[h=2]Description:[/h]
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
cyclicAMP -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
[19],
[8].
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-
CFTRexhibited a markedly reduced
ATP ase 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
[37],
[33].
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}.