Fast, repetitive light-activation of CaV3.2 using channelrhodopsin 2.
Channelrhodopsin-2 (ChR2) is a light-gated ion channel that is successfully used in neurosciences to depolarize cells with blue light. In this regard control of membrane voltage with light opens new perspectives for the characterization of ion channels and the search for inhibitors or modulators. Here, we report a control of membrane potential with ChR2 and the potassium channel mTrek for the purpose of screening for ion channel specific drugs. To verify principle we have chosen the voltage gated calcium channel Ca(V)3.2 as potential drug target. For this purpose we transfected the ChR2 gene into a HEK293T-cell line that permanently expresses Ca(V)3.2 and the K-channel mTrek. The resting potential was adjusted with low concentration of extracellular potassium ions whereas transient depolarization was achieved by activation of ChR2 with short pulses of blue light. Calcium ion influx through Ca(V)3.2 was monitored by observing fura-2 fluorescence. This approach allowed a repetitive activation of Ca(V)3.2. The Ca(2+) influx was specifically blocked by the inhibitor mibefradil. Since this assay is genetically-encoded, it may be employed for a variety of voltage-gated calcium channels and should be applicable to multi-well reader formats for high-throughput screening.
Prigge
Matthias
M
Institute for Biology, Experimental Biophysics, Humboldt Universität zu Berlin, Germany. Matthias_Prigge@web.de
Rösler
Anton
A
Hegemann
Peter
P
D000818:Animals; D002121:Calcium Channel Blockers / Q000494:pharmacology; D020747:Calcium Channels, T-Type / Q000378:metabolism*; D002460:Cell Line; D000075402:Channelrhodopsins; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D015640:Ion Channel Gating; D007473:Ion Channels / Q000187:drug effects*; D008027:Light; D008564:Membrane Potentials; D020748:Mibefradil / Q000494:pharmacology; D051379:Mice; D024683:Potassium Channels, Tandem Pore Domain / Q000378:metabolism; D014162:Transfection
C504883:Cacna1h protein, mouse; D002121:Calcium Channel Blockers; D020747:Calcium Channels, T-Type; D000075402:Channelrhodopsins; D007473:Ion Channels; D024683:Potassium Channels, Tandem Pore Domain; C103742:potassium channel protein TREK-1; D020748:Mibefradil