Guangyu Wang 1,2,3, Rheeann Linsley 4 and Yohei Norimatsu 5

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1 External Zn 2+ binding to cysteine-substituted cystic fibrosis transmembrane conductance regulator constructs regulates channel gating and curcumin potentiation Guangyu Wang 1,2,3, Rheeann Linsley 4 and Yohei Norimatsu 5 1 Department of Physiology and Pharmacology, Oregon Health & Sciences University, Portland, OR, US 2 Department of Drug Research and Development, Institute of iophysical Medico-chemistry, Reno, NV, US 3 Department of Physiology and Membrane iology, University of California School of Medicine, Davis, C, US 4 Truman State University, Kirksville, MO, US 5 Department of Physiology, Kirksville College of Osteopathic Medicine,.T. Still University, Kirksville, MO, US Keywords C transporter; extracellular loop; intracellular loop; long-range gating coupling; metal disturbance Correspondence G. Wang, Department of Physiology and Membrane iology, University of California School of Medicine, Davis, C 95616, US Tel/Fax: gary.wang1@gmail.com The -dependent Zn 2+ inhibition of / H and /H CFTR was done in 21 and 22 and a part of data was first presented in abstract and poster forms (iophys J 22, 82, 239a; 47th iophysical Society nnual Meeting ddendum, 23, Pos L59, p16). (Received 1 July 215, revised 2 May 216, accepted 11 May 216) doi:1.1111/febs The cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is activated by TP binding-induced dimerization of nucleotidebinding domains, the interaction between the phosphorylated regulatory (R) domain and the curcumin-sensitive interface between intracellular loop (ICL) 1 and ICL4, and the resultant inward-to- outward reorientation of transmembrane domains. lthough transmembrane helices (TM) 2 and TM11 link the ICL1 ICL4 interface with the interface between extracellular loop (ECL) 1 and ECL6, it is unknown whether both interfaces are gating-coupled during the reorientation. Herein, and mutations were used to engineer two Zn 2+ bridges near and at the ECL1 ECL6 interface, respectively, and the gating effects of a Zn 2+ disturbance at the ECL1 ECL6 interface on the stimulatory ICL1/ICL4-R interaction were determined. The results showed that both Zn 2+ bridges inhibited channel activity in a dose- and -dependent manner, and the inhibition was reversed by a washout or suppressed by thiol-specific modification. Interestingly, their -dependent Zn 2+ inhibition was weakened at higher Zn 2+ concentrations, their Zn 2+ affinity was stronger in the resting state than in the activated state, and their activation current noises were decreased by external Zn 2+ binding. More importantly, the external Zn 2+ inhibition was reversed by internal curcumin in the construct but not in the mutant. Therefore, although both Zn 2+ bridges may promote channel closure, external Zn 2+ may disturb the ECL1 ECL6 interface and thus prevent the stimulatory ICL1/ICL4-R interaction and curcumin potentiation via a gating coupling between these two interfaces. bbreviations 2-ME, 2-mercaptoethonal; C, TP-binding cassette; CF, cystic fibrosis; CFTR, cystic fibrosis transmembrane conductance regulator; ECL, extracellular loop; IMX, 3-isobutyl-methylxanthine; ICL, intracellular loop; MTSES, sodium (2-sulfonatoethyl)methanethiosulfonate; MTSET +, 2-(trimethylammonium)ethyl methanethiosulfonate bromide; ND, nucleotide-binding domain; NEM, N-ethylmaleimide; NPP-M, 5-nitro-2-(3-phenylpropylamino)benzamide; PK, protein kinase ; R, regulatory; RR, rectification ratio; TEVC, two-electrode voltage clamp; TMD, transmembrane domain; TM, transmembrane The FES Journal 283 (216) ª 216 Federation of European iochemical Societies

2 G. Wang et al. Gating-coupled transmembrane helixes of CFTR Introduction The cystic fibrosis transmembrane conductance regulator (CFTR) is the product of the gene that is mutated in cystic fibrosis (CF). It belongs to a superfamily of TPbinding cassette (C) transporters but functions as an anion-selective chloride channel with five distinct domains [1,2]. Two transmembrane domains (TMD) 1 and 2, each with six transmembrane (TM) a-helices linked with six extracellular loops (ECLs) and four intracellular loops (ICLs), form a channel pore. Their gating reorientation between an inward-facing conformation and an outward -facing one is regulated by TPbinding/hydrolysis at the interface of two cytosolic nucleotide-binding domains (ND 1 and ND 2 ) and phosphorylation of a unique cytosolic regulatory (R) domain by protein kinase (PK) [3 8]. The highly conserved ICL1/ICL4 ND1 and ICL2/ICL3 ND2 swapping interactions in both TMD reorientations, as indicated by crystallography studies of bacterial C transporters TM287-TM288 and Sav1866 [9,1] and confirmed by crosslinking experiments [11 13], may facilitate TP-dependent gating regulation [3]. On the other hand, three R ICL3 interactions prevent channel opening by prohibiting TP binding-induced ND1 ND2 dimerization or the PK-triggered ICL1/ICL4 R interaction [4 8]. They include the H-bond between the OH group of unphosphorylated S768 of the R domain and the imidazole group of H95 of ICL3 [5], the asymmetric electrostatic interaction between K946 of ICL3 and D835, D836, and E838 of the R domain [6], and the Fe 3+ bridge between H95 and H954 of ICL3 and C832, D836, H775, and phosphorylated S768 of the R domain [4]. Thus, ICLs play a pivotal role in channel gating. In support of the stimulatory ICL1/ICL4 R interactions, sufficient Fe 3+ at the ICL3 R interface suppresses curcumin or NPP-M potentiation which requires the ICL1/ICL4 R interactions [7,8]. ecause both the ECL1 ECL6 interface and the ICL1 ICL4 interface are highly conserved in both inward- and outward-facing TMDs and linked via transmembrane (TM) 2 and TM11 [9,1], and several negatively charged residues such as D11, D112, E115, E116, E1124, and reside at the ECL1 ECL6 interface while curcumin-sensitive and F178 or and R166 are paired at the ICL1 ICL4 interface (Fig. 1), we test if an engineered Zn 2+ bridge disturbs the ECL1 ECL6 interface and thus affects the ICL1 ICL4 interface and then the stimulatory ICL1/ICL4 R interaction and curcumin potentiation via a gating coupling between these two interfaces. Zn 2+ has been successfully used to deduce spatial relationships of protein structures due to the highly restricted structural requirements for metal R166 Inward C1122 F178 R166 Outward C334 E1124 F178 D11 D112 Fig. 1. The spatial relationship between the ECL1 ECL6 interface and the ICL1 ICL4 interface. Negatively charged residues are located around the ECL1 ECL6 interface of CFTR, which is linked to the curcumin-sensitive ICL1 ICL4 interface via TM2 (cyan) and TM11 (yellow) in the inward (left) and outward-facing (right) TMDs, based on the crystal structures of bacterial transporters TM287 TM288 [9] and Sav1866 [1], respectively. (blue) is close to (red) in the inward-facing TMDs but aside from it in the outward-facing TMDs. In contrast, (blue) is circled by E1124,, D11, D112, E115, and E116 (red) at the ECL1 ECL6 interface in both inward- and outward-facing TMDs. The curcumin-sensitive and F178 (red), and and R166 (blue) at the ICL1 ICL4 interface are close to each other in both orientations. coordination [14,15]. The most common Zn 2+ ligands in proteins are the imidazole side chain of histidine, the sulfhydryl side chain of cysteine, and the acidic side chains of glutamate and aspartate. Their logk 1 (K 1, stability constants for Zn 2+ ) are 6.63, 9.8, 5.45, and 5.84, respectively. The distances between Zn 2+ and its surrounding ligands are well characterized as in structural Zn 2+ sites [14 18]. The most common Zn 2+ coordination geometry is tetrahedral. Generally, high-affinity Zn 2+ sites with a K i value ranging from 1 nm to 1 lm employ three or four amino acid side chains, while low-affinity Zn 2+ sites with a K i value ranging from 2 lm to 1 mm comprise one or two water molecules in addition to three or two side chains [15]. t least two side chains are necessary to stabilize Zn 2+ within a protein. Therefore, an endogenous or engineered Zn 2+ bridge can regulate gating of an ion channel by restricting a movement of side chains [19,2]. In this study, external Zn 2+ was without any effect on wild-type (WT) CFTR The FES Journal 283 (216) ª 216 Federation of European iochemical Societies 2459

