Regulation of cystic fibrosis transmembrane conductance regulator single-channel gating by bivalent PDZ-domain-mediated interaction

Abstract

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-dependent protein kinase- and ATP-regulated chloride channel, the activity of which determines the rate of electrolyte and fluid transport in a variety of epithelial tissues. Here we describe a mechanism that regulates CFTR channel activity, which is mediated by PDZ domains, a family of conserved protein-interaction modules. The Na(+)/H(+) exchanger regulatory factor (NHERF) binds to the cytoplasmic tail of CFTR through either of its two PDZ (PDZ1 and PDZ2) domains. A recombinant fragment of NHERF (PDZ1-2) containing the two PDZ domains increases the open probability (P(o)) of single CFTR channels in excised membrane patches from a lung submucosal gland cell line. Both PDZ domains are required for this functional effect, because peptides containing mutations in either domain are unable to increase channel P(o). The concentration dependence of the regulation by the bivalent PDZ1-2 domain is biphasic, i.e., activating at lower concentrations and inhibiting at higher concentrations. Furthermore, either PDZ domain alone or together is without effect on P(o), but either domain can competitively inhibit the PDZ1-2-mediated stimulation of CFTR. Our results support a molecular model in which bivalent NHERF PDZ domains regulate channel gating by crosslinking the C-terminal tails in a single dimeric CFTR channel, and the magnitude of this regulation is coupled to the stoichiometry of these interactions.

Figure 1

Specific interaction of CFTR and NHERF. (A) Yeast two-hybrid analysis of CFTR–NHERF interaction. Full-length NHERF is shown at the top. NHERF cDNA isolated from the two-hybrid screen () and other deletion constructs are aligned beneath the full-length NHERF. Interaction strength of NHERF constructs with the C terminus (1,411–1,480) of CFTR are summarized based on induction of reporter genes β-galactosidase (β-gal) and Leu-2 (Leu). PDZ domains used in all subsequent experiments are marked (*). (B) Coimmunoprecipitation of CFTR and NHERF from CHO cells. Extracts (Input) from control cells and cells expressing HA-tagged NHERF and/or CFTR were immunoprecipitated (IP) with anti-HA antibodies, and the immunoprecipitates were blotted by using anti-CFTR or anti-HA antibodies, as indicated. Input lanes contain 5% of extract used for immunoprecipitation. (C) GST-affinity binding assays. [³⁵S]methionine-labeled in vitro-translated products (I) and the GST-PDZ1–2 () “pull-down” products (P) of wild-type CFTR (WT) and mutant CFTR (L1480A) are indicated. (D) Specificity of PDZ1 and PDZ2 for CFTR C terminus. The terminal 3 aa of CFTR (1,411–1,480) and the NHERF PDZ domains are indicated.

Figure 2

Kinetics of CFTR–NHERF PDZ domain interactions. Experimental data (dots) represent average of repeated injections of analyte for each concentration, as indicated. Global fit of the data to a simple bimolecular reaction is shown by solid lines. (A) Kinetic-response data for GST–PDZ1 binding to CFTR. (B) Kinetics-response data for GST–PDZ2 binding to CFTR. (C) Association (k_a) and the dissociation (k_d) rate constants for binding of CFTR C terminus to PDZ1 and PDZ2 domains. (D) Sensorgram overlays for various GST–PDZ1–2 fusion proteins binding to CFTR C terminus at a concentration of 50 nM. Wild-type PDZ1–PDZ2 (–2), PDZ1K19A–PDZ2 (1*–2), PDZ1–PDZ2 K158A, K159A (1–2*) and PDZ1K19A–PDZ2 K158A, K159A (1*–2*), are indicated.

Figure 3

Effect of NHERF PDZ-domain-mediated interactions on CFTR channel activity. (A) Single-channel current traces of CFTR before and after addition of 40 nM PDZ1–2. The closed level is indicated by arrowheads. (B) P_o of CFTR before and after addition of 40 nM PDZ1–2 (solid lines) or PDZ1*–2* (dashed line). Each trace represents individual experiments. (C) Mean ratio of CFTR P_o after and before addition of 40 nM of various PDZ peptides. * Indicates significant difference of P_o (P < 0.01) after addition of peptide. PDZ1–2 (–2), PDZ1*–PDZ2* (1*–2*), PDZ1*–PDZ2 (1*–2), PDZ1–PDZ2* (1–2*), PDZ1 (1), PDZ2 (2), and PDZ1 and PDZ2 mixed together (1&2) are indicated. Number of experiments indicated in parentheses.

Figure 4

Biphasic regulation of CFTR by bivalent PDZ-domain-mediated interactions. (A) An equilