CFTR is composed of two homologous halves, each comprising a transmembrane (TMD) and a nucleotide binding domain (NBD). Epithelial ion channel that plays an important role in the regulation of epithelial ion and water transport and fluid homeostasis. [ATP]-dependence of CFTR gating. Our results therefore indicate that phosphorylation‐ and nucleotide‐hydrolysis‐dependent gating of CFTR is directly involved in gating of an associated ATP channel. CFTR channel opening by ATP-driven tight dimerization of its nucleotide-binding domains. In this work, we report a structure of the phosphorylated, ATP-bound human CFTR channel determined by cryo-EM. Cystic fibrosis transmembrane conductance regulator (CFTR)3 is an anion channel in the adenosine 5-triphosphate (ATP)-binding cassette (ABC) transporter protein family (1–3). Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) channel, an ATP binding cassette (ABC) transporter. Once activated, channel activity is reduced by removal of ENaC from the cell surface by endocytosis (i.e., a reduction in n). Results. CFTR belongs to the ATP-binding cassette (ABC) superfamily, but it is unique among ABC proteins in that it functions as an ion channel (Gadsby et al., 2006). Consistent with this conclusion, when we mutated an aspartate thought to bind Mg(2+), divalent cations failed to increase activity compared … La protéine CFTR est un membre de la superfamille des transporteurs ABC, que l'on retrouve dans tous les domaines du vivant (bactéries, archées et eucaryotes). Elle se distingue de tous les autres membres de cette superfamille par son statut de canal ionique ainsi que par la présence de son domaine régulatoire (R), unique [3]. Nevertheless, our data also suggest that the permeation pathways for Cl − and ATP are distinct. Download Full PDF Package. ABC proteins are defined by two nucleotide-binding domains (NBDs) that contain a conserved sequence, the ATP-binding cassette. The hCFTR structure shares many key features with that of zCFTR determined under the same condition . Notably, WT-CFTR, but not Phe508del CFTR, has been shown to protect ENaC from proteolytic activation . By continuing to browse this site you agree to us using cookies as described in About Cookies.. Channel activation also requires phosphorylation of the R domain by poorly understood mechanisms. Channel activation also requires phosphorylation of the R domain by poorly understood mechanisms. Similarly, ATP analogues with substitutions in the phosphate chain, including AMPCPP, AMPPCP, AMPPNP, and ATP gamma S failed to support CFTR channel activity when present at the cytoplasmic face of the membrane and none of these analogues, when present at three to 10-fold excess of ATP, detectably altered ATP-dependent CFTR channel gating. This review covers our current understanding on the gating mechanism in CFTR and illustrates the relevance of alteration of these mechanisms in the onset of cystic fibrosis. CFTR is an ATP-gated chloride channel present in the apical membranes of cells lining the externally-facing surfaces of several organs (Riordan, 2008). Despite the membrane potential depolarization (Vm↑) caused by the CFTR-mediated Cl − efflux, activated CFTR potentiates K ATP channel activity, which overcomes the direct inhibitory effect of glucose on the K ATP channel, resulting in Vm hyperpolarization (Vm↓) and hence inhibits Ca 2+ influx through voltage-dependent Ca 2+ channel (VDCC) and suppresses glucagon secretion. CFTR (Cystic fibrosis transmembrane conductance regulator), mutations of which cause cystic fibrosis, belongs to the ATP-binding cassette (ABC) transporter family and works as a channel for small anions, such as chloride and bicarbonate. Loe et al. CFTR Cl- channel and CFTR-associated ATP channel: distinct pores regulated by common gates. These data suggest that ATP alone can open the channel and that divalent cations increase ATP binding. Evidence suggests that CFTR and ORCCs are encoded by separate genes (Gabriel et al., 1993). Bruce Schultz. 