title: Generation and characterization of a cell line with inducible expression of Ca(v)3.2 (T-type) channels.
abstract: Establishment of stable cell lines that constitutively express Ca(2+) channels at high density and that are useful for in vitro studies may be complicated by problems with seal quality and duration during whole-cell patch-clamp electrophysiology. The current studies describe the generation and characterization of cells that express the human alpha1H T-type Ca(2+) channel under the control of a tetracycline-inducible expression system. Western blot and immunostaining studies revealed that expression of the alpha1H protein occurred only in the presence of tetracycline. Using the whole-cell patch-clamp method, the cells displayed peak inward currents of 1.15 +/- 0.14 nA in response to voltage-clamp steps. The T-type Ca(2+) current was inhibited by the T-type Ca(2+) channel antagonist, mibefradil, with an IC(50) of 160 nM. This cell line, with inducible channel expression, sealed with longer duration during whole-cell patch-clamp recording when compared with a cell line that constitutively expresses the alpha1H Ca(2+) channel. Ca(2+) influx through this channel could also be detected after the addition of extracellular Ca(2+). The amount of Ca(2+) influx was dependent on the [Ca](o) with an EC(50) of 4 mM. The Ca(2+) influx was also inhibited by mibefradil with a potency (IC(50) = 183 nM) similar to that observed in the voltage-clamp studies. Overall, this inducible alpha1H Ca(2+) channel-expressing cell line is useful for the study of human T-type Ca(2+) channel function, and offers advantages over a similar cell line that constitutively expresses the channel.
authors.affiliation: Department of Molecular Pharmacology, Merck Research Laboratories, West Point, PA 19486, USA. menghang_xia@merck.com
authors.forename: Menghang
authors.initials: M
authors.lastname: Xia
authors.forename: John P
authors.initials: JP
authors.lastname: Imredy
authors.forename: Vincent P
authors.initials: VP
authors.lastname: Santarelli
authors.forename: Hongyu Annie
authors.initials: HA
authors.lastname: Liang
authors.forename: Cindra L
authors.initials: CL
authors.lastname: Condra
authors.forename: Paul
authors.initials: P
authors.lastname: Bennett
authors.forename: Kenneth S
authors.initials: KS
authors.lastname: Koblan
authors.forename: Thomas M
authors.initials: TM
authors.lastname: Connolly
mesh_terms: D002118:Calcium / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000494:pharmacology*; D020747:Calcium Channels, T-Type / Q000187:drug effects / Q000502:physiology*; D018929:Cell Culture Techniques / Q000379:methods; D002460:Cell Line; D002478:Cells, Cultured; D006801:Humans; D015640:Ion Channel Gating / Q000187:drug effects; D007668:Kidney / Q000187:drug effects / Q000196:embryology / Q000502:physiology*; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology*; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D013752:Tetracycline / Q000494:pharmacology*
chemical_list: C479952:CACNA1H protein, human; D002121:Calcium Channel Blockers; D020747:Calcium Channels, T-Type; D011994:Recombinant Proteins; D013752:Tetracycline; D002118:Calcium