Regulation of CFTR gating (normal and CF)
The Cystic fibrosis transmembrane conductance regulator
(CFTR) is a chloride channel that belongs to the
ATP-binding cassette (ABC) superfamily [1].
Mutations in CFTR-encoding gene cause cystic fibrosis (CF),
a genetic disease characterized by defective transport of chloride ions across several
epithelial tissues [2], [3]. CFTR
has two nucleotide binding domains (NBD1 and NBD2) that control channel gating by binding
and hydrolysis of ATP. Upon dimerization of the two NBDs of
CFTR in a head-to-tail configuration, two
ATP-binding pockets (ABP1 and ABP2) are formed with the
ATP molecules sandwiched at the interface [4].
Each ABP plays a different role in CFTR gating; ABP2 is the
site critical for the ATP-dependent opening of the
CFTR channel, whereas ATP
binding to ABP1 is believed to contribute to the stability of the open channel
conformation [5], [6]. CFTR also
has a large centrally localized regulatory domain (R domain) that is a special feature of
this ABC protein [7], [8], [9], [10].
Pyrophosphate is the product of the reaction of
cAMP synthesis from ATP.
Pyrophosphate is well-known for its ability to modulate
CFTR gating. Together with ATP,
Pyrophosphate can lock CFTR in
a stable open state. It has been reported that high concentrations of
Pyrophosphate lead to inhibition of CTFR gating. However, it
is unclear if Pyrophosphate inhibits the process directly or
indirectly through its ability to chelate Mg2+ [11], [12], [13]. It is shown that
Pyrophosphate potentiates CFTR
only in human, but not mouse [14].
CFTR channel activity is modulated by phosphorylation by
cyclic AMP-dependent Protein
kinase A (PKA) [15]. Two gating modes have been
reported: CFTR channel open bursts are long in the presence
of PKA, but shorten upon PKA
removal, presumably reflecting rapid partial CFTR
dephosphorylation [16]. The mechanisms by which
ATP and PKA together regulate
CFTR channel gating are complex and controversial. Thus,
phosphorylated CFTR behaves as a conventional ligand-gated
channel employing cytoplasmic ATP as a readily available
cytoplasmic ligand [17]. ATP binding leads to
channel opening whereupon its hydrolysis prompts channel closing, and phosphorylation
acts like a switch to drive gating of the transmembrane ion pore [18].
CFTR phosphorylation affects
ATP binding and not the subsequent steps of hydrolysis and
channel opening [8], [19].
AMP-activated protein kinase (AMPK) can also
phosphorylate CFTR and thus lead to reduced secretion of
chloride ions by inhibition of the channel activity without affecting the number of
CFTR channels in the plasma membrane. The exact molecular
mechanism of this event is unknown [20], [21], [22].
G551D is the third overall most common CF mutation with a worldwide frequency of ~3%.
This mutation is associated with a severe phenotype characterized by pulmonary
dysfunction and pancreatic insufficiency [23], [24]. G551 is
located in the signature sequence of NBD1that, together with the Walker A and B motifs of
NBD2, forms ABP2, a critical site for channel opening by ATP
[5]. G551D mutation more likely hampers conformational changes at ABP2 that
facilitate NBD dimerization, i.e., channel opening by ATP
[25]. G551D-CFTR exhibited a markedly reduced
ATPase activity [26], [27].
Furthermore, G551D-CFTR does not respond to
ADP or changes in Mg('2+)
concentration. The residual low activity of G551D-CFTR
represents ATP-independent gating events [28], [29].
Micromolar Cd('2+) and
Zn('2+) can dramatically increase the activity of
G551D-CFTR in the absence of
ATP. This effect of Cd('2+) and
Zn('2+) is not seen in wild-type channels [30].
Some CFTR potentiators are examined as correctors of gating. These potentiators are
Anthracene-9-carboxylic acid (9-Anthroic acid) [31], Phloxine B [32], [33],
benzimidazolone analogs NS004 [34] and
NS1619, Genistein [35], [36], [37],
7-n-Butyl-6-(4-hydroxyphenyl)[5H]pyrrolo[2,3-b]pyrazine (Aloisine
A) [38],
2-(2-(1H-indol-3-yl)-N-methylacetamido)-N-(4-isopropylphenyl)-2-phenylacetamide
(PG01),
N-cycloheptyl-6-(N-ethyl-N-phenylsulfamoyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide
(SF01), sulfonamide
6-(N-ethyl-N-phenylsulfamoyl)-N-(2-methoxybenzyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide
(SF03) [39],
Capsaicin [40],
Curcumin [41],
and VX-770 (Vertex Pharmaceuticals Inc.)