3 Gating-coupled transmembrane helixes of CFTR G. Wang et al. and thus a /H mutation could be used to engineer a Zn 2+ bridge to disturb this ECL1 ECL6 interface, while a /H mutation near the ECL1 ECL6 interface was exploited to generate a control Zn 2+ bridge, and the gating effects of a Zn 2+ disturbance on the stimulatory ICL1/ICL4 R interaction were investigated. Our results showed that the engineered Zn 2+ bridge with either /H or /H may promote CFTR channel closure possibly by changing the conformation near the external channel gate. However, only the ECL1 Zn 2+ ECL6 bridge may affect the ICL1 ICL4 interface and then the gating-regulatory ICL1/ICL4 R interaction which is required for channel activation by PK and subsequent channel potentiation by curcumin. ccordingly, the gatingcoupled ECL1 ECL6 and ICL1 ICL4 interfaces may take an important role in PK-dependent channel activation and potentiation. Results /H mutation generates an inhibitive Zn 2+ site There are several endogenous cysteines, glutamates, and aspartates, and a histidine at the external side of WT CFTR. Therefore, we first examined the sensitivity of a WT CFTR-expressing oocyte to external Zn 2+ by determining the whole-oocyte current. fter WT CFTR was activated by exposing the oocyte to 1 lm isoproterenol (to activate the coexpressed b-adrenergic receptor) and 1 mm IMX (a PDE inhibitor), 2-mercaptoethonal (2-ME) was employed to promote the free state of a cysteine or to remove endogenous metal ions. fter 2-ME was washed out, introduction of Zn 2+ (5 lm to 5 mm) to the extracellular side was without any effect on the conductance and the reversal potential of WT CFTR (Figs 2,D and 3C). Therefore, WT CFTR was insensitive to extracellular Zn 2+ and could serve as a good negative control to examine the effect of an engineered Zn 2+ disturbance on channel gating. The positive charge of R334 in TM6, located in the outer vestibule of the conduction pore, increases the local concentration and thus promotes anion flow through the pore [21]. To promote the free-thiol state of the target cysteine, CFTR was pretreated with 2-ME after it was stimulated by 1 lm isoproterenol and 1 mm IMX. t steady activation 5 lm external Zn 2 inhibited its channel conductance by 54.47% in a voltage-independent manner and the inhibition was reversed by a washout (Fig. 2,D), suggesting that Zn 2+ acts at the extracellular surface of the protein. In addition, Zn 2+ had no effect on the reversal potential. In stark contrast, R334Q CFTR, like WT CFTR, was unaffected by external Zn 2 even at 5 mm concentrations (Fig. 3C). Furthermore, in agreement with the previous report [21], external MTSET + (1 lm) increased the channel conductance by about twofold by modifying the SH group of CFTR, while external MTSES (1 lm) inhibited it by about 84% (Fig. 3). However, pretreatment of both MTS reagents clearly abolished the Zn 2+ effect (Fig. 3). Finally, external Zn 2 (5 lm) also inhibited the conductance of R334H CFTR by about 38.8% in a voltage-independent manner but had no significant effect on the reversal potential, and the inhibition was reversed by a washout (Fig. 4,C). Thus, the single Cys- or His-substituted CFTR constructs at position 334 acquired a Zn 2+ - binding site and /H may be a Zn 2+ -liganding residue. It is noteworthy that the rectification ratio (RR) of R334H CFTR was significantly decreased possibly by a mutation-induced conformational change (Fig. 4,). Zn 2+ inhibition of /H CFTR needs two Zn 2+ - liganding protein residues Figure 2E indicates that the Zn 2+ inhibition of CFTR was enhanced with the increasing Zn 2+ concentration. The fit of the Zn 2+ dose response of CFTR with a Hill equation indicated K i of 31 lm, n of.7, and maximal inhibition of 85.5% (Fig. 2E), suggesting that a Zn 2+ block of CFTR can be described as a high-affinity and reversible binding event that inhibited most of channel activity. The kinetics of block was also consistent with the reversible binding of Zn 2+ at a single site (Fig. 2D). Zn 2+ also suppressed more conductance of R334H CFTR at high Zn 2+ concentration. similar Zn 2+ dose response of R334H CFTR was well fitted with a Hill plot with K i of 483 lm and n of 1 and maximal inhibition of 77% (Fig. 4D). ecause both potency and efficacy of Zn 2+ inhibition of R334H CFTR were less than those of CFTR, the maximal Zn 2+ inhibition of /H CFTR may reflect the maximal Zn 2+ -binding probability or the maximal fraction of Zn 2+ -inactivated channels. ecause at high concentration in the perfusate can act as a weak ligand for Zn 2+ (log K 1 =.43, log K 2 =.61, log K 3 =.53, log K 4 =.2) [22], we test whether binding to /H-Zn 2+ attenuates the channel conductance as it would introduce a net negative charge in the outer vestibule. To examine the effect on the Zn 2+ inhibition of /H CFTR, 246 The FES Journal 283 (216) ª 216 Federation of European iochemical Societies

