Live cell imaging of mutant and wild-type GABA-A receptor trafficking using a novel reporter protein

Sun, 11/08/2009 - 1:22pm

Efficient signaling in the brain requires precise regulation and targeting of cell surface ion-channels. Mutations in these channels associated with inherited diseases can cause improper targeting and reduced surface expression. Thus, the development of tools to readily label and follow the trafficking of ion-channels in living cells in real time is essential.  The GABA-A receptor (GABAAR) mediates the majority of inhibition in the brain and mutations in this receptor linked to human epileptic syndromes are reported to reduce surface expression.  To examine cell surface expression and trafficking of the GABAAR, we tagged the receptor with a novel reporter protein (HaloTag ) and visualized the tagged receptor by covalently tethering HaloTag specific fluorescent ligands in living cells and in vitro (Los & Wood, Methods Mol Biol 2007, 356: 195-208). The HaloTag protein was inserted into the N-terminus of the GABAAR ?2L subunit (?2L-vHT) and expressed with wild-type α1 and β2 subunits to form functional GABAARs. The ?2L-vHT fusion protein had the expected molecular weight, expressed well in HeLa cells and Xenopus oocytes, and was efficiently labeled with the HaloTag ligands. Functional activity of GABAARs expressed in Xenopus oocytes was assayed using two-electrode voltage clamp. 







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