Pflugers Arch - Eur J Physiol DOI 10.1007/s00424-014-1501-7

ION CHANNELS, RECEPTORS AND TRANSPORTERS

State-dependent blocker interactions with the CFTR chloride channel: implications for gating the pore Paul Linsdell

Received: 3 February 2014 / Revised: 10 March 2014 / Accepted: 11 March 2014 # Springer-Verlag Berlin Heidelberg 2014

Abstract Chloride permeation through the cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channel is subject to voltage-dependent open-channel block by a diverse range of cytoplasmic anions. However, in most cases the ability of these blocking substances to influence the pore opening and closing process has not been reported. In the present work, patch clamp recording was used to investigate the statedependent block of CFTR by cytoplasmic Pt(NO2)42− ions. Two major effects of Pt(NO2)42− were identified. First, this anion caused fast, voltage-dependent block of open channels, leading to an apparent decrease in single-channel current amplitude. Secondly, Pt(NO2)42− also decreased channel open probability due to an increase in interburst closed times. Interestingly, mutations in the pore that weakened (K95Q) or strengthened (I344K, V345K) interactions with Pt(NO2)42− altered blocker effects both on Cl− permeation and on channel gating, suggesting that both these effects are a consequence of Pt(NO2)42− interaction with a single site within the pore. Experiments at reduced extracellular Cl− concentration hinted that Pt(NO2)42− may have a third effect, possibly increasing channel activity by interfering with channel closure. These results suggest that Pt(NO2)42− can enter from the cytoplasm into the pore inner vestibule of both open and closed CFTR channels, and that Pt(NO2)42− bound in the inner vestibule blocks Cl− permeation as well as interfering with channel opening and, perhaps, channel closure. Implications for the location of the channel gate in the pore, and the operation of this gate, are discussed. Keywords Chloride channel . Cystic fibrosis transmembrane conductance regulator . Ion channel pore . Open-channel block . Site-directed mutagenesis . State-dependent block P. Linsdell (*) Department of Physiology and Biophysics, Dalhousie University, PO Box 15000, Halifax, NS B3H 4R2, Canada e-mail: [email protected]

Introduction Cystic fibrosis (CF) is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) [22]. CFTR is a member of the ATP-binding cassette family of membrane transport proteins [6]. It functions as a phosphorylation-regulated, ATP-gated anion channel in the apical membrane of many different types of epithelial cells [9]. As with other ABC proteins, CFTR is comprised of two transmembrane domains (TMDs) and two cytoplasmic nucleotide binding domains (NBDs); CFTR also contains a unique regulatory (R) domain that is the site of channel regulation by phosphorylation [13]. The NBDs are the site of channel regulation by ATP binding and hydrolysis [12, 14]. ATP binding promotes NBD dimerization and initiates a “burst” of channel openings interrupted only by very brief, ATP-independent closures; ATP hydrolysis then leads to NBD separation and terminates the burst, following which the channel closes into a longer-lived interburst closed state [5, 12–14]. Mechanistically speaking, a propagated conformational change initiated by ATP interaction with the NBDs is presumed to somehow control the function of a “gate” within the TMDs that opens and closes to allow or prevent the movement of Cl− and other small anions through the channel pore [19]. The NBDs and TMDs are physically and functionally linked by long intracellular loops (ICLs) that join individual membrane spanning α-helices (TMs) [15], presumably allowing gating information initiated in the NBDs to be transmitted to the TMDs. The structure of the TMDs forming the channel pore, and the location and mechanism of the gate in the pore are not well understood. The open-channel pore is thought to contain a relatively narrow region that is the main determinant of ion selectivity, and which is flanked by wider outer and inner vestibules [19]. The inner vestibule is thought to be both deeper and wider than the outer vestibule in open channels,

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and is lined by the cytoplasmic ends of TMs 1, 6, 11 and 12 [19]. While closing of the channel pore is thought to be associated with localized structural changes in the inner vestibule [1, 2, 7, 25, 26, 29], narrow pore region [26, 27], and outer vestibule [3, 26, 27, 33], the location of the functionally important channel gate, and the mechanism by which this gate alternately allows or prevents the flow of Cl− ions, are not known [19]. The inner vestibule is also the site of channel inhibition by a large number of negatively charged substances that act as open-channel blockers [20]. These blockers enter into the channel pore from its cytoplasmic end in a voltage- and extracellular [Cl−]-dependent fashion to temporarily occlude Cl− permeation through the open channel [20]. Many different kinds of anionic open-channel blockers have been shown to interact with a single positively charged amino acid side chain located in the inner vestibule, K95 from TM1, such that blocker binding affinity is greatly weakened when this positive charge is neutralized by mutagenesis [18, 20, 36]. Interestingly, the functionally important positive charge of K95 can be “transplanted” to other, nearby sites lining the inner vestibule by mutagenesis; in particular I344 and V345 (TM6) [8] and S1141 (TM12) [36] are able to host this charge and retain normal blocker binding as well as other functional pore properties. Furthermore, when the number of positive charges lining this part of the inner vestibule is increased from one (as in wild type) to two (for example in the channel mutants I344K, V345K, and S1141K), blocker potency is increased [8, 36]. This strengthened interaction with blockers is particularly striking for the small, divalent tetranitroplatinate anion (Pt(NO2)42−) [8, 36], leading to the suggestion that a second fixed positive charge in the inner vestibule favors the attraction of multivalent anions, relative to monovalent anions such as Cl−, to the pore [8]. While the interaction of channel blockers with the openchannel permeation pathway has been studied in detail, the relationship between block and gating (opening and closing) of the channel pore has received little attention. One blocker, Au(CN)2−, was shown to affect both permeation and gating [21]. However, these two effects may show different molecular bases, such that effects on gating most likely reflect a nonpore-mediated action [25]. Similarly, a number of substances including 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) [5, 30], genistein [17], and phloxine B [4] have been shown to produce open-channel block and also to affect gating by acting at distinct sites that are likely outside of the channel pore. Here I show that the previously described open-channel blocker Pt(NO2)42− has complex effects on CFTR channel gating. Taking advantage of mutations that both weaken and strengthen Pt(NO2)42− binding in the inner vestibule of the pore, it is shown that Pt(NO2)42− binding to a single site in the pore affects both Cl− permeation and channel gating. It is proposed that blocker binding within the inner vestibule