Many of these potentiators (e.g., Phloxine B [32], [33], benzimidazolone analogs NS004,
Genistein [36], [37],
9-Anthroic acid [31]) can both stimulate, and
inhibit wild-type and mutant CFTR channel activity in a
dose-dependent manner. Phosphorylation status of CFTR is a
very important for action of potentiators on this channel,
Different CFTR domains may be important for action of different potentiators. It is
suggested, that Phloxine B, benzimidazolone analogs
NS004 and NS1619, and
Genistein stimulate CFTR via
interaction with NBD2 domain, and inhibit CFTR via binding
to NBD1 domain or by occluding the pore [33], [37].
9-Anthroic acid binding sites for potentiating and
inhibitory effects on CFTR channels are located outside of
the R-domain [31].
Curcumin strongly activates
G551D-CFTR channel. Stimulatory effect of
Curcumin does not require dimerization of the two NBDs.
However, stimulation by curcumin is nonetheless strongly
dependent on prior phosphorylation of the channel by PKA,
even though neither ATP nor NBD2 are required for this
activation. Thus, induction of CFTR opening can be carried
out via an alternative mechanism that bypasses the normal requirement for
ATP binding and NBD dimerization [41].
VX-770 (Vertex Pharmaceuticals Inc.), an investigational
oral potentiator, is designed to act directly on the malfunctioning
CFTR protein to help restore the balance of salt and water.
Clinical development of VX-770 is currently focused on a
subset of CF patients who have a G551D CFTR mutation
{http://www.vpharm.com/current-projects/drug-candidates/vx-770.html}.
References:
- Riordan JR, Rommens JM, Kerem B, Alon N, Rozmahel R, Grzelczak Z, Zielenski J, Lok S, Plavsic N, Chou JL
Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA.
Science (New York, N.Y.) 1989 Sep 8;245(4922):1066-73
- Frizzell RA
Functions of the cystic fibrosis transmembrane conductance regulator protein.
American journal of respiratory and critical care medicine 1995 Mar;151(3 Pt 2):S54-8
- Welsh MJ, Smith AE
Molecular mechanisms of CFTR chloride channel dysfunction in cystic fibrosis.
Cell 1993 Jul 2;73(7):1251-4
- Vergani P, Lockless SW, Nairn AC, Gadsby DC
CFTR channel opening by ATP-driven tight dimerization of its nucleotide-binding domains.
Nature 2005 Feb 24;433(7028):876-80
- Zhou Z, Wang X, Liu HY, Zou X, Li M, Hwang TC
The two ATP binding sites of cystic fibrosis transmembrane conductance regulator (CFTR) play distinct roles in gating kinetics and energetics.
The Journal of general physiology 2006 Oct;128(4):413-22
- Bompadre SG, Hwang TC
Cystic fibrosis transmembrane conductance regulator: a chloride channel gated by ATP binding and hydrolysis.
Sheng li xue bao : [Acta physiologica Sinica] 2007 Aug 25;59(4):431-42
- Ostedgaard LS, Baldursson O, Welsh MJ
Regulation of the cystic fibrosis transmembrane conductance regulator Cl- channel by its R domain.
The Journal of biological chemistry 2001 Mar 16;276(11):7689-92
- Baker JM, Hudson RP, Kanelis V, Choy WY, Thibodeau PH, Thomas PJ, Forman-Kay JD
CFTR regulatory region interacts with NBD1 predominantly via multiple transient helices.
Nature structural & molecular biology 2007 Aug;14(8):738-45
- Chen TY, Hwang TC
CLC-0 and CFTR: chloride channels evolved from transporters.
Physiological reviews 2008 Apr;88(2):351-87
- Hegedus T, Serohijos AW, Dokholyan NV, He L, Riordan JR
Computational studies reveal phosphorylation-dependent changes in the unstructured R domain of CFTR.
Journal of molecular biology 2008 May 16;378(5):1052-63
- Gunderson KL, Kopito RR
Effects of pyrophosphate and nucleotide analogs suggest a role for ATP hydrolysis in cystic fibrosis transmembrane regulator channel gating.