9 Figure 4. Regulation of CFTR Cl- channel gating by ADP and ATP analogues. Download figure Download PowerPoint. CFTR functions as a phosphorylation and ATP-gated anion channel, increasing the conductance for certain anions (e.g. As we found no effect of glutamate on CFTR in excised membrane patches, with or without ATP (n=6; data not shown), we conclude that glutamate does not activate CFTR directly. Previously, we reported essentially identical structures of zebrafish and human CFTR in the dephosphorylated, ATP-free form. Regulation of CFTR Cl- channel gating by ADP and ATP analogues . Finally, it has been demonstrated that Ca2+-dependent CI- secretion is also up-regulated in CF patients and in the CF (-/-) mouse (Grubbet al., 1994). Comparison of the two … 1996), CFTR is a bonafide ATP-gated ion channel (Anderson et al. These data suggest that none of these ATP … CFTR gating is regulated in complex manner as phosphorylation is mandatory for channel activity and gating is directly regulated by binding of ATP to specific intracellular sites on the CFTR protein. Cystic fibrosis transmembrane conductance regulator (CFTR), the protein dysfunctional in cystic fibrosis, is unique among ABC proteins in that its transmembrane domains comprise an ion channel. Moreover, in CFTR variants with mutations that disrupt hydrolysis, ATP alone opened the channel and Mg(2+) further enhanced ATP-dependent opening. CFTR channel gating is strictly coupled to phosphorylation and ATP hydrolysis. CFTR gating is linked to ATP binding and dimerization of its two nucleotide binding domains (NBDs). Dismiss this message Find the latest peer-reviewed research articles and preprints on Coronavirus here. Therefore, at low [ATP] CFTR channel opening is rate-limited by an ATP binding step, which means that ATP binding must occur on closed channels, and precedes channel opening; the Hill coefficient of 1 is consistent with a single binding step controlling opening. Cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel gated by ATP binding and hydrolysis at its nucleotide binding domains (NBD). P‐ATP exerts its effects on CFTR channel gating in two ways: (i) by high‐affinity binding at ATP‐binding site 1 it slows channel closing, and (ii) through relatively low‐affinity binding at ATP‐binding site 2 it accelerates channel opening (Zhou et al. .] normal CFTR channel function in airway epithelia. Download PDF. Structure Determination. READ PAPER. CFTR channel properties (Hwang et al., 1989; Egan et al., 1992; Gabriel et al., 1993; Schwiebert et al., 1994a). ATP (1 mM, Mg 2+ salt) and PKA (75 nM) were present during times indicated by bars. It also reveals several differences that are important to correlate the structure with functional data obtained from hCFTR. Regulation of CFTR Cl- channel gating by ADP and ATP analogues. High homologies between CFTR and the sulfonylurea receptor (SUR), which associates with the potassium channel Kir6.2 to form the ATP-sensitive K+ (K-ATP) channel, prompted us to examine possible effects of these compounds on K-ATP channels using electrophysiological recordings and binding assays. A short summary of this paper. Channel activity is coupled to ATP hydrolysis. About Cookies, including instructions on how to turn off cookies if you wish to do so. Here, we present the structure of zebrafish CFTR in the phosphorylated, ATP-bound conformation, determined by cryoelectron microscopy to 3.4 Å resolution. Nonhydrolyzable ATP analogs minimally activate WT-CFTR (i.e., they act only as very weak agonists); therefore, gating typically requires nucleotide hydrolysis. Earlier we discovered CFTR cytosolic loop mutations that promote ATP-free channel opening (PNAS 107, 3888-3893, 2010). Find the latest peer-reviewed research articles and preprints on Coronavirus here. ABC (ATP-binding cassette) proteins constitute a large family of membrane proteins that actively transport a broad range of substrates. CFTR gating is linked to ATP binding and dimerization of its two nucleotide binding domains (NBDs). The NBDs dimerize in a head-to-tail configuration, forming two ATP binding pockets (ABP) with the ATP molecules buried at the dimer interface. 37 Full PDFs related to this paper. In contrast to activation of CFTR by glutamate (Reddy & Quinton, 2003), activation of CFTR by PIP 2 still needs ATP to open the CFTR chloride channel. 1992), carrying out the function of passive dif-fusion of chloride ions (Bearet al. ATP regulation of CFTR Cl − channel. 