Abstract
The relative distribution of ClC-1 between the surface and transverse tubular system (TTS) membranes in skeletal muscle fibers is still a matter of debate. A useful approach to investigate this issue is to transfect plasmids coding for fluorescently-tagged transgenic channels into the fibers of FDB muscles (by in vivo electroporation), and assess the intracellular targeting of the expression products. Using a human clone (hClC-1), we recently observed expression of EYFP-hClC-1 at both the TTS and surface membranes, while another group, using a murine clone (mClC-1) reported expression of EGFP-mClC-1 only at the sarcolemma. Since these differences could have originated from divergent experimental conditions, we compared the expression of both clones under identical conditions. The hClC-1 (NM_013491.2) and mClC-1 (NM_013491.2) were subcloned in rRcCMV and pcDNA3.1 vectors, respectively. The localization of tagged-ClC-1 was assessed by using two-photon laser scanning microscopy. As sarcomeric localization markers we used a second harmonic generation (SHG) band that marks the location of myosin, and di-8-ANEPPS fluorescence that marks the location of TTS. Action potentials and chloride currents were recorded from dissociated FDB fibers using a two-microelectrode system. We found that, as reported previously with hClC-1, mClC-1 is targeted to both the TTS and surface membranes. In addition, the overexpression of either ClC-1 clone resulted in significant reductions in the input resistance, and a corresponding significant increase in the chloride currents, recorded from the muscle fibers. Overall, our results demonstrate that transgenic ClC-1 are functionally expressed at the surface and TTS membranes of skeletal muscles fibers. We thank Drs. J. Lueck, U. of Iowa, and C. Fahlke, Medizinischen Hochschule Hannover, Germany, for sharing hClC-1 and mClC-1, respectively. This work was supported by NIH grants AR047664, AR041802, and AR054816.
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