The Journal of biological chemistry 1994 Jul 29;269(30):19349-53
- Carson MR, Winter MC, Travis SM, Welsh MJ
Pyrophosphate stimulates wild-type and mutant cystic fibrosis transmembrane conductance regulator Cl- channels.
The Journal of biological chemistry 1995 Sep 1;270(35):20466-72
- Csanady L, Chan KW, Nairn AC, Gadsby DC
Functional roles of nonconserved structural segments in CFTR's NH2-terminal nucleotide binding domain.
The Journal of general physiology 2005 Jan;125(1):43-55
- Scott-Ward TS, Cai Z, Dawson ES, Doherty A, Da Paula AC, Davidson H, Porteous DJ, Wainwright BJ, Amaral MD, Sheppard DN, Boyd AC
Chimeric constructs endow the human CFTR Cl- channel with the gating behavior of murine CFTR.
Proceedings of the National Academy of Sciences of the United States of America 2007 Oct 9;104(41):16365-70
- Howell LD, Borchardt R, Kole J, Kaz AM, Randak C, Cohn JA
Protein kinase A regulates ATP hydrolysis and dimerization by a CFTR (cystic fibrosis transmembrane conductance regulator) domain.
The Biochemical journal 2004 Feb 15;378(Pt 1):151-9
- Csanady L, Nairn AC, Gadsby DC
Thermodynamics of CFTR channel gating: a spreading conformational change initiates an irreversible gating cycle.
The Journal of general physiology 2006 Nov;128(5):523-33
- Aleksandrov AA, Chang X, Aleksandrov L, Riordan JR
The non-hydrolytic pathway of cystic fibrosis transmembrane conductance regulator ion channel gating.
The Journal of physiology 2000 Oct 15;528 Pt 2:259-65
- Basso C, Vergani P, Nairn AC, Gadsby DC
Prolonged nonhydrolytic interaction of nucleotide with CFTR's NH2-terminal nucleotide binding domain and its role in channel gating.
The Journal of general physiology 2003 Sep;122(3):333-48
- Vais H, Zhang R, Reenstra WW
Dibasic phosphorylation sites in the R domain of CFTR have stimulatory and inhibitory effects on channel activation.
American journal of physiology. Cell physiology 2004 Sep;287(3):C737-45
- Hallows KR, Raghuram V, Kemp BE, Witters LA, Foskett JK
Inhibition of cystic fibrosis transmembrane conductance regulator by novel interaction with the metabolic sensor AMP-activated protein kinase.
The Journal of clinical investigation 2000 Jun;105(12):1711-21
- Hallows KR, McCane JE, Kemp BE, Witters LA, Foskett JK
Regulation of channel gating by AMP-activated protein kinase modulates cystic fibrosis transmembrane conductance regulator activity in lung submucosal cells.
The Journal of biological chemistry 2003 Jan 10;278(2):998-1004
- Hallows KR, Kobinger GP, Wilson JM, Witters LA, Foskett JK
Physiological modulation of CFTR activity by AMP-activated protein kinase in polarized T84 cells.
American journal of physiology. Cell physiology 2003 May;284(5):C1297-308
- Cutting GR, Kasch LM, Rosenstein BJ, Zielenski J, Tsui LC, Antonarakis SE, Kazazian HH Jr
A cluster of cystic fibrosis mutations in the first nucleotide-binding fold of the cystic fibrosis conductance regulator protein.
Nature 1990 Jul 26;346(6282):366-9
- Kerem BS, Zielenski J, Markiewicz D, Bozon D, Gazit E, Yahav J, Kennedy D, Riordan JR, Collins FS, Rommens JM
Identification of mutations in regions corresponding to the two putative nucleotide (ATP)-binding folds of the cystic fibrosis gene.
Proceedings of the National Academy of Sciences of the United States of America 1990 Nov;87(21):8447-51
- Bompadre SG, Li M, Hwang TC
Mechanism of G551D-CFTR (cystic fibrosis transmembrane conductance regulator) potentiation by a high affinity ATP analog.
The Journal of biological chemistry 2008 Feb 29;283(9):5364-9
- Ramjeesingh M, Ugwu F, Stratford FL, Huan LJ, Li C, Bear CE
The intact CFTR protein mediates ATPase rather than adenylate kinase activity.