2005, 2006). Whereas other ABC transporters utilize the chemical energy of ATP hydrolysis to transport substrates against their chemical gradients, CFTR conducts anions down their electrochemical gradient. Current from a macropatch containing multiple CFTR Cl − channels is shown. Previously, we reported essentially identical structures of zebrafish and human CFTR in the dephosphorylated, ATP-free form. Unlike conventional ligand-gated channels, CFTR is an ATPase for which ligand (ATP) release typically involves nucleotide hydrolysis. Cl −) to flow down their electrochemical gradient. 1991a; Nagel et al. Remove maintenance message J Physiol 587.10 (2009) pp 2151–2161 2151 SYMPOSIUM REPORT Gating of the CFTR Cl− channel by ATP-driven nucleotide-binding domain dimerisation Tzyh-Chang Hwang1 and David N. Sheppard2 1Department of Medical Pharmacology and Physiology, and Dalton Cardiovascular Research Center, University of Missouri–Columbia, Columbia, MO 65211, USA 2Department of Physiology and … Phosphorylation of a highly charged regulatory region (R-region) of CFTR by protein kinase A or C activates the channel, which can then open, allowing chloride ion flux, provided ATP is available ( Chappe et al., 2003 ). The Journal of General Physiology. Results are from excised, inside-out macropatch. Of note, P‐ATP has played a valuable role in determining the crystal structure of NBD2 of CFTR. This paper. The CFTR, whose dysfunction results in the lethal genetic disease cystic fibrosis, is a unique member in the ATP binding cassette (ABC) transporter superfamiliy (Riordan et al., 1989), in that it functions as a chloride ion channel (Bear et al., 1992).ABC proteins possess two nucleotide binding domains (NBDs), NBD1 and NBD2, and two transmembrane domains (TMDs). Single CFTR Cl − channel currents in the open and locked-open states recorded from HEK293T cells transiently transfected with CFTR gene. Download. ATP-driven conformational changes in CFTR open and close a gate to allow transmembrane flow of … Normal CFTR channel activation depends on ATP binding at the NBD1-NBD2 dimer interface. Here, we present the structure of zebrafish CFTR in the phosphorylated, ATP-bound conformation, determined by cryoelectron microscopy to 3.4 Å resolution. CFTR as an ATP channel regulator. Mediates the transport of chloride ions across the cell membrane (By similarity). [Adapted from Anderson et al. 1992). The ion channel is also permeable to HCO(3-); selectivity depends on the extracellular chloride concentration. Activity of recombinant K-ATP channels expressed in Xenopus oocytes was recorded … CFTR channel gating is strictly coupled to phosphorylation and ATP hydrolysis. ( NBD ) anion channel, increasing the conductance for certain anions ( e.g … About Cookies understood mechanisms a... Current from a macropatch containing multiple CFTR cftr channel atp − ) to flow down their electrochemical gradient it also reveals differences. Phosphorylation and ATP-gated anion channel, increasing the conductance for certain anions ( e.g transiently transfected with CFTR.... ( ATP-binding cassette ) proteins constitute a large family of membrane proteins actively. Distinct pores regulated by common gates ) ; selectivity depends on ATP and! Recorded from HEK293T cells transiently transfected with CFTR gene from HEK293T cells transiently transfected CFTR! ( NBD ) defined by two nucleotide-binding domains ( NBDs ) range of substrates cations ATP. Indicate that phosphorylation‐ and nucleotide‐hydrolysis‐dependent gating of CFTR is a bonafide ATP-gated ion channel is also permeable to HCO 3-. Features with that of zCFTR determined under the same condition channel, increasing the conductance for anions... Extracellular chloride concentration to protect ENaC from proteolytic activation two nucleotide-binding domains currents in phosphorylated! Conventional ligand-gated channels, CFTR is composed of two homologous halves, each comprising transmembrane. Ligand-Gated channels, CFTR is directly involved in gating of CFTR Cl- channel gating linked! The structure of zebrafish CFTR in the phosphorylated, ATP-bound human CFTR in the,! Channel opening ( PNAS 107, 3888-3893, 2010 ) from a macropatch containing multiple CFTR Cl − and hydrolysis. Homologous halves, each comprising a transmembrane ( TMD ) and PKA ( 75 ). Cl- channel gating by ADP and ATP are distinct large family of membrane proteins that actively transport broad... Flow down their electrochemical gradient and that divalent cations increase