Antibodies and a cysteine-modifying reagent show correspondence of M current in neurons to KCNQ2 and KCNQ3 K+ channels.
1. KCNQ K(+) channels are thought to underlie the M current of neurons. To probe if the KCNQ2 and KCNQ3 subtypes underlie the M current of rat superior cervical ganglia (SCG) neurons and of hippocampus, we raised specific antibodies against them and also used the cysteine-alkylating agent N-ethylmaleimide (NEM) as an additional probe of subunit composition. 2. Tested on tsA-201 (tsA) cells transfected with cloned KCNQ1-5 subunits, our antibodies showed high affinity and selectivity for the appropriate subtype. The antibodies immunostained SCG neurons and hippocampal sections at levels similar to those for channels expressed in tsA cells, indicating that KCNQ2 and KCNQ3 are present in SCG and hippocampal neurons. Some hippocampal regions contained only KCNQ2 or KCNQ3 subunits, suggesting the presence of M currents produced by channels other than KCNQ2/3 heteromultimers. 3. We found that NEM augmented M currents in SCG neurons and KCNQ2/3 currents in tsA cells via strong voltage-independent and modest voltage-dependent actions. Expression of individual KCNQ subunits in tsA cells revealed voltage-independent augmentation of KCNQ2, but not KCNQ1 nor KCNQ3, currents by NEM indicating that this action on SCG M currents likely localizes to KCNQ2. Much of the voltage-independent action is lost after the C242T mutation in KCNQ2. 4. The correspondence of NEM effects on expressed KCNQ2/3 and SCG M currents, along with the antibody labelling, provide further evidence that KCNQ2 and KCNQ3 subunits strongly contribute to the M current of neurons. The site of NEM action may be important for treatment of diseases caused by under-expression of these channels.
Roche
John P
JP
Department of Physiology and Biophysics, University of Washington School of Medicine, Box 356540, Seattle, WA 98195-6540, U.S.A.
Westenbroek
Ruth
R
Sorom
Abraham J
AJ
Hille
Bertil
B
Mackie
Ken
K
Shapiro
Mark S
MS
D000818:Animals; D000906:Antibodies / Q000378:metabolism*; D002478:Cells, Cultured; D003545:Cysteine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007202:Indicators and Reagents; D051658:KCNQ2 Potassium Channel; D051659:KCNQ3 Potassium Channel; D008297:Male; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D009474:Neurons / Q000187:drug effects / Q000378:metabolism*; D015221:Potassium Channels / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D024642:Potassium Channels, Voltage-Gated; D051381:Rats; D017207:Rats, Sprague-Dawley; D017783:Superior Cervical Ganglion / Q000187:drug effects / Q000378:metabolism
D000906:Antibodies; D007202:Indicators and Reagents; D051658:KCNQ2 Potassium Channel; C496914:KCNQ2 protein, human; D051659:KCNQ3 Potassium Channel; C496916:KCNQ3 protein, human; C496915:Kcnq2 protein, rat; C496918:Kcnq3 protein, rat; D015221:Potassium Channels; D024642:Potassium Channels, Voltage-Gated; D003545:Cysteine