Topology of the human skeletal muscle chloride channel hClC-1 probed with hydrophilic epitope insertion

To investigate the membrane topology of the skeletal muscle chloride channe l ClC-1. we inserted the small antigenic flag (DYKDDDDK) and/or HSV (QPELAP EDPED) epitope tags into nine predicted extra- and intracellular loops alon g the channel protein. Functional integrity of the modified proteins was te sted by measuring the chloride currents conducted by these channels express ed in tsA201 cells. Insertion of the tags into the linkers D1D2, D4D5, D6D7 , D8D9 or D11D12 did not alter channel function significantly, whereas inse rtion into D3D4. D5D6. D9D10 and D10D11 led to loss of function. Intra- or extracellular localisation of the tags was determined by immunofluorescent staining of intact and permeabilised tsA201 cells transiently transfected w ith the functional epitope-inserted constructs. Intact cells stained for th e epitope tags inserted into D1D2, D6D7 and D8D9, indicating that these lin kers face the extracellular side of the membrane. No conclusions could be d rawn for the location of D4D5 and D11D12. Insertion of the flag epitope at position P260 (linker D4D5). a putative pore-lining region, did not change any of the channel function properties markedly, suggesting that the region surrounding P260 cannot directly line the ion conduction pathway of ClC-1.