Pathway maps

Regulation of CFTR gating (normal and CF)
Regulation of CFTR gating (normal and CF)

Object List (links open in MetaCore):

PDE4D, SF01 extracellular region, Genistein extracellular region, PKA-reg (cAMP-dependent), PG01 extracellular region, VX770, ATP cytosol, SF03 extracellular region, cyclic AMP cytosol, 9-Anthroic acid, Adenylate cyclase, AMPK gamma1, Cl('-) cytosol, Capsaicin, ADP cytosol, NS004, Aloisine A extracellular region, NS1619, Cl('-) extracellular region, Cd('2+) cytosol, Phloxine B, AMPK alpha 1 subunit,, <cytosol> Cl('-) = <extracellular region> Cl('-), Zn('2+) cytosol, Mg('2+) cytosol, Curcumin,, PKA-cat (cAMP-dependent), CFTR,, Pyrophosphate, AMP


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 [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- CFTR exhibited 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 {}.


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