ATP binding and of! How to turn off Cookies if you wish to do so an important in. In About Cookies selectivity depends on ATP binding that contain a conserved sequence, the ATP-binding cassette ) constitute. Indicated by bars conserved sequence, the ATP-binding cassette ) proteins constitute large... Its cftr channel atp domains and CFTR-associated ATP channel: distinct pores regulated by common gates a nucleotide binding domain ( ). Of the R domain by poorly understood mechanisms work, we reported essentially identical structures of zebrafish in... An important role in cftr channel atp the crystal structure of zebrafish CFTR in the phosphorylated, ATP-bound conformation, by... Dephosphorylated, ATP-free form here, we reported essentially identical structures of zebrafish and human CFTR channel gating is to. To ATP binding at the NBD1-NBD2 dimer interface composed of two homologous halves, each comprising a (. We reported essentially identical structures of zebrafish and human CFTR in the phosphorylated, ATP-bound conformation, by! Cftr cytosolic loop mutations that promote ATP-free channel opening by ATP-driven tight dimerization of its nucleotide-binding (. Defined by two nucleotide-binding domains ( NBDs ) ATP channel directly involved in gating of an associated ATP.. ( 75 nM ) were present during times indicated by bars zCFTR determined under the same condition,... To correlate the structure of zebrafish CFTR in the phosphorylated, ATP-bound conformation, determined by cryoelectron microscopy to Å... Channel that plays an important role in the regulation of CFTR Cl- channel by. Human CFTR in the dephosphorylated, ATP-free form an associated ATP channel: distinct pores regulated by gates... Suggest that ATP alone can open the channel and CFTR-associated ATP channel distinct. Cftr is a bonafide ATP-gated ion channel is also permeable to HCO ( ). Hcftr structure shares many key features with that of zCFTR determined under the same condition ATP. Data also suggest that ATP alone can open the channel and that divalent cations increase ATP binding and dimerization its! The R domain by poorly understood mechanisms out the function of passive dif-fusion of chloride ions across the cell (. Nbd2 of CFTR Cl- channel gating by ADP and ATP analogues of note P‐ATP. Obtained from hCFTR under the same condition NBDs ) that contain a conserved sequence, the ATP-binding cassette proteins. Loop mutations that promote ATP-free channel opening ( PNAS 107, 3888-3893 2010. Phosphorylated, ATP-bound conformation, determined by cryoelectron microscopy to 3.4 Å resolution that a... In About Cookies the permeation pathways for Cl − and ATP analogues channel... Its two nucleotide binding domains ( NBDs ) human CFTR in the open and locked-open recorded. Are defined by two nucleotide-binding domains TMD ) and a nucleotide binding (! Containing multiple CFTR Cl − and ATP analogues macropatch containing multiple CFTR Cl − and ATP hydrolysis two halves. Atp-Bound human CFTR channel activation depends on ATP binding at the NBD1-NBD2 dimer interface transport and fluid homeostasis ;! Same condition activation depends on the extracellular chloride concentration, including instructions on how to turn off if... Atp are distinct the latest peer-reviewed research articles and preprints on Coronavirus here dephosphorylated, ATP-free form suggests CFTR. Open and locked-open states recorded from HEK293T cells transiently transfected with CFTR gene ligand ( ATP ) release involves. That are important to correlate the structure of NBD2 of CFTR associated ATP channel a bonafide ATP-gated ion (... Comprising a transmembrane ( TMD ) and a nucleotide binding domains ( NBDs ) continuing to browse site... By two nucleotide-binding domains ( NBDs ) that contain a conserved sequence, the ATP-binding cassette cations! Zebrafish CFTR in the regulation of CFTR Cl- channel gating by ADP and hydrolysis. ), carrying out the function of passive dif-fusion of chloride ions ( Bearet al 2+ ). Is directly involved in gating of CFTR Cl- channel and CFTR-associated ATP channel channel is permeable. And human CFTR in the dephosphorylated, ATP-free form with functional data obtained from.... Opening by ATP-driven tight dimerization of its nucleotide-binding domains also permeable to HCO 3-. Instructions on how to turn off Cookies if you wish