4 G. Wang et al. Gating-coupled transmembrane helixes of CFTR 8 I, µ Control 5 um Zn2 2 V,mV Control 5 um Zn2 I, µ 3 V, mv WT D E % block of g 5 µm Zn 2+ Washout % block of g Zn2 Zn(NO3)2 t,min WT +NO [Zn 2+ ], mm C 11 I, µ NO3 5 5 um Zn(NO3)2 V, mv F #/Zn 2+ site 2 Hill plot [Zn 2+ ], mm Fig. 2. Zn 2+ inhibition of the conductance (g )ofaxenopus oocyte expressing WT and CFTR. The channel was stimulated by 1 lm isoproterenol and 1 mm IMX. 2-ME was used and then washed out before the addition of Zn 2+ to promote the free-thiol state of a cysteine. (, ) I V curves of () WT and () CFTR in the absence (black line) and presence (red line) of 5 lm Zn 2. The rectification ratio (RR) = (g E rev+25 mv )/g E rev-25 mv ). For WT, RR = (N = 3) and (N = 3) in the absence and presence of 5 lm Zn 2, respectively. For, RR =.93.1 (N = 6) and.89.1 (N = 6) in the absence and presence of 5 lm Zn 2, respectively. (C) I V curves of CFTR in the absence (black line) and presence (red line) of 5 lm Zn(NO 3 ) 2. RR =.93.1 (N = 5) and.91.1 (N = 5) in the absence and presence of 5 lm Zn(NO 3 ) 2, respectively. (D) Time courses of the conductance of WT and CFTR in response to 5 lm Zn 2+ and washout. (E) Zn 2+ dose responses in the presence of external and NO 3 in the perfusate. The solid lines are the Hill-fitting plots. In the presence of,%g max = 85.46, K i = lm, n =.7; in the absence of, %g max = 69.17, K i = lm, n =.94 (N = 3 4, P <.5, from unpaired Student s t-test). (F) The binding stoichiometry of #/-Zn 2+ as a function of [Zn 2+ ]. The solid line is the Hill plot (n = 1, K i = 33.8 lm, ( /Zn 2+ ) max = 2.1, ( / Zn 2+ ) min =.47). all in the perfusate was replaced with nonliganding NO 3 and the Zn(NO 3 ) 2 solution was applied. Figures 2C and 4 show that external NO 3 had only slight effects on the macroscopic I V plots of / H CFTR. It is interesting that 5 and 5 lm Zn 2+ still reversibly suppressed the channel conductance of and R334H CFTR, respectively, but to a less extent (by 21.4% and 15.%, respectively) and without any effect on the reversal potential (Figs 2C and 4). These findings demonstrated that may participate in the Zn-/H complex to exert an additional suppressive charge effect on the conductance. The fit of the Zn 2+ dose responses with the Hill equation gave K i of 11 lm, n of.9, and maximal inhibition of 69% for CFTR (Fig. 2E), but K i of 1.5 mm, n of 1, and maximal inhibition of 64% for R334H CFTR (Fig. 4D). Therefore, these -independent K i values suggest that at least another liganding residue is required for the Zn 2+ inhibition of /H CFTR because at least two side chains are necessary to stabilize Zn 2+ within a protein. Figures 2F and 4E demonstrate that the relative effect on channel conductance, which was normalized to the number of bound to one /H-Zn 2+ site using the maximal inhibition of the channel conductance by Zn (NO 3 ) 2 (assumed as the maximal Zn 2+ -binding probability), was weakened with an increasing Zn 2+ concentration. The resultant Hill-fitting plot indicates that at most may be bound to one /Hsite. It is interesting that the -dependent Zn 2+ The FES Journal 283 (216) ª 216 Federation of European iochemical Societies 2461

5 Gating-coupled transmembrane helixes of CFTR G. Wang et al. C g at V m = E rev (µs) g at V m = E rev (µs) µm Isoproterenol + 1 mm IMX 2-ME Zn 2+ 2-ME MTSET Zn 2+ 2-ME µm Isoproterenol + 1 mm IMX Time (min) Zn 2+ MTSES Zn Time (min) WT +MTSES +MTSET R334Q % Zn 2+ block of g Fig. 3. Premodification of CFTR with 1 lm MTSET + or MTSES suppressed Zn 2+ (5 lm) block. Exposure of an oocyte expressing CFTR to the channel-impermeant reagent, () stimulatory MTSET + or () inhibitory MTSES, blocked the Zn 2+ inhibition of CFTR after the channel was stimulated by 1 lm isoproterenol and 1 mm IMX and pretreated with 2-ME to promote the free-thiol state of the target cysteine. (C) Percentage Zn 2+ inhibition of the conductance of CFTR constructs (N = 3; P <.5 versus WT CFTR, from unpaired Student s t-test). Zn 2+ inhibition of CFTR was similar to that of R334H CFTR although the Zn 2+ -binding affinity for R334H CFTR was lower than that for CFTR (Figs 2E,F and 4D,E). Thus, the weakened -dependent Zn 2+ inhibition of /H CFTR at the higher Zn 2+ concentration may not be due to a decrease in the number of bound to Zn 2+ -/ H but to Zn 2+ binding-induced channel closure. is a liganding residue for the Zn 2+ inhibition of CFTR ecause at most may be bound to Zn 2+ - /H (Figs 2F and 4E), another liganding residue is required for the tetrahedral structure of the inhibitive Zn 2+ coordination. s at the extracellular end of TM12 is close to in the inward-facing conformation (Fig. 1), we test if is a liganding residue for the Zn 2+ inhibition of CFTR. Figure 5 shows that DTT only slightly increased the conductance of / CFTR after it was stimulated by 1 lm external isoproterenol and 1 mm IMX. fter DTT was washed out, this double mutant was insensitive to 5 lm Zn 2 (Fig. 5). ccordingly, we propose as a ligand for the Zn 2+ inhibition of CFTR. ecause the putative -Zn 2+ - bridge is near the ECL1 ECL6 interface, it can act as a control Zn 2+ disturbance. /H mutation also produces an inhibitive Zn 2+ site T1122 is located at the extracellular end of TM11 (Fig. 1). Our data demonstrated that after CFTR was stimulated by 1 lm isoproterenol and 1mM IMX and pretreated with 2-ME, external Zn 2 (5 lm) was without any effect on the reversal potential of CFTR but inhibited its conductance by about 7.7% (Fig. 6), and the inhibition was reversed by a washout or suppressed by pretreatment with inhibitory MTSET + and MTSES (Fig. 7). In contrast, the conductance of T1122V CFTR was not altered by 5 lm Zn 2+ (Fig. 7C). Similarly, Zn 2 (5 lm) also decreased the channel conductance of T1122H CFTR by about 55.2% (Fig. 8). Thus, the /H mutation generated an inhibitive Zn 2+ site at the ECL1 ECL6 interface and /H may serve as a Zn 2+ -liganding residue. Zn 2+ inhibition of /H CFTR also needs at least two Zn 2+ -liganding protein residues Once in the perfusate was replaced with NO 3, application of 5 lm external Zn(NO 3 ) 2 also inhibited the conductance of both and T122H CFTR by about 62.1% and 56.3%, respectively (Figs 6 and 8). lthough T122C/H CFTR exhibited dose-dependent Zn 2+ inhibition, it is interesting that the Zn 2+ inhibition of and T1122H CFTR was weakly - dependent and -independent, respectively (Figs 6C and 8C). The Hill equation-fitted Zn 2+ dose responses of CFTR indicated small changes in K i from 7.3 to 18.7 lm, n from.8 to.9, and maximal inhibition from 85% to 88% upon substitution of with external NO 3 in the perfusate (Fig. 6C). However, the Hill equation-fitted Zn 2+ dose responses of T1122H CFTR showed constant K i of 3 lm, n of 1, and maximal inhibition of about 87% no matter whether conducting was present or not (Fig. 8C) The FES Journal 283 (216) ª 216 Federation of European iochemical Societies

