and to native M currents in dissociated rat superior cervical ganglion (SCG) neurons, by comparing the block of these currents produced by tetraethylammonium (TEA) with the block of currents generated by a tandem KCNQ3/2 construct. TEA concentration-inhibition curves against coexpressed KCNQ2 plus KCNQ3 currents, and against native M currents in SCG neurons from 6-week-old [postnatal day 45 (P45)] rats, were indistinguishable from those for the expressed tandem construct, and fully accorded with a 1:1 stoichiometry. Inhibition curves in neurons from younger (P17) rats could be better fitted assuming an additional small proportion of current carried by KCNQ2 homomultimers. Single-cell PCR yielded signals for KCNQ2, KCNQ3, and KCNQ5 mRNAs in all SCG neurons tested from both P17 and P45 rats. Quantitative PCR of whole-ganglion mRNA revealed stable levels of KCNQ2 and KCNQ5 mRNA between P7 and P45, but excess and incrementing levels of KCNQ3 mRNA. Increasing levels of KCNQ3 protein between P17 and P45 were confirmed by immunocytochemistry. We conclude that coexpressed KCNQ2 plus KCNQ3 cDNAs generate channels with 1:1 (KCNQ2:KCNQ3) stoichiometry in CHO cells and that native M channels in SCG neurons adopt the same conformation during development, assisted by the increased expression of KCNQ3 mRNA and protein.
Hadley
Jennifer K
JK
Department of Pharmacology, University College London, London WC1E 6BT, United Kingdom.
Passmore
Gayle M
GM
Tatulian
Lucine
L
Al-Qatari
Mona
M
Ye
Fei
F
Wickenden
Alan D
AD
Brown
David A
DA
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D004305:Dose-Response Relationship, Drug; D006801:Humans; D007150:Immunohistochemistry; D051656:KCNQ Potassium Channels; D051658:KCNQ2 Potassium Channel; D051659:KCNQ3 Potassium Channel; D007700:Kinetics; D009474:Neurons / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D026902:Potassium Channel Blockers / Q000494:pharmacology; D015221:Potassium Channels / Q000096:biosynthesis* / Q000187:drug effects / Q000235:genetics; D024642:Potassium Channels, Voltage-Gated; D021122:Protein Subunits / Q000096:biosynthesis / Q000187:drug effects / Q000235:genetics; D012333:RNA, Messenger / Q000096:biosynthesis; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017783:Superior Cervical Ganglion / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D019789:Tetraethylammonium / Q000494:pharmacology*; D014162:Transfection
D051656:KCNQ Potassium Channels; D051658:KCNQ2 Potassium Channel; C496914:KCNQ2 protein, human; D051659:KCNQ3 Potassium Channel; C496916:KCNQ3 protein, human; C412231:KCNQ5 protein, human; C496915:Kcnq2 protein, rat; C496918:Kcnq3 protein, rat; C501028:Kcnq5 protein, rat; D026902:Potassium Channel Blockers; D015221:Potassium Channels; D024642:Potassium Channels, Voltage-Gated; D021122:Protein Subunits; D012333:RNA, Messenger; D019789:Tetraethylammonium