The CFTR channel is an essential mediator of electrolyte transport across epithelial tissues. CFTR opening is promoted by ATP binding and dimerization of its two nucleotide binding domains (NBDs). Phosphorylation of its R domain (e.g. by PKA) is also required for channel activity. The CFTR structure is unsolved but homology models of the CFTR closed and open states have been produced based on the crystal structures of evolutionarily related ABC transporters. These models predict the formation of a tetrahelix bundle of intracellular loops (ICLs) during channel opening. Here we provide evidence that residues E267 in ICL2 and K1060 in ICL4 electrostatically interact at the interface of this predicted bundle to promote CFTR opening. Mutations or a thiol modifier that introduced like charges at these two positions substantially inhibited ATP-dependent channel opening. ATP-dependent activity was rescued by introducing a second site gain of function (GOF) mutation that was previously shown to promote ATP-dependent and ATP-independent opening (K978C). Conversely, the ATP-independent activity of the K978C GOF mutant was inhibited by charge- reversal mutations at positions 267 or 1060 either in the presence or absence of NBD2. The latter result indicates that this electrostatic interaction also promotes unliganded channel opening in the absence of ATP binding and NBD dimerization. Charge-reversal mutations at either position markedly reduced the PKA sensitivity of channel activation implying strong allosteric coupling between bundle formation and R domain phosphorylation. These findings support important roles of the tetrahelix bundle and the E267-K1060 electrostatic interaction in phosphorylation-dependent CFTR gating.
Background: Multidrug resistance proteins (MRPs) and the cystic fibrosis transmembrane conductance regulator (CFTR) are thermodynamically distinct ATP-binding cassette (ABC) transporters. Results: Structural elements that couple ATP binding to channel opening in the CFTR channel also facilitate MRP drug export. Conclusion: MRPs and CFTR share components of a conserved activation mechanism. Significance: Allosteric hot spots suggest mechanistic similarities between thermodynamically distinct ABC transporters.
The protein O-linked β-N-acetylglucosamine (O-GlcNAc) modification plays an important role in skeletal muscle development and physiological function. In this study, bitransgenic mice were generated that overexpressed NCOAT(GK), an O-GlcNAcase-inactive spliced variant of the O-GlcNAcase gene, specifically in skeletal muscle using the muscle creatine kinase promoter. Expression of the chimeric enhanced green fluorescent protein-NCOAT(GK) transgene caused an increase of cellular O-GlcNAc levels, along with the accumulation and activation of proapoptotic factors in muscles of bitransgenic mice. The consequence of overexpressing the transgene for a 2-wk period was muscle atrophy and, in some cases, resulted in the death of male mice. Muscle atrophy is a common complication of many diseases, some of which correlate markedly with high cellular O-GlcNAc levels, such as diabetes. Our study provides direct evidence linking muscle atrophy and the disruption of O-GlcNAcase activity.
The ABCC transporter subfamily includes pumps, the long and short multidrug resistance proteins (MRPs), and an ATP-gated anion channel, the cystic fibrosis transmembrane conductance regulator (CFTR). We show that despite their thermodynamic differences, these ABCC transporter subtypes use broadly similar mechanisms to couple their extracellular gates to the ATP occupancies of their cytosolic nucleotide binding domains. A conserved extracellular phenylalanine at this gate was a prime location for producing gain of function (GOF) mutants of a long MRP in yeast (Ycf1p cadmium transporter), a short yeast MRP (Yor1p oligomycin exporter), and human CFTR channels. Extracellular gate mutations rescued ATP binding mutants of the yeast MRPs and CFTR by increasing ATP sensitivity. Control ATPase-defective MRP mutants could not be rescued by this mechanism. A CFTR double mutant with an extracellular gate mutation plus a cytosolic GOF mutation was highly active (single-channel open probability >0.3) in the absence of ATP and protein kinase A, each normally required for CFTR activity. We conclude that all 3 ABCC transporter subtypes use similar mechanisms to couple their extracellular gates to ATP occupancy, and highly active CFTR channels that bypass defects in ATP binding or phosphorylation can be produced.
Following a 100 km race creatine kinase (CK) creatine kinase MB (CKMB) activities were serially measured in well trained athletes and compared with enzyme activities in patients with acute myocardial infarction (AMI). The half-time of disappearance of CK (CKt1/2) was 1.75 +/- 0.70 days in runners who trained within the 1st week after the race, and was 0.81 +/- 0.18 days in patients with AMI, P less than 0.005. CKt1/2 in runners was shorter (1.17 +/- 0.28 days) when no training was performed in the first postrace week. CKt1/2 was linearly correlated with age (P less than 0.01) in the runners but not in the patients. CKMBt1/2 was 1.30 and 1.11 days in two runners and 0.56 +/- 0.10 days in patients with AMI (P less than 0.05). In line with histologic and enzymatic findings in skeletal muscle of long distance runners as reported by other workers, our kinetic data provide further evidence that CK and CKMB are released from muscular compartments in runners other than those in patients with AMI.
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