6 G. Wang et al. Gating-coupled transmembrane helixes of CFTR 5 I, µ.5 mm Zn2 1 V, mv R334H 7 3 I, µ NO3.5 mm Zn(NO3)2 V,mV R334H 9 C D E.5 mm Zn 2+ 5 t, min 6 8 WT R334H+NO3 R334H+ Washsout % block of g #/Zn 2+ site % block of g [Zn 2+ ], mm Zn2 Zn(NO3)2 R334H R334H Hill plot [Zn 2+ ], mm Fig. 4. Zn 2+ inhibition of the conductance of a Xenopus oocyte expressing R334H CFTR. The channel was stimulated by 1 lm isoproterenol and 1 mm IMX. () I V curves of R334H CFTR in the absence (black line) and presence (red line) of 5 lm Zn 2. RR =.79.1 (N = 6) and.75.1 (N = 6) in the absence and presence of 5 lm Zn 2, respectively. () I V curves of R334H CFTR in the absence (black line) and presence (red line) of 5 lm Zn(NO 3 ) 2.RR=.73.2 (N = 6) and.72.1 (N = 6) in the absence and presence of 5 lm Zn(NO 3 ) 2, respectively. (C) Time courses of the conductance of WT and R334H CFTR in response to 5 lm Zn 2+ and washout. (D) Zn 2+ dose responses in the presence of external and NO 3 in the perfusate. The solid lines are the Hill plots. In the presence of, %g max = 76.88, K i = lm, n = 1; in the absence of, %g max = 64.28, K i = mm, n = 1(N = 3, P <.5 versus untreated, from unpaired Student s t-test). (E) The binding stoichiometry of #/R334H-Zn 2+ as a function of [Zn 2+ ]. The solid line is the Hill-fitting plot (n = 1, K i = 267 lm, ( /Zn 2+ ) max = 2.4, ( /Zn 2+ ) min =.52). Thus, the Zn 2+ block of /H CFTR can be described as a reversible binding event that diminished channel conductance. Their higher apparent affinities for Zn 2+ suggest at least another protein residue is necessary for Zn 2+ inhibition. Figure 6D shows that at most 2.4 may be bound to one -Zn 2+ site and the effect was still decreased with an increasing Zn 2+ concentration and finally disappeared. Thus, Zn 2+ binding to may promote channel closure or the number of liganding residues for the Zn 2+ inhibition of may be increased at the higher Zn 2+ concentration. In contrast, because the Zn 2+ inhibition of T1122H CFTR was -independent (Fig. 8C), no may be bound to T1122H-Zn 2+ or the channel may be closed by Zn 2+ so that the effect may not be observed. n ECL1-Zn 2+ -ECL6 bridge primarily inhibits CFTR activity ecause /H at ECL6 is close to D11 at ECL1 of inward-facing TMDs (Fig. 1), we hypothesize that a primary Zn 2+ bridge between ECL1 and ECL6 inhibit /H CFTR activity. To examine this hypothesis, we determined the Zn 2+ sensitivity of /H CFTR constructs. Figure 9 shows that DTT increased the conductance of /D11 CFTR after it was stimulated by 1 lm external isoproterenol and 1 mm IMX. fter DTT was washed out, this double mutant was more insensitive to 1 lm Zn 2 than CFTR. Thus, D11 may be a primary liganding residue for the Zn 2+ inhibition of CFTR. In other words, a primary -Zn 2+ -D11 bridge at the ECL1 ECL6 interface may inhibit channel activity and then allow binding to further decrease the channel conductance as a negative charge at the outer vestibule. s the -dependent Zn 2+ inhibition of CFTR was significantly weakened at the higher Zn 2+ concentration (Fig. 6C), we also examined the effect of 5 lm Zn 2+ on /D11 CFTR. Figure 9 indicates that this double mutant was still sensitive to 5 lm Zn 2+ although the efficacy was significantly decreased. ccordingly, additional liganding residues may bind to the -Zn 2+ -D11 The FES Journal 283 (216) ª 216 Federation of European iochemical Societies 2463

7 Gating-coupled transmembrane helixes of CFTR G. Wang et al. 1 µm Isoproterenol + 1 mm IMX g at V m =E rev (µs) 8 DTT 5 µm Zn 2+ Inh / / Time (min) % Zn 2+ block of g Fig. 5. is a liganding residue for the Zn 2+ inhibition of CFTR. The channel was stimulated by 1 lm isoproterenol and 1 mm IMX. DTT was used and then washed out before the addition of Zn 2+ to promote the free-thiol state of a cysteine. () Time course of the conductance of / CFTR in response to 5 lm Zn 2+ and washout. Inh172 is a CFTR-specific blocker. () Percentage Zn 2+ (5 lm) inhibition of the conductance of CFTR constructs (N = 4 8; P <.5 versus CFTR, from unpaired Student s t- test). bridge at a high Zn 2+ concentration. s T1122H CFTR exhibited -independent Zn 2+ inhibition, we further examined the Zn 2+ sensitivity of T1122H/ D11 CFTR. Figure 9 shows that 5 lm Zn 2+ still inhibited this double mutant by about 23.5%, less than that of T1122H CFTR (Fig. 8C). In sharp contrast, the T1122H/E116 or T1122H/E115 CFTR failed to alter the sensitivity to 5 lm Zn 2+. Therefore, the binding of other liganding residues such as to the T1122H-Zn 2+ -D11 bridge may result in no - dependent Zn 2+ inhibition of T1122H CFTR. ecause T1122H/Y19F was also insensitive to 1 or 5 lm Zn 2+ (Fig. 9), we propose that the OH group of Y19 may also bind to the T1122H-Zn 2+ -D11 bridge to increase the inhibitory efficacy. In any way, the primary inhibitory /H-Zn 2+ -D11 bridge at the ECL1 ECL6 interface may allow us to test the gating coupling between the ECL1 ECL6 interface and the ICL1 ICL4 interface. Zn 2+ inhibition of and /H CFTR is state-dependent ecause the -dependent Zn 2+ inhibition of /H or CFTR was attenuated with the increasing Zn 2+ dose (Figs 2F, 4E and 6D), the Zn 2+ inhibition of /H or /H CFTR may result from channel closure. In this case, more Zn 2+ inhibition would be observed in a closed state. To test this hypothesis, we employed isoproterenol to determine the Zn 2+ inhibition of or T1122H CFTR in the absence and presence of 1 mm IMX, which causes low and high activation of CFTR, respectively (Fig. 9). Figure 1 indicates that in the presence of 1 lm isoproterenol and 1 mm IMX, Zn 2+ (1 lm) only inhibited the conductance of or T1122H CFTR by 27.1% and 18.9%, respectively. However, removal of IMX more than doubled the Zn 2+ (1 lm) inhibition of or T1122H CFTR. Thus, these data are in agreement with the proposal that Zn 2+ binding to /H or /H CFTR may close the channel. To further support this proposal, Zn 2+ (5 lm) was introduced in the pipette solution before 1.5 mm TP and unitsml 1 PK were applied to the cytoplasmic side of inside-out patch to activate CFTR constructs. Figure 11 shows that WT CFTR was activated with 1.5 mm TP and 48 unitsml 1 PK regardless of the presence of Zn 2+ at the external side (Fig. 11). In contrast, once CFTR was activated with 2464 The FES Journal 283 (216) ª 216 Federation of European iochemical Societies