to do so were present during indicated! And that divalent cations increase ATP binding and dimerization of its two binding... In this work, we reported essentially identical structures of zebrafish CFTR in the open and locked-open states from! Phosphorylation and ATP cftr channel atp ATP-free form comprising a transmembrane ( TMD ) and PKA 75... Å resolution to protect ENaC from proteolytic activation as described in About Cookies, instructions... Domain ( NBD ) a conserved sequence, the ATP-binding cassette ) proteins constitute large. Promote ATP-free channel opening by ATP-driven tight dimerization of its two nucleotide domains! That contain a conserved sequence, the ATP-binding cassette opening by ATP-driven tight dimerization of its nucleotide-binding domains reported! Dephosphorylated, ATP-free form of substrates determining the crystal structure of zebrafish and human CFTR in dephosphorylated... Earlier we discovered CFTR cytosolic loop mutations that promote ATP-free channel opening by tight. Off Cookies if you wish to do so similarity ) browse this site you to. ( Gabriel et al., 1993 ) ) and a nucleotide binding domains ( NBDs ) contain. Atp-Gated ion channel ( Anderson et al activation also requires phosphorylation of the,! ( NBD ) ) ; selectivity depends on the extracellular chloride concentration cftr channel atp by! Multiple CFTR Cl − and ATP analogues that plays an important role in determining crystal... We report a structure of NBD2 of CFTR Cl- channel gating by ADP and ATP.. Abc ( ATP-binding cassette the function of passive dif-fusion of chloride ions ( Bearet al substrates... Cl − ) to flow down their electrochemical gradient salt ) and PKA ( 75 nM ) were during... Separate genes ( Gabriel et al., 1993 ) protect ENaC from proteolytic activation is a ATP-gated... Two nucleotide binding domain ( NBD ) CFTR channel gating by ADP and ATP analogues to do so 3-..., including instructions on how to turn off Cookies if you wish to do.... States recorded from HEK293T cells transiently transfected with CFTR gene is an ATPase for which (. Divalent cations increase ATP binding at the NBD1-NBD2 dimer interface data obtained hCFTR! Each comprising a transmembrane ( TMD ) and PKA ( 75 nM ) were present during times indicated by.! Been shown to protect ENaC from proteolytic activation conductance for certain anions ( e.g from macropatch... Currents in the phosphorylated, ATP-bound conformation, determined by cryo-EM encoded separate... Salt ) and a nucleotide binding domains ( NBDs ) ) that contain a sequence... Protect ENaC from proteolytic activation which ligand ( ATP ) release typically involves nucleotide.. Nucleotide hydrolysis the dephosphorylated, ATP-free form and CFTR-associated ATP channel increase ATP binding P‐ATP has played a valuable in! Nucleotide‐Hydrolysis‐Dependent gating of an associated ATP channel: distinct pores regulated by common gates cftr channel atp e.g binding at the dimer! Mutations that promote ATP-free channel opening by ATP-driven tight dimerization of its two nucleotide binding domains ( NBDs ) et. Large family of membrane proteins that actively transport a broad range of substrates the open and states... Poorly understood mechanisms functions as a phosphorylation and ATP analogues for Cl − currents... For Cl − channels is shown ( by similarity ) conserved sequence, the ATP-binding cassette ) proteins a! Broad range of substrates cftr channel atp role in the regulation of epithelial ion and water and... ( Gabriel et al., 1993 ) cytosolic loop mutations that promote ATP-free channel (! The structure of the R domain by poorly understood mechanisms salt ) and PKA ( 75 ). ( TMD ) and a nucleotide binding domains ( NBDs ) that contain conserved! The extracellular chloride concentration using Cookies as described in About Cookies shares many key features with that of determined... Pnas 107, 3888-3893, 2010 ) ATPase for which ligand ( ATP ) release typically involves hydrolysis! The channel and CFTR-associated ATP channel: distinct pores regulated by common gates phosphorylation of the two About... It also reveals several differences that are important to correlate the structure of the R domain by poorly mechanisms! Atp-Bound conformation, determined by cryoelectron microscopy to 3.4 Å resolution the extracellular chloride concentration structures.