When excised inside-out membrane patches are bathed in symmetrical Cl−-rich solutions, the current-voltage (I-V) relationship of macroscopic cystic fibrosis transmembrane conductance regulator (CFTR) Cl− currents inwardly rectifies at large positive voltages. To investigate the mechanism of inward rectification, we studied CFTR Cl− channels in excised inside-out membrane patches from cells expressing wild-type human and murine CFTR using voltage-ramp and -step protocols. Using a voltage-ramp protocol, the magnitude of human CFTR Cl− current at +100 mV was 74 ± 2% (n = 10) of that at −100 mV. This rectification of macroscopic CFTR Cl− current was reproduced in full by ensemble currents generated by averaging single-channel currents elicited by an identical voltage-ramp protocol. However, using a voltage-step protocol the single-channel current amplitude (i) of human CFTR at +100 mV was 88 ± 2% (n = 10) of that at −100 mV. Based on these data, we hypothesized that voltage might alter the gating behavior of human CFTR. Using linear three-state kinetic schemes, we demonstrated that voltage has marked effects on channel gating. Membrane depolarization decreased both the duration of bursts and the interburst interval, but increased the duration of gaps within bursts. However, because the voltage dependencies of the different rate constants were in opposite directions, voltage was without large effect on the open probability (Po) of human CFTR. In contrast, the Po of murine CFTR was decreased markedly at positive voltages, suggesting that the rectification of murine CFTR is stronger than that of human CFTR. We conclude that inward rectification of CFTR is caused by a reduction in i and changes in gating kinetics. We suggest that inward rectification is an intrinsic property of the CFTR Cl− channel and not the result of pore block.
The cystic fibrosis transmembrane conductance regulator (CFTR) is a Cl ؊ channel gated by ATP-driven nucleotide-binding domain (NBD) dimerization. Here we exploit species differences between human and murine CFTR to investigate CFTR channel gating. Using homologous recombination, we constructed human-murine CFTR (hmCFTR) chimeras with sequences from NBD1, NBD2, or the regulatory domain (RD) of human CFTR replaced by the equivalent regions of murine CFTR. The gating behavior of hmRD and human CFTR were indistinguishable, whereas hmNBD1 and hmNBD2 had subtle effects on channel gating, prolonging both burst duration and interburst interval. By contrast, hmNBD1؉2, containing both NBDs of murine CFTR, reproduced the gating behavior of the subconductance state of murine CFTR, which has dramatically prolonged channel openings. The CFTR potentiator pyrophosphate (PPi) enhanced human, hmRD, and hmNBD1 CFTR Cl ؊ currents, but not those of hmNBD2, hmNBD1؉2, and murine CFTR. By analyzing the rate-equilibrium free-energy relationships of chimeric channels, we obtained snapshots of the conformation of the NBDs during ATP-driven dimerization. Our data demonstrate that the conformation of NBD1 changes before that of NBD2 during channel opening. This finding suggests that NBD dimerization does not proceed by a symmetric tweezer-like motion, but instead in an asymmetric fashion led by NBD1. We conclude that the NBDs of murine CFTR determine the unique gating behavior of its subconductance state, whereas NBD2 controls channel potentiation by PPi.ATP-binding cassette transporter ͉ chloride ion channel ͉ cystic fibrosis ͉ recombinational cloning ͉ rate-equilibrium free-energy relationships M utation of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl Ϫ channel causes the genetic disease cystic fibrosis (CF) (1). CFTR is composed of two membranespanning domain (MSD)-nucleotide-binding domain (NBD) motifs linked by a unique regulatory domain (RD) (1). The MSDs assemble to form a low-conductance (6-to 10-pS) anion-selective pore (2). The RD contains multiple consensus phosphorylation sites, phosphorylation of which is a prerequisite for channel opening (2). The NBDs form a head-to-tail dimer with two ATP-binding sites located at the dimer interface (3). ATP binds tightly to one ATP-binding site (site 1; formed by the Walker A and B motifs of NBD1 and the LSGGQ motif of NBD2), whereas ATP is hydrolyzed rapidly at the other ATP-binding site (site 2; formed by the Walker A and B motifs of NBD2 and the LSGGQ motif of NBD1) (4, 5). Anion flow through the CFTR pore is gated by the interaction of ATP with sites 1 and 2 powering NBD dimerization and, hence, conformational changes in the MSDs (5).A powerful strategy to investigate structure-function relationships is to exploit functional differences between homologues from divergent species. In previous work, we demonstrated that the gating behavior of murine CFTR (mCFTR) differs from that of human CFTR (hCFTR) in several important respects (6, 7). First, mCFTR opens for prolon...
Niflumic acid is widely used to inhibit Ca(2+) -activated Cl(-) channels. However, the chemical structure of niflumic acid resembles that of diphenylamine-2-carboxylate, a drug that inhibits the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel. To investigate how niflumic acid inhibits CFTR Cl(-) channel, we studied recombinant wild-type human CFTR in excised inside-out membrane patches. When added to the intracellular solution, niflumic acid caused a concentration- and voltage-dependent decrease of CFTR Cl(-) current with half-maximal inhibitory concentration (K(i)) of 253 microM and Hill co-efficient of approximately 1, at -50 mV. Niflumic acid inhibition of single CFTR Cl(-) channels was characterized by a very fast, flickery block that decreased dramatically current amplitude without altering open-probability. Consistent with these data, spectral analysis of CFTR Cl(-) currents suggested that channel block by niflumic acid was described by the closed <--> open <--> blocked kinetic scheme with blocker on rate (k(on)) = 13.9 x 10(6) M(-1)s(-1), off rate (k(off))=3348 s(-1) and dissociation constant (K(d)) = 241 microM, at -50 mV. Based on these data, we tested the effects of niflumic acid on transepithelial Cl(-) secretion and cyst growth using type I MDCK epithelial cells. Niflumic acid (200 microM) inhibited cAMP-stimulated, bumetanide-sensitive short-circuit current by 55%. Moreover, the drug potently retarded cyst growth. We conclude that niflumic acid is an open-channel blocker of CFTR that inhibits Cl(-) permeation by plugging the channel pore. It or related agents might be of value in the development of new therapies for autosomal dominant polycystic kidney disease.
I-salicylate. Based on substate analogue studies, exoglycosidase digestions, and co-chromatography with fucosylated standards, the product of the reaction with pNP-LNB was Fuc␣1, 2Gal1,3GlcNAc-pNP. The cFTase preferred substrates with a Gal1,3 linkage, and thus its acceptor substrate specificity resembles the human Secretor-type ␣1,2-FTase. Afucosyl isoforms of the FP21 glycoprotein, GP21-I and GP21-II, were purified from the cytosol of a Dictyostelium mutant and found to be substrates for the cFTase, which exhibited an apparent K m of 0.21 M and an apparent V max of 460 nmol/min/mg protein toward GP21-II. The highly purified cFTase was inhibited by the reaction products Fuc␣1,2Gal1,3GlcNAc-pNP and FP21-II. FP21-I and recombinant FP21 were not inhibitory, suggesting that acceptor substrate specificity is based primarily on carbohydrate recognition. A cytosolic location for this step of FP21 glycosylation is implied by the isolation of the cFTase from the cytosolic fraction, its high affinity for its substrates, and its failure to be detected in crude membrane preparations.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.