The Biochemical journal 2008 Jun 1;412(2):315-21
- Cheung JC, Kim Chiaw P, Pasyk S, Bear CE
Molecular basis for the ATPase activity of CFTR.
Archives of biochemistry and biophysics 2008 Aug 1;476(1):95-100
- Bompadre SG, Sohma Y, Li M, Hwang TC
G551D and G1349D, two CF-associated mutations in the signature sequences of CFTR, exhibit distinct gating defects.
The Journal of general physiology 2007 Apr;129(4):285-98
- Aleksandrov L, Aleksandrov A, Riordan JR
Mg2+ -dependent ATP occlusion at the first nucleotide-binding domain (NBD1) of CFTR does not require the second (NBD2).
The Biochemical journal 2008 Nov 15;416(1):129-36
- Wang X, Bompadre SG, Li M, Hwang TC
Mutations at the signature sequence of CFTR create a Cd(2+)-gated chloride channel.
The Journal of general physiology 2009 Jan;133(1):69-77
- Ai T, Bompadre SG, Sohma Y, Wang X, Li M, Hwang TC
Direct effects of 9-anthracene compounds on cystic fibrosis transmembrane conductance regulator gating.
Pflugers Archiv : European journal of physiology 2004 Oct;449(1):88-95
- Cai Z, Sheppard DN
Phloxine B interacts with the cystic fibrosis transmembrane conductance regulator at multiple sites to modulate channel activity.
The Journal of biological chemistry 2002 May 31;277(22):19546-53
- Melin P, Norez C, Callebaut I, Becq F
The glycine residues G551 and G1349 within the ATP-binding cassette signature motifs play critical roles in the activation and inhibition of cystic fibrosis transmembrane conductance regulator channels by phloxine B.
The Journal of membrane biology 2005 Dec;208(3):203-12
- Gribkoff VK, Champigny G, Barbry P, Dworetzky SI, Meanwell NA, Lazdunski M
The substituted benzimidazolone NS004 is an opener of the cystic fibrosis chloride channel.
The Journal of biological chemistry 1994 Apr 15;269(15):10983-6
- Illek B, Fischer H, Santos GF, Widdicombe JH, Machen TE, Reenstra WW
cAMP-independent activation of CFTR Cl channels by the tyrosine kinase inhibitor genistein.
The American journal of physiology 1995 Apr;268(4 Pt 1):C886-93
- Al-Nakkash L, Hu S, Li M, Hwang TC
A common mechanism for cystic fibrosis transmembrane conductance regulator protein activation by genistein and benzimidazolone analogs.
The Journal of pharmacology and experimental therapeutics 2001 Feb;296(2):464-72
- Derand R, Bulteau-Pignoux L, Becq F
Comparative pharmacology of the activity of wild-type and G551D mutated CFTR chloride channel: effect of the benzimidazolone derivative NS004.
The Journal of membrane biology 2003 Jul 15;194(2):109-17
- Noel S, Faveau C, Norez C, Rogier C, Mettey Y, Becq F
Discovery of pyrrolo[2,3-b]pyrazines derivatives as submicromolar affinity activators of wild type, G551D, and F508del cystic fibrosis transmembrane conductance regulator chloride channels.
The Journal of pharmacology and experimental therapeutics 2006 Oct;319(1):349-59
- Pedemonte N, Sonawane ND, Taddei A, Hu J, Zegarra-Moran O, Suen YF, Robins LI, Dicus CW, Willenbring D, Nantz MH, Kurth MJ, Galietta LJ, Verkman AS
Phenylglycine and sulfonamide correctors of defective delta F508 and G551D cystic fibrosis transmembrane conductance regulator chloride-channel gating.
Molecular pharmacology 2005 May;67(5):1797-807
- Ai T, Bompadre SG, Wang X, Hu S, Li M, Hwang TC
Capsaicin potentiates wild-type and mutant cystic fibrosis transmembrane conductance regulator chloride-channel currents.
Molecular pharmacology 2004 Jun;65(6):1415-26
- Wang W, Bernard K, Li G, Kirk KL
Curcumin opens cystic fibrosis transmembrane conductance regulator channels by a novel mechanism that requires neither ATP binding nor dimerization of the nucleotide-binding domains.
The Journal of biological chemistry 2007 Feb 16;282(7):4533-44