interferes with the local conformational changes that underlie channel opening and closing.

Materials and methods Experiments were carried out on baby hamster kidney cells transiently transfected with CFTR, as described in detail previously [36]. Mutations were introduced using the QuikChange site-directed mutagenesis system (Agilent Technologies, Santa Clara, CA, USA) and verified by DNA sequencing. Some experiments were carried out with channels containing the NBD2 mutation E1371Q, which this laboratory [25–27, 29, 36] and others [2, 3] have used to abolish ATPdependent channel gating and lock CFTR channels in the open state. Where the properties of different channel pore variants (wild type, K95Q, I344K, V345K) have been directly compared in wild type and E1371Q backgrounds, the wildtype background is referred to as “1371E” to indicate that the endogenous glutamate residue is present at this position. Macroscopic and single-channel CFTR currents were recorded using patch clamp recordings from inside-out membrane patches, as described in detail recently [36]. Following patch excision and recording of background currents, CFTR channels were activated by exposure to protein kinase A catalytic subunit (PKA; 20 nM) plus MgATP (1 mM or, where indicated (Fig. 1) 50 μM) in the intracellular solution. In some cases (see Fig. 1), patches were subsequently treated with 2 mM sodium pyrophosphate (PPi) to stabilize the channel open state. For both macroscopic and single-channel recordings, both intracellular (bath) and extracellular (pipette) solutions contained (in millimolar): 150 NaCl, 2 MgCl2, 10 N-tris[hydroxymethyl] methyl-2-aminoethanesulfonate, pH 7.4. For experiments at low extracellular [Cl−] (Fig. 6), NaCl in the pipette solution was replaced by Na gluconate. Current traces were filtered at 50–100 Hz using an eight-pole Bessel filter, digitized at 250 Hz–1 kHz, and analyzed using pCLAMP software (Molecular Devices, Sunnyvale, CA, USA). Measurement of single-channel and macroscopic current amplitudes, and construction of leak-subtracted macroscopic current–voltage (I-V) relationships were carried out as described in detail recently [36]. Different concentrations of potassium tetranitroplatinate (K2Pt(NO2)42−) were applied directly to the cytoplasmic face of inside-out patches from stock solutions made up in normal intracellular solution. Blocker concentration-inhibition relationships were fitted by the equation:  
nH  Fractional unblocked current ¼ 1= 1 þ PtðNO2 Þ4 =K D

where KD is the apparent blocker dissociation constant and nH the slope factor or Hill coefficient. Under most circumstances nH was very close to unity, except for experiments using

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Fig. 1 Channel gating affects block by intracellular Pt(NO2)42−. (A, B) Example leak-subtracted macroscopic I-V relationships for wild-type CFTR in the presence of 1 mM ATP (A) or following maximization of channel open probability by treatment with PPi (2 mM; B). In each case, currents were recorded before (control) and after the addition of 100 μM and 1 mM Pt(NO2)42− to the intracellular (bath) solution. (C, D) Mean fraction of control current remaining after addition of different concentrations of Pt(NO2)42− at a membrane potential of −100 mV (C) or + 50 mV (D), in channels activated by 1 mM ATP (black filled circle), 1 mM ATP plus 2 mM PPi (white empty circle) or 50 μM ATP (filled down-pointing triangle) as indicated. Data have been fitted as described in “Materials and methods”. (E) Mean KD values obtained from such fits

at different membrane potentials. Data have been fitted as described in “Materials and methods”. (F) Example leak-subtracted macroscopic I-V relationships for E1371Q-CFTR in the presence of 1 mM ATP, recorded before (control) and after the addition of 100 μM and 1 mM Pt(NO2)42− to the intracellular solution. (G, H) Mean data from experiments with E1371Q channels under the same conditions used for wild-type channels in (C–E). (I–K) Mean values of KD (at 0 mV in I and at +50 mV in J) and zδ (K) obtained from fits of KD-V relationships such as those shown in (E) and (H), for both wild type and E1371Q channels under different nucleotide conditions as indicated. Asterisks indicate a significant difference from the same channel construct under 1 mM ATP conditions (P