8 G. Wang et al. Gating-coupled transmembrane helixes of CFTR 5 um Zn NO3 5 um Zn(NO3) I, µ I, µ V, mv V, mv C % block of g D - #/Zn 2+ site 8 4 Zn2 Zn(NO3) [Zn 2+ ], mm 2 Hill plot [Zn 2+ ], mm Fig. 6. Zn 2+ inhibition of the conductance (g ) of a Xenopus oocyte expressing CFTR. The channel was stimulated by 1 lm isoproterenol and 1 mm IMX. 2-ME was used and then washed out before the addition of Zn 2+ to promote the free-thiol state of a cysteine. () I V curves of CFTR in the absence (black line) and presence (red line) of 5 lm Zn 2.RR= (N = 6) and (N = 6) in the absence and presence 5 lm Zn 2, respectively. () I V curves of CFTR in the absence (black line) and presence (red line) of 5 lm Zn(NO 3 ) 2.RR= (N = 3) and (N = 3) in the absence and presence of 5 lm Zn(NO 3 ) 2, respectively. (C) Zn 2+ dose responses in the presence of external and NO 3 in the perfusate. The solid lines are the Hill-fitting plots. In the presence of, %g max = 84.76, K i = lm, n =.85; in the absence of, %g max = 88.44, K i = lm, n =.93 (N = 3 4, P <.5 versus untreated, from unpaired Student s t-test). (D) The binding stoichiometry of #/-Zn 2+ as a function of [Zn 2+ ]. The solid line is the Hill plot (n = 1, K i = 4 lm, ( /Zn 2+ ) max = 2.4, ( /Zn 2+ ) min = ). 1.5 mm TP and 48 unitsml 1 PK, and upregulated with 2 lm N-ethylmaleimide (NEM) [4], the current noise was also increased with activation. However, the application of 5 lm Zn 2+ to the extracellular side of CFTR completely suppressed the TP/PK-induced increases in the current amplitude and noise (Fig. 11,D). similar result was observed with CFTR (Fig. 11C,D). ecause the pipette solution contained 1 mm EGT (stability constant for Zn 2+, log K = 12.6 at 2 C) [23], free Zn 2+ concentration smaller than 1 lm was enough to completely inhibit channel activity in the resting state. Thus, the Zn 2+ affinity for or CFTR in the resting state may be higher than that (K i = lm) in the activated state (Figs 2E and 6C). Taken together, the external /H-Zn 2+ - or /H-Zn 2+ -D11 bridge may promote CFTR closure. Internal curcumin reverses the Zn 2+ inhibition of not but CFTR ecause the -Zn 2+ -D11 bridge and the -Zn 2+ - bridge reside at and near the ECL1 ECL6 interface, respectively, and the ECL1 ECL6 interface is linked with the curcumin-sensitive ICL1 ICL4 interface via TM2 and TM11, we test if the former can prevent internal curcumin potentiation via the gating coupling between two interfaces while the latter cannot. Figure 11 shows that internal curcumin potentiated activity by about 2% in the absence of Zn 2+ at the extracellular side and the total CFTR-mediated current was finally blocked by Inh172 (1 lm) or glibenclamide (2 lm). In the presence of Zn 2+ (< 1 lm) at the extracellular side, internal curcumin slowly reversed the Zn 2+ inhibition of CFTR and increased the current noise (Fig. 11,D). oth the curcumin-reversed current and the curcumin-increased current noise were finally inhibited by Inh172 (1 lm) and glibenclamide (2 lm). In sharp contrast, internal curcumin only similarly increased CFTR activity in the absence of extracellular Zn 2+ (Fig. 11C) but failed to significantly reverse the Zn 2+ inhibition or to increase the current noise of CFTR in the presence of external Zn 2+ (< 1 lm) (Fig. 11C,D). The CFTR-medicated current was finally blocked by Inh172 (1 lm) or glibenclamide (2 lm). Therefore, the ECL1-Zn 2+ - ECL6 bridge may disturb the ICL1 ICL6 interface and thus prevent the stimulatory ICL1/ICL4 R interactions. The FES Journal 283 (216) ª 216 Federation of European iochemical Societies 2465

9 Gating-coupled transmembrane helixes of CFTR G. Wang et al. C g at V m =E rev (µs) g at V m =E rev ( µs ) WT +MTSES +MTSET T1122V Discussion 1 µm Isoproterenol + 1 mm IMX 2-ME 1 µm Isoproterenol + 1 mm IMX 2-ME Zn 2+ MTSET Zn Time (min) Zn 2+ MTSES Zn Time (min) % Zn 2+ block of g Fig. 7. Premodification of CFTR with 1 lm MTSET + or MTSES suppressed Zn 2 (5 lm) block. The channel was stimulated by 1 lm isoproterenol and 1 mm IMX and pretreated with 2-ME to promote the free-thiol state of the target cysteine. Exposure of an oocyte expressing CFTR to the channelimpermeant reagent () MTSET + or () MTSES decreased the channel conductance and blocked the Zn 2+ inhibition of CFTR. (C) Percentage Zn 2+ inhibition of the conductance of CFTR constructs (N = 3 4; P <.5 versus WT CFTR, from unpaired Student s t-test). Gating rearrangements of the CFTR pore is controlled by several domain domain interactions. Once the R domain is phosphorylated by PK to release its regulatory extension from ND1 and its C-terminus from ICL3, the highly conserved ICL1/ICL4 ND1 and ICL2/ICL3 ND2 swapping interactions in both inward- and outward -facing TMDs facilitate channel opening by TP binding-induced ND1 ND2 dimerization [9 13,24 27]. However, the released R domain from ICL3 can bind to the highly conserved ICL1 ICL4 interface for optimal channel opening by promoting the tight tetrahelix bundle-induced inwardto- outward reorientation of TMDs and the TP binding-induced ND1 ND2 dimerization [3 8]. C 5 um Zn2 7 V, mv NO I, µ T1122H I, µ 5 um Zn(NO3)2 V, mv % block of g 8 4 Zn2 Zn(NO3)2 T1122H T1122H [Zn 2+ ], mm Fig. 8. Zn 2+ inhibition of the conductance (g )ofaxenopus oocyte expressing T1122H CFTR. The channel was stimulated by 1 lm isoproterenol and 1 mm IMX. () I V curves of T1122H CFTR in the absence (black line) and presence (red line) of 5 lm Zn 2. RR = (N = 3) and (N = 3) in the absence and presence of 5 lm Zn 2 ; respectively. () I V curves of T1122H CFTR in the absence (black line) and presence (red line) of 5 lm Zn (NO 3 ) 2. RR = (N = 4) and (N = 4) in the absence and presence of 5 lm Zn(NO3) 2, respectively. (C) Zn 2+ dose responses in the presence of external and NO 3 in the perfusate. The solid lines are the Hill-fitting plots. In the presence of,%g max = 86.42, K i = lm, n = 1; in the absence of,%g max = 89.4, K i = lm, n = 1(N = 3 6). Sufficient Fe 3+ binding to the ICL3 R interface can prevent the stimulatory ICL1/ICL4 R interaction and the resultant curcumin or NPP-M potentiation [7,8]. In this study, the externally engineered / H-Zn 2+ - bridge near the ECL1 ECL6 interface may directly inactivate the channel possibly by stabilizing the inward-facing TMDs while the externally engineered /H-Zn 2+ -D11 bridge at the ECL The FES Journal 283 (216) ª 216 Federation of European iochemical Societies

