Proceedings of The Physiological Society
University of Oxford (2011) Proc Physiol Soc 23, PC71
Role of cysteine residues in the normal operation and assembly of K+-Cl- cotransporter isoform 2 (KCC2)
G. Carpentier1, L. Caron1, M. Jacob-Wagner1, P. Isenring1
1. Laval University, Quebec, Quebec, Canada.
The K+-Cl- cotransporters (KCCs) are polytopic membrane proteins that belong to the cation-Cl- cotransporter (CCC) family. They are known to regulate intracellular K+ and Cl- concentration as well as cell volume. Their transport activity is sensitive to N-ethylmaleimide (NEM), a thiol reagent that alkylates available sulfhydryl groups, and their C-terminus can interact with other CCCs to form oligomers. These features suggest that a number of cysteine residues within the KCCs play an important role in carrier activity and/or assembly. To determine whether this is the case, and which residues might be involved, we have exploited a mutagenic approach using rat KCC2 as a model. Cysteine residues substituted were those predicted to be cytoplasmic and, thus, belong to the N- or C-terminus. All cysteine-to-glycine substitutions (C8G, C742G, C854G and C982G) were generated by site-directed mutagenesis and heterologously expressed. In Xenopus laevis oocytes, two of the substitutions (C8G or C742G) produced no effects. However, C854G increased Rb+ transport by 30.9 % ± 0.1 and C982G decreased transport by 88.0 % ± 2.0 % (n=5) without altering cell surface protein expression as determined through biotinylation studies (n=3). They were therefore characterized in greater detail using the same expression system. Interestingly, they responded as did wild type (wt) carrier to the inhibitory effect of 250 µM furosemide (% decreases in activity were of 94.7 ± 0.3 for C854G, 91.4 ± 0.3 for C982G and 94.0 ± 0.4 for wt; n=3) but were more sensitive to the inhibitory effect 1 mM NEM (% decreases were of 32.8 ± 0.8 for C854G and 12.3 ± 0.3 for and C982G compared to 6.9 ± 1.1 for wt; n=3). To determine whether mutation-induced changes in NEM-sensitive transport activity could be due to changes in homoologomeric assembly, coimmunoprecipitation studies (n=2) were carried out using epitope-tagged carriers. Results showed that the quantity of carriers precipitated with wt KCC2 was lower for mutant C854G than for wt KCC2 or mutant C982G. Studies are underway to determine whether C854 and C982 are reactive to other thiol-specific reagents (such as MTSEA) and whether they can form disulphide bounds between each other or other residues. Lastly, FRAP studies in KCC2-transfected HEK-293 cells showed that NEM increased post-bleach fluorescence recovery time in a concentration-dependent manner (21.7 ± 1.1 sec compared to 15.2 ± 0.5 sec at 1 mM NEM). Among other possibilities, these results suggest that NEM decreases carrier mobility in cell membranes as would occur with higher order structures. Studies are also underway to determine the effect of C854G and C982G on fluorescent recovery time. Collectively, the data suggest that C854 and C982 play an important role in KCC2 function, perhaps by supporting homo- and/or heterooligomeric structure formation.
Where applicable, experiments conform with Society ethical requirements