Potassium channels lost during harvesting of epithelial cells are restored with a kinetics that depends on channel species.
The polarized distribution of K(+) channels in MDCK cells is lost upon harvesting and restored upon re-seeding. Using semi-quantitative PCR, in the present work we find that (i) Cells do not "wait" for the normal recycling of membrane proteins to restore their lost channels, but trigger their replacement, suggesting that the membrane has a way of engaging the nucleus. (ii) Replacement channels do not come from an internal reservoir, as it is the case with Na(+), K(+)-ATPase, but requires a de novo synthesis. (iii) Replacement is not an all-or-none response, since mRNA for MaxiK channels increases by 8-fold after re-seeding, but those for Kv1.6 and Kv1.7 are not affected by harvesting/re-seeding. (iv) TEA, charybdotoxin and iberiotoxin fail to trigger the replacement response in mature monolayers, suggesting that replacement is not due to suppression of channel function. (v) MDCK cells have a typical transporting epithelial phenotype (TEP) consisting of tight junctions (TJs) plus polarity. Although the polarized distribution of K-channels is a prominent attribute of TEP, blocking their function does not perturb the development of TEP, as gauged through the development of TJs, nor level of expression (Western blot) and distribution (confocal microscopy) of occludin, and claudins 1, 3 and 7.
García-Villegas
Refugio
R
Department of Physiology, Biophysics and Neurosciences, Cinvestav-IPN México City, México. rgarciav@fisio.cinvestav.mx
Escamilla
Juan
J
Fiorentino
Rosana
R
Cereijido
Marcelino
M
D000818:Animals; D001483:Base Sequence; D002118:Calcium / Q000494:pharmacology; D002460:Cell Line; D017124:Conserved Sequence; D004285:Dogs; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism*; D005784:Gene Amplification / Q000235:genetics; D005786:Gene Expression Regulation / Q000187:drug effects; D007700:Kinetics; D008969:Molecular Sequence Data; D010641:Phenotype; D026902:Potassium Channel Blockers / Q000494:pharmacology; D015221:Potassium Channels / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics; D016415:Sequence Alignment; D014158:Transcription, Genetic / Q000235:genetics
D026902:Potassium Channel Blockers; D015221:Potassium Channels; D012333:RNA, Messenger; D002118:Calcium