The H-loop in the second nucleotide-binding domain of the cystic fibrosis transmembrane conductance regulator is required for efficient chloride channel closing

Abstract

The cystic fibrosis transmembrane conductance regulator (CFTR) is an ATP-binding cassette (ABC) transporter that functions as a cAMP-activated chloride channel. The recent model of CFTR gating predicts that the ATP binding to both nucleotide-binding domains (NBD1 and NBD2) of CFTR is required for the opening of the channel, while the ATP hydrolysis at NBD2 induces subsequent channel closing. In most ABC proteins, efficient hydrolysis of ATP requires the presence of the invariant histidine residue within the H-loop located in the C-terminal part of the NBD. However, the contribution of the corresponding region (H-loop) of NBD2 to the CFTR channel gating has not been examined so far. Here we report that the alanine substitution of the conserved dipeptide HR motif (HR-->AA) in the H-loop of NBD2 leads to prolonged open states of CFTR channel, indicating that the H-loop is required for efficient channel closing. On the other hand, the HR-->AA substitution lead to the substantial decrease of CFTR-mediated current density (pA/pF) in transfected HEK 293 cells, as recorded in the whole-cell patch-clamp analysis. These results suggest that the H-loop of NBD2, apart from being required for CFTR channel closing, may be involved in regulating CFTR trafficking to the cell surface.

Fig. 1

The H-loop region in nucleotide-binding domains of CFTR and other human ABC transporters. A. Multiple sequence alignment showing conserved amino acids within the H-loop region of the N-terminal (N) or C-terminal (C) NBDs of different human ABC proteins. MRP1 (ABCC1) and MRP2 (ABCC2) – multidrug resistance related proteins 1 and 2; MDR1 (ABCB1) and MDR3 (ABCB4) – multidrug resistance proteins 1 and 3; SUR1 (ABCC8) – sulfonylurea receptor 1. The position of the H-loop region in the amino acid sequence of the full-length proteins is indicated on the left. B. The sequence of the H-loop region in CFTR constructs with HR→AA and Δ1395–1403 mutations.

Fig. 2

Maturation analysis of the CFTR mutants. The autoradiograph shows wild-type and mutant CFTR proteins that were expressed in HEK 293 cells, immunoprecipitated using monoclonal anti-CFTR antibody, radiolabeled with ³²P using protein kinase A (PKA) and electrophoretically separated by SDS-PAGE. Band C corresponds to the fully glycosylated mature CFTR protein, whereas the non-glycosylated or core-glycosylated proteins migrate as bands A and B, respectively [80].

Fig. 3

Exemplary single channel traces from HEK 293 cells expressing WT (A), HR→AA (B), Δ1395–1403 (C) or Δ508 (D) CFTR. The horizontal bars, placed to the left of the individual records, indicate closed states of the channel (baseline level). The voltage was stepped from −100 to +100 mV at 10 mV increments for 2 seconds. Signal was filtered at 280 Hz. Both pipette and bath solutions contained 129 mM Tris-HCl and 16 mM TEA-Cl. Bath solution was supplemented with 250 μM 8-CPT-cAMP.

Fig. 4

The subconductance states recorded in patches expressing the wild-type CFTR channel. Potential −80 mV, signal was filtered at 50 Hz. A. An exemplary single channel trace showing closed (C) state with large (O1) and small (O2) subconductance openings. B. A diagram showing the distribution of amplitudes for all openings recorded at potential −80 mV.

Fig. 5

The outwardly rectified chloride channel (ORCC) activity in patches expressing the wild type CFTR channel. A. An exemplary single channel recording showing both CFTR and ORCC activity. The first part of the upper trace, enhanced in the lower trace, shows CFTR openings, while the remaining part of the upper trace corresponds to ORCC activity, characterized by large amplitude of channel openings. Potential was +50 mV and signal was filtered at 500 Hz (upper trace) or 50 Hz (lower trace). The horizontal bars, placed to the right of the individual records, indicate closed states of the channel (baseline level). B. The linear I-V relationship for the CFTR channel. C. The I-V relationship for the large conductance channel showing outward rectification (ORCC).

Fig. 6

The kinetics of a whole-cell currents in exemplary HEK 293 cells transfected with a wild-type (WT, triangles), HR→AA (squares) or Δ1395–1403 (circles) CFTR. At time zero 250 μM 8-CPT-cAMP was added to the bath. Holding potential was −30 mV. Once in 60 seconds the potential was changed to +100 mV and the current was recorded after 345 ms.

Fig. 7

The CFTR- and ORCC-specific currents in HEK 293 cells expressing different variants of CFTR. A-C. The I-V relationship for currents recorded in whole-cell patch-clamp experiments performed on HEK 293 cells expressing WT (A, n=10), HR→AA (B, n=8), and Δ1395–1403 (C, n=5) CFTR channels. Currents were activated by 250 μM 8-CPT-cAMP and after reaching the maximum amplitude they were blocked with 500 μM DIDS, and then with 500 μM DIDS and 100 μM glibenclamide. In some experiments 5 minutes after activation 100 μM glibenclamide was added followed by 500 μM DIDS and 100 μM glibenclamide. Holding potential was −30 mV; voltage steps from −100 mV to +100 mV with 10 mV increments; duration of 345 ms; no leak subtraction. The outward rectification, seen in the 8-CPT-cAMP-stimulated currents generated by all three CFTR variants, lead to increased current densities at positive (+100 mV) voltages, as compared to the corresponding values at negative (−100 mV) voltages. Rectification was stronger for the wild-type CFTR (A, about three-fold increase) than for both mutants (B and C, less than two-fold increase). D. Average chloride current density (pA/pF) of CFTR (glibenclamide-sensitive), ORCC (DIDS-sensitive), and total chloride channels measured in HEK 293 cells expressing WT and mutant CFTR: HR→AA (n=8), Δ1395–1403 (n=5), Δ508 (n=7). Potential 80 mV. * denotes significant (p<0.01) change of current density, as compared with WT.