10 G. Wang et al. Gating-coupled transmembrane helixes of CFTR Fig. 9. Identification of liganding residues for the Zn 2+ inhibition of /H CFTR. The channel was stimulated by 1 lm isoproterenol and 1mM IMX. DTT was used and then washed out before the addition of Zn 2+ to promote the free-thiol state of a cysteine. () Time course of the conductance of /D11 and T1122H/Y19F and T1122H/E115 CFTR in response to 1 or 5 lm Zn 2+ and washout. Inh172 is a CFTR-specific blocker. () Percentage Zn 2+ inhibition of the conductance of /H CFTR constructs (N = 3 6; P <.5 versus or T1122H, from unpaired Student s t-test). ECL6 interface may inactivate the channel and prohibit internal curcumin potentiation also by indirectly affecting the PK- and curcumin-sensitive ICL1/ICL4 interface. Thus, the ECL1 ECL6 interface, gatingcoupled with the ICL1 ICL4 interface via TM2 and TM11, may regulate PK-dependent channel gating and potentiation. Zn 2+ binding to /H or /H CFTR inactivates the channel from both open and closed states CFTR is a channel. can bind to Zn 2+ to form [Zn 4 ] 2 in the presence of a large amount of in the perfusate. However, the Zn 2+ inhibition of / H or /H CFTR is not ascribed to a block of the channel pore by [Zn 4 ] for three reasons. First, external Zn 2 (5 mm) did not alter the channel activity of WT or R334Q or T1122V CFTR (Figs 2,D, 3C and 7C). Second, the Zn 2+ inhibition of both and CFTR was suppressed by modification of or with the thiol-specific MTS reagents or by missense alanine substitutions of nearby liganding residues (Figs 3, 5, 7 and 9). Third, the Zn 2+ inhibition of /H or /H CFTR was also observed when was replaced with nonliganding NO 3 in the perfusate (Figs 2, 4, 6 and 8). However, the different Zn 2+ inhibition of /H or CFTR in the presence of external and NO 3 is not due to the allosteric regulation of the channel pore by external NO 3 because external NO 3 had no significant effect on the channel conductance of /H or /H (Figs 2C, 4, 6 and 8). More importantly, no different Zn 2+ inhibition of T1122H CFTR was found upon replacement of with NO 3 (Fig. 8C). These observations are consistent with the finding that external NO 3 only slightly affects DR CFTR gating [28]. Therefore, the effect on the Zn 2+ inhibition of /H or CFTR may result from the decrease in the single channel The FES Journal 283 (216) ª 216 Federation of European iochemical Societies 2467

11 Gating-coupled transmembrane helixes of CFTR G. Wang et al. C D Fig. 1. State-dependent Zn 2+ inhibition of and T1122H CFTR. (, C) Time courses of the Zn 2+ inhibition of () CFTR and (C) T1122H CFTR after the channel was stimulated by 1 lm isoproterenol with (top) or without (bottom) 1 mm IMX. DTT was used and then washed out before the addition of Zn 2+ to promote the free-thiol state of a cysteine. ( & D) Percentage Zn 2+ (1 lm) inhibition of () and (D) T1122H in the presence of 1 lm isoproterenol (N = 3 8; P <.5 versus additional IMX, from unpaired Student s t-test). conductance when binding to the /H-Zn 2+ - or -Zn 2+ -D11 bridge introduced a net negative charge at the outer vestibule of CFTR (Figs 2D,E, 4C,D, 6C,D, 12,). To this end, not only R334 but also T1122 may be vestibule-lining. Supporting this proposal, both T1115C and S1118C CFTR exhibit state- and side-dependent activity inhibition by MTSES [29]. It is interesting that MTSET + and MTSES increased and decreased the conductance of CFTR, respectively (Fig. 3), consistent with the previous report about vestibule-lining R334 [21,3]. However, both MTSET + and MTSES inhibited CFTR activity (Fig. 7). The MTSET + -induced inhibition in the conductance of CFTR may be due to a possible inhibitive electrostatic attraction between the positively charged MTSET + abduct and a nearby negatively charged residue such as D11 (Figs 9 and 12). It should be noteworthy that and D11 may not line the outer vestibule of CFTR because their modification with MTS reagents had no effect on the channel conductance [31,32]. However, Y19 has been reported as vestibule-lining [33]. On the other hand, when the concentration of Zn 2 was increased, more should be bound to the /H-Zn 2+ - or D11-Zn 2+ - bridge so that the effect on their Zn 2+ inhibition would be enhanced with the increasing concentration of Zn 2. However, /H and CFTR constructs exhibited the weakened dependence of Zn 2+ inhibition with the increasing Zn 2 concentration (Figs 2F, 4E and 6C). Therefore, the channel open probability may be decreased by the external -Zn 2+ -/H or D11-Zn 2+ - bridge so that some effects on the conductance may not be observed when the channel is closed (Fig. 12,). This proposal is supported by the findings that external Zn 2+ more dramatically inhibited the activity of or /H CFTR in the resting state than 2468 The FES Journal 283 (216) ª 216 Federation of European iochemical Societies

12 G. Wang et al. Gating-coupled transmembrane helixes of CFTR TP PK (36 U ml 1) 1 µm Inh172 TP PK (U ml 1) 1 µm Mixing Inh172 WT 1 s 2 p 2 p 2 min WT + 5 µm Zn 2+ 2 µm Glibenclamide 2 µm PK (U ml 1) TP NEM µm Curcumin PK (U ml 1) TP µm Curcumin 1 µm Inh172 4 min 5 p 1 µm Inh172 2 µm Glibenclamide + 5 µm Zn 2+ 2 min 5 p 2 µm Glibenclamide C PK (U ml 1) 5 µm TP µm Curcumin NEM 2 µm TP 6 PK 6 (U ml 1) NEM 5 µm Curcumin 4 min 2 p 2 µm Glibenclamide 1 µm Inh µm Zn 2+ 1 µm Inh172 2 min 2 p D I TP/PK /I Curcumin, % 2 4 N TP/PK /N Control N Curcumin /N Control Fig. 11. Effects of external Zn 2+ (< 5 lm) on channel activation by TP/PK and potentiation by curcumin. In the absence (left) and presence (right) of Zn 2+ at the extracellular side, inside-out macroscopic currents of () WT, () or (C) CFTR across an excised HEK-293T patch were treated with 1.5 mm MgTP and unitsml 1 PK and 5 lm curcumin. The holding potential was 6 mv. Inh172 (1 lm) or glibenclamide (2 lm) was used to block the total CFTR-mediated current. rrows indicate the time when each reagent was added. (D) Left: relative currents evoked by TP and PK. The control was the curcumin-potentiated current (N = 3 5; P <.1 versus no Zn 2+, from unpaired Student s t-test). Middle: the TP/PK-induced changes in current noise ratios of and in the absence and presence of external Zn 2+ (< 5 lm) (N = 3 5; P <.5 versus no Zn 2+, from unpaired Student s t-test). Right: the curcumin-induced changes in current noise ratios of and in the absence and presence of external Zn 2+ (< 5 lm) (N = 3 5; P <.5 versus no Zn 2+, from unpaired Student s t-test). The FES Journal 283 (216) ª 216 Federation of European iochemical Societies 2469

13 Gating-coupled transmembrane helixes of CFTR G. Wang et al. Partially Inactivated C334 D11 T1122 C334 R166 Open osed Inactivated F178 F178 C334 R166 C334 F178 R166 R334 D11 C1122 D11 R166 F178 C1122 R166 D11 F178 C1122 R166 D11 C1122 F178 C334 D11 T1122 C334 9 o 9 o 9 o C R334 D11 C1122 Y19 D11 H1122 D11 D11 Y19 R166 9 o F178 C1122 H1122 R166 D11 D11 Y19 F178 9 o C1122 H1122 R166 9 o D11 C1122 D11 H1122 Y19 F178 Y19 D11 H o D11 H1122 Y19 D11 Y19 9 o H o D11 Y19 H1122 Fig. 12. Tentative molecular mechanisms of the Zn 2+ inhibition of (), (), and (C) T1122H CFTR. The highly conserved ICL1 ICL4 interface, which may be gating-coupled with the ECL1 ECL4 interface via TM2 (cyan) and TM11 (yellow), serves as an active site for the stimulatory binding of the phosphorylated R domain (not shown) [8].,,, and D11/Y19 are disperse in the open state and cluster together in the closed state. Either () the -Zn 2+ - bridge or () the -Zn 2+ -D11 bridge or (C) the T1122H/-Zn 2+ -D11/Y19 bridge may inactivate the channel from the closed state or partially inactivate the channel from the open state. The open and partially inactivated states are conducting while the closed and inactivated states are nonconducting. binding to either () the -Zn 2+ - bridge or () the -Zn 2+ -D11 bridge in the partially inactivated state may further decrease the channel conductance as a net negative charge at the outer vestibule (blue circle) of CFTR. The open states were based on McjD while the closed and inactivated states were based on TM287 TM288 [9,39]. The curcumin-sensitive ICL1/ICL4 interface may not be affected by () the -Zn 2+ - bridge. However, (, C) the /H-Zn 2+ -D11 bridge in the closed state may induce a conformational change at both ECL1 ECL6 and ICL1 ICL4 interfaces and thus prevent the R domain from binding to the ICL1 ICL4 interface for channel opening. in the activated state (Figs 1 and 11), and that external Zn 2+ binding to or CFTR suppressed the TP/PK-induced increase in the current noise (Fig. 11 D). ltogether, the /H-Zn 2+ - or D11-Zn 2+ -/H bridge may promote channel closure. s external Zn 2+ binding to 247 The FES Journal 283 (216) ª 216 Federation of European iochemical Societies

14 G. Wang et al. Gating-coupled transmembrane helixes of CFTR or CFTR in the resting state completely prevented channel activation but the maximal Zn 2+ inhibition of or in the activated state was smaller than 1% (Figs 2E, 6C, and 11 D), we propose that external /H-Zn 2+ - and D11- Zn 2+ -/H bridges may completely inactivate the channel from the closed state but may partially inactivate the channel from the open state (Fig. 12). Thus, the total -independent Zn 2+ inhibition of the activated /H or /H CFTR channel may reflect a complicated overlap of these four states (Figs 2E, 4D, 6C, 8C and 12). s the partially inactivated channel is still conducting, binding to the Zn 2+ bridge in this state may further decrease the permeation as a net negative charge in the outer vestibule (Figs 2E, 4D, 6C and 12,). ecause no -dependent Zn 2+ inhibition was found for /H CFTR at the higher Zn 2+ concentration, Y19 and at the ECL1 ECL6 interface may finally bind to the /H-Zn 2+ -D11 bridge (Figs 6C, 8C, 9 and 12C). The ECL1 ECL6 interface is gating coupled to the ICL1/ICL4 R interaction The channel pore of CFTR is gated by the reorientation between the inward- and outward -facing TMDs. lthough either inward- or outward -facing TMDs of CFTR have been crystalized no matter whether TP is bound or free at the ND1 ND2 interface [34,35], recent functional studies demonstrated that ND1 and ND2 are separated from each other only in the closed state [36]. Furthermore, the channel gate was found between amino acid residues 337 and 344 along TM6, which may also serve as the selectivity filter for CFTR [3,31,37]. ecause /H at the extracellular end of pore-lining TM6 [21,3] and at the extracellular end of pore-lining TM12 [31] are separated from each other in the outward -facing TMDs but close to each other in the inward-facing TMDs (Figs 1 and 12), the /H-Zn 2+ - bridge may promote the outward -to-inward reorientation of TMDs for channel closure (Figs 1 12). In addition, not TP/PK but internal curcumin still reversed the Zn 2+ inhibition of CFTR (Fig. 11,D). Thus, the /H-Zn 2+ - bridge near the ECL1 ECL6 interface may not affect the ICL1 ICL4 interface. In this case, the / H-Zn 2+ - bridge may only partially inactivate the channel once the R domain binds to the ICL1/ICL4 R interface for channel opening (Figs 2E,F and 4D,E). On the other hand, once Zn 2+ bridges /H with in the inward-facing closed state, it may be difficult for the R domain to bind to the ICL1 ICL4 interface unless curcumin stabilizes the ICL1/ICL4 R interaction (Fig. 11,D) and thus promotes channel opening possibly by separating the ECL1 ECL6 interface from the /H-Zn 2+ - bridge (Fig. 12). In the case of the Zn 2+ inhibition of /H CFTR, although the ECL1 ECL6 interface is highly conserved in the Sav1866-based outward-facing CFTR homology model (Fig. 1), this model is inconsistent with pore-lining TM1 and TM11 [29,33,38]. In this regard, Corradi et al. [39] proposed a McjD-based CFTR homology model as the open state to include TM1 as pore-lining. ecause this model indicates that D11 and Y19 are disperse from /H (Fig. 12,C), it is possible that a movement between ECL1 and ECL6 may be required for channel activation and thus the restriction of the movement by the Zn 2+ bridge at the interface may inhibit /H CFTR activity. Moreover, as the Zn 2+ inhibition of CFTR was not reversed by TP/ PK and internal curcumin (Fig. 11C,D), we propose that /H may be still separated from D11 and Y19 in the closed state but external Zn 2+ binding to /H and D11 and Y19 may pull them together so as to inactivate the channel by disturbing the gatingregulatory ICL1 ICL4 interface and thus preventing the stimulatory binding of the R domain or curcumin to this interface (Figs 12,C and 13). For example, the highly conserved and F178, or and R166 in both TMDs orientations facilitate the stimulatory binding of the R domain and curcumin to the ICL1 ICL4 interface [8]. However, external Zn 2+ binding to the ECL1 ECL6 may induce a relative movement at the ICL1 ICL4 interface so as to stop curcumin binding (Figs 12,C and 13). Thus, the /H-Zn 2+ -D11 bridge may not only induce a direct gating rearrangement at the ECL1 ECL6 interface but also disturb the ICL1 ICL4 interface which is required for optimal channel opening and curcumin potentiation. Experimental procedures Molecular biology ll the mutants based on WT human CFTR were produced using the luescript CFTR cdn templates and the QuikChangeTM site-directed mutagenesis kit (Stratagene, San Diego, C, US) and confirmed by automated sequencing. and CFTR mutants were also subcloned into the pcdn3 mammalian expression vector (Invitrogen, Carlsbad, C, US). The CFTR crn for Xenopus oocyte injection were synthesized using the in vitro transcription kit, mmessage mmachine (mbion Inc., ustin, TX, US). The transcription products were purified, and the quality and quantity of the transcripts were assessed on the agarose gel. The FES Journal 283 (216) ª 216 Federation of European iochemical Societies 2471

