The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) has been shown to function as a cyclic AMP-dependent chloride channel by many laboratories using diverse model systems. Our studies of the purified, reconstituted protein provided th3 first direct evidence that the CFTR molecule can function as a chloride channel when phosphorylated by the PKA. Our studies of the purified, reconstituted protein provided the first direct evidence that the CFTR molecule can function as a sodium chloride channel when phosphorylated by PKA. Further, our reconstitution system provided direct evidence that purified CFTR can also function as an ATPase and that this activity is coupled to gating of the CFTR chloride channel. Recently, it has been postulated """"""""Linsdell and Hanrahan) that the ATPase activity of CFTR may be coupled to a distinct transpor6t mechanism, namely the energy-dependent transport of organic anions such as glutamate and glutathione. As this novel transport may have tremendous biological significance, it is important that we determine if it constitutes an intrinsic property of CFTR and further, to assess the mechanism underlying this activity. Overall Goal: to determine if purified CFTR, reconstituted into phospholipid liposomes, mediates the transport of organic anions using an energy dependent, """"""""pump"""""""" mechanism. Experimental Approach: We have recently developed a novel method for the purification of CFTR which permits the preparation of relatively large quantities of purified protein (approximately 1 mg/litre Sf9 cells). We will use purified CFTR protein, reconstituted into large unilamellar phospholipid liposomes (> 500 nm in diameter) to assess MgATP-dependent, concentrative uptake of radiolabeled glutamate (one of the organic anion substrates identified by Linsdell et al.). Uptake will be monitored over time using a rapid filtration apparatus, similar to that described by Doige et al. in studies of the pump function of purified P-glycoprotein. Predictions: If glutamate is transported through CFTR via an active pump mechanism, we expect that ATP, but not the non-hydrolyzable analogue, AMP-PNP, will stimulate liposome accumulation of this putative substrate. Further, as the ATPase activity of CFTR is stimulated by PKA phosphorylation, we expect that the intraliposomal accumulation of glutamate will also be stimulated by phosphorylation. Finally, interruption the catalytic cycle by the addition of transition state analogues, should lead to dissipation of pump generated concentration gradients.
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