Mouse renal inner medullary collecting duct cells (mIMCD-3 cell-line) express several members of the CLCA protein family (including mCLCA1) that mediate Cl– channel activity (Stewart et al. 2001) The voltage-dependent Cl– channel mCLC-5 is also expressed in mIMCD-3 cells within acidic endosomes (Sayer et al. 2001). Here we report subcellular localisation of CLCA1 and correlate this to that of mCLC-5.
mIMCD-3 cells were transiently transfected alone or in combination with expression vectors for GFP fusion proteins for mCLCA1 (Sayer et al. 2000) and mCLC-5 (Sayer et al. 2001) (pCDNA3.1/mCLCA1CT-RFP, or pDsRed1/mCLC-5CT, Clontech), using Lipofectamine 2000 (Life Technologies). Positive transfectants were identified using a Leica confocal laser imaging microscope equipped (CLSM) with a Kr-Ar laser by their green/red fluorescence and analysed 24-48 h post-transfection. In order to identify plasma membrane in mCLCA1-GFP transfectants, cells were preincubated for 2 min at room temperature with TRITC-wheat germ agglutinin (WGA) in phosphate-buffered saline (PBS) or complete medium (Ham’s F12/DMEM) at 50 mg ml-1. Alternatively, in order to identify acidic endosmomes, cells were incubated with 75 nM lysotracker red (Molecular Probes) for 30 min in PBS or complete medium. Optical sections of positive transfectants were collected to allow analysis of co-localisation of mCLCA1-GFP fluorescence with plasma membrane, acidic endosomes and mCLC-5.
mCLCA1-GFP fluorescence at 24/48 h post-transfection was primarily associated with intracellular vesicular structures. A minor proportion of CLCA1-GFP fluorescence was co-localised with TRITC-WGA, confirming the presence of mCLCA1-GFP at the plasma membrane. mCLCA1-GFP fluorescence showed separation from both acidic endosomes and mCLC5-GFP, with only a minor overlap.The presence of mCLCA1 at the plasma membrane is consistent with mCLCA1 conferring Cl– channel activity or acting as a Cl– channel regulator. The virtual separation of mCLC-5 from mCLCA1 suggests that CLCA proteins could not compensate for loss of CLC-5 function in IMCD cells.
This work was supported by the NCKRF and the NKRF