15 Gating-coupled transmembrane helixes of CFTR G. Wang et al. Fig. 13. The tentative molecular mechanism of the Zn 2+ -dependent curcumin potentiation of CFTR. The ECL1 ECL4 interface may be gating-coupled with the highly conserved ICL1 ICL4 interface via TM2 (cyan) and TM11 (yellow). The specific binding of the phosphorylated R domain to the ICL1 ICL4 interface is required for channel opening by PK and potentiation by curcumin [8]. However, the binding of Zn 2+ to the ECL1/ECL4 interface induces a conformational change at both interfaces so that the R domain cannot bind to the ICL1/ICL4 interface for channel opening and curcumin potentiation. Cell culture and transfection Human embryonic kidney (HEK)-293T cells were cultured in Fe 3+ -containing Dulbecco s modified Eagle s medium (Mediatech, Manassas, V, US) supplemented with 1% fetal bovine serum and 1 mm penicillin/streptomycin and were transiently transfected with and CFTR mutants cdn using the Lipofectamine transfection kit (Invitrogen). For patch-clamp recordings, the transfected cells were transferred to plastic coverslips and used 1 4 days postseeding. Preparation of Xenopus oocytes Oocytes were harvested from anesthetized Xenopus laevis toads and manually defolliculated after incubation in a 2mgmL 1 collagenase-containing bath for h. The harvesting of Xenopus oocytes was conducted following national guidelines for animal experiments. The oocytes were kept in a 18 C incubator overnight in modified arth s solution (MSH) containing 88 mm Na, 1 mm K,.82 mm MgSO 4,.33 mm Ca(NO 3 ) 2 4H 2 O,.41 mm Ca 2 2H 2 O, 2.4 mm NaHCO 3, 5 mm HEPES-Na, 5 mm HEPES-H +, and 15 mgl 1 gentamicin or 25 mgl 1 amikicin. On the second day, the oocytes were injected with crn encoding WT CFTR and mutants, each in 5 nl sterile water with or without the crn encoding human b 2 -adrenergic receptor. Generally, oocytes were incubated at 18 C for 3 7 days before electrophysiological recording. Electrophysiological recordings Individual oocytes were continuously perfused (3 mlmin 1 ) at room temperature with Frog Ringer solution containing 98 mm Na, 2 mm K, 1 mm Mg 2, 1.8 mm Ca 2, 2.5 mm HEPES-Na, and 2.5 mm HEPES- H +, ph7.4. The -free solution contained: 98 mm NaNO 3,2mM KNO 3,1mM Mg(NO 3 ) 2, 1.8 mm Ca(NO 3 ) 2, 2.5 mm HEPES-Na, and 2.5 mm HEPES-H +, ph7.4. The two-electrode voltage clamp system (TEVC-2; Dagan Corporation, Minneapolis, MN, US) was used to measure the macroscopic currents. Oocytes were normally kept under open circuit condition in experimental chambers. Typically, the membrane potential was ramped from 12 to + 6 mv over a period of 1.8 s in order to acquire the macroscopic I V plots. CFTR was activated by 1 lm isoproterenol in the absence or presence of 1 mm 3-isobutylmethylxanthine (IMX, a PDE inhibitor) until a steady current was obtained. ll the CFTR constructs were pretreated with 2-mercaptoethanol (2-ME) or DTT to remove potential disulfide bonds or metal contamination. MTS reagents (Toronto Research Chemicals, Toronto, Canada) were stored at 2 C and were dissolved in a bath solution within 3 s prior to each experiment. Generally, positively charged MTSET + (1 lm) and negatively charged MTSES (1 lm) were applied for 4 1 min until modification reached a steady state. Zn 2 or Zn(NO 3 ) 2 solution was applied to the perfusion solution to block the conductance of resting or activated CFTR. ecause the stock Zn 2 solution was prepared by adding several doses of a Na solution, [Zn 4 ] 2 was majorly applied in the presence of. Generally, the inhibition of CFTR conductance by Zn 2+ was reversed easily. Patch-clamp analysis HEK-293T cells expressing or CFTR channels were recorded in the inside-out configuration using an xon 2 amplifier (xon Instruments, Foster City, C, US). CFTR currents were recorded in symmetrical solutions containing 14 mm N-methyl-D-glutamine chloride, 3mM Mg 2,1mMEGT, and 1 mm TES (ph7.3). The resulting resistance of borosilicate patch pipette was 3 4MΩin the bath solution. Inside-out macroscopic currents were evoked by 1.5 mm MgTP and unitsml The FES Journal 283 (216) ª 216 Federation of European iochemical Societies