Inhibition of protein kinase CK2 closes the CFTR Cl channel, but has no effect on the cystic fibrosis mutant deltaF508-CFTR

Abstract

Background: Deletion of phenylalanine-508 (DeltaF508) from the first nucleotide-binding domain (NBD1) in the wild-type cystic fibrosis (CF) transmembrane-conductance regulator (wtCFTR) causes CF. However, the mechanistic relationship between DeltaF508-CFTR and the diversity of CF disease is unexplained. The surface location of F508 on NBD1 creates the potential for protein-protein interactions and nearby, lies a consensus sequence (SYDE) reported to control the pleiotropic protein kinase CK2.

Methods: Electrophysiology, immunofluorescence and biochemistry applied to CFTR-expressing cells, Xenopus oocytes, pancreatic ducts and patient biopsies.

Results: Irrespective of PKA activation, CK2 inhibition (ducts, oocytes, cells) attenuates CFTR-dependent Cl(-) transport, closing wtCFTR in cell-attached membrane patches. CK2 and wtCFTR co-precipitate and CK2 co-localized with wtCFTR (but not DeltaF508-CFTR) in apical membranes of human airway biopsies. Comparing wild-type and DeltaF508CFTR expressing oocytes, only DeltaF508-CFTR Cl(-) currents were insensitive to two CK2 inhibitors. Furthermore, wtCFTR was inhibited by injecting a peptide mimicking the F508 region, whereas the DeltaF508-equivalent peptide had no effect.

Conclusions: CK2 controls wtCFTR, but not DeltaF508-CFTR. Others find that peptides from the F508 region of NBD1 allosterically control CK2, acting through F508. Hence, disruption of CK2-CFTR interaction by DeltaF508-CFTR might disrupt multiple, membrane-associated, CK2-dependent pathways, creating a new molecular disease paradigm for deleted F508 in CFTR.

Fig. 1

CK2 colocalisation with wild-type, but not ΔF508 CFTR 4% paraformaldehyde-fixed human ciliated nasal airway epithelial cells from wild type (a) and Pseudomonas-free homozygous ΔF508 CF (b) subjects stained with anti-CFTR rhodamine and anti-CK2α FITC. Images were acquired with a Zeiss LSM510 microscope using identical laser output and gain and are typical of samples from at least three separate individuals from each group. Scale bar is 5 μm. Controls omitting primary antibodies were blank at the same settings.

Fig. 2

CFTR, but not ΔF508-CFTR associates with CK2 (a) Co-immunoprecipitation (IP) of CK2 with CFTR using identical input protein content of membranes from cystic fibrosis bronchial epithelia (CFBE) and human bronchial epithelia (HBE). (b) 1% octyl-gluco-side extracted HBE membranes overlaid onto dot blots of wild-type/ΔF508 peptide (GTIKENIIFGVSYDEYRYR/GTIKENIIGVSYDEYRYR; labelled as KENIIF/KENII, respectively), and probed for CK2α or PKC (both Santa Cruz). Bottom panels omit primary antibody and fourth PKC panel is positive control; unrelated peptide is from NBD2 (QRVGLLGRTGSGKSTLL); results shown are representative of n = 3 experiments.

Fig. 3

Luminal TBB inhibits secretin-stimulated bicarbonate secretion in pancreatic ducts (a) Representative traces from an inhibitor stop experiment using a guinea pig pancreatic duct exposed to a basolateral solution containing amiloride (0.2 mM) and H₂DIDS (0.5 mM) for 3 min. (b) Summary data showing the initial rates of transmembrane HCO₃⁻ efflux (−JB⁻) measured over 60 s. Measurements were made, in the absence and presence of a 6-10 minute pre-treatment of pancreatic ducts with TBB (40 μM), from control ducts (albumin; 1% w/v; the vehicle for secretin) or ducts treated with secretin (Sec; 10 nM) in the basolateral solution. Data are means + SEM (control, n = 4; TBB, n = 10; secretin, n = 5; secretin + TBB, n = 3). The asterisk indicates a value that is significantly different from the control value (P < 0.05).

Fig. 4

TBB inhibits CFTR channel gating in intact cells, but not excised membrane patches (a) Top, representative recording of two CFTR Cl⁻ channels in a cell-attached (CA) membrane patch from a C127 cell expressing wild-type human CFTR. During the periods indicated by the bars, forskolin (20 μM) and TBB (10 μM) were present in the bath solution. (b, c) Middle, representative recording of a single CFTR Cl⁻ channel in an excised (EX) inside-out membrane patch in the absence (b) and presence (c) of TBB (10 μM). ATP (1 mM) and PKA (75 nM) were continuously present in the intracellular solution. Dotted lines indicate where channels are closed and downward deflections correspond to channel openings. (d) 5 s portions indicated by numbered arrows in (a), (b) and (c) are shown on an expanded time scale (right). (e, f) Bottom Values of open probability (P_o, (e)) and current amplitude (i, (f)) of CFTR Cl⁻ channels recorded in cell-attached and excised inside-out membrane patches made in the absence and presence of TBB (10 μM). Columns and error bars are means + SEM (CA, n = 4-12; EX, n = 6). Data from cell-attached membrane patches in the presence of TBB (10 μM) are shown in consecutive 10 s intervals for 80 s (P_o) and 30 s (i). The asterisks and double crosses indicate values that are significantly different from the control values (P < 0.05).

Fig. 5

CK2 inhibition blocks wild-type, but not ΔF508-CFTR Cl⁻ currents in Xenopus oocytes (a) Data for oocytes expressing wild-type human CFTR. (b) Equivalent data for oocytes expressing ΔF508 CFTR. Left, time-courses of cAMP-stimulated Cl⁻ currents. During the periods indicated by the bars, IBMX (1 mM) + forskolin (Fors; 2 μM) and TBB (1 μM) were added to the bath solution. Oocytes were voltage-clamped from −90 to +30 mV, in steps of 10 mV, each 1 s. Middle, effects of TBB on cAMP-stimulated Cl⁻ currents ((a) # and * indicate significant differences from control and IBMX/fors, respectively, P < 0.05; (b) * indicates significant difference from control, P < 0.05; n = 7 for (a) and (b)). Right, effects of poly E:Y peptide 4:1 (10 μM) on cAMP-stimulated Cl⁻ currents. Data are means + SEM ((a) control, n = 8; poly E:Y, n = 13; (b) control, n = 7; poly E:Y, n = 12); * indicates significant difference in magnitude of IBMX/fors-activated conductance, P < 0.05. When oocytes expressing wild-type CFTR were pre-treated with TBB (1 μM) prior to activating CFTR Cl⁻ currents with cAMP agonists (IBMX (1 mM) and forskolin (2 μM)) current magnitude was blunted severely. The magnitude of activated CFTR Cl⁻ conductance in these oocytes (22 ± 4 μS, n = 8) is equivalent to the residual CFTR Cl⁻ conductance following the inhibition of activated CFTR Cl⁻ currents by TBB (1 μM) (Fig. 5a, middle panel).

Fig. 6

Demonstration of the interaction of CK2 and CFTR using a TBB-insensitive CK2 construct and CFTR peptides (a) Oocytes were injected with cRNA encoding either CK2 alone (left two panels) or CK2 with wild-type CFTR (right two panels), stimulated with IBMX/forskolin and subsequently exposed to TBB (1 μM). CK2-dm indicates the double mutant form of CK2α that is unaffected by the CK2 inhibitor, TBB. The Y-axis shows CFTR conductance (G_CFTR) normalised to each individual pre-TBB value, and is expressed as a fraction of the IBMX (1 mM) and forskolin (2 μM) stimulated level. Data are means + SEM (CK2 alone, n = 4; CK2-dm, n = 4; CK2 + CFTR, n = 8; CK2-dm + CFTR, n = 20). The co-expression of CK2 with CFTR generated cAMP-stimulated CFTR Cl⁻ conductances in the expected range (30 - 50 μS). (b) Wild-type (KENIIF), but not ΔF508 (KENII) peptide inhibits CFTR Cl⁻ current. Xenopus oocytes expressing wild-type human CFTR were injected with 100 nM of GTIKENIIFGVSYDEYRYR (wild-type, WT, KENIIF) or GTIKENIIGVSYDEYRYR (ΔF508, KENII) peptides or water control, 20 h prior to stimulation with IBMX (1 mM) and forskolin (2 μM). Data are means + SEM, n = 23. NS indicates not significantly different from water control, * indicates significantly different from control, P < 0.01.

Fig. 7

Mutation of S511-CFTR abrogates CFTR inhibition by TBB, but does not disrupt CFTR channel gating (a) Single-channel analysis of CFTR constructs expressed in BHK cells. Left, representative single-channel recordings show the gating behaviour of the indicated CFTR constructs in the presence of ATP (1 mM) and PKA (75 nM) at −50 mV in the presence of a large Cl⁻ concentration gradient ([Cl⁻]_int = 147 mM; [Cl⁻]_ext = 10 mM). The dotted lines indicate where channels are closed and downward deflections correspond to channel openings. Right, quantification of single-channel current amplitude (i) and P_o. Values are means + SEM (n = 6, except S511A where n = 4). The asterisk indicates a value that is significantly different from wild-type CFTR, P < 0.05). No channel activity was detected in 8 membrane patches excised from BHK cells expressing ΔF508-S511D grown at 37 °C. (b) TBB insensitivity of S511D-CFTR. S551D and S511D-ΔF508-CFTR constructs were expressed in Xenopus oocytes and tested for TBB (1 μM) sensitivity in the presence of cAMP agonists. CFTR Cl⁻ conductance was determined as described in Figure 5 and the Methods. Data are means + SEM (n = 7). It was not possible to study the construct S511A-CFTR in Xenopus oocytes because for unknown reasons oocytes expressing this construct all died immediately after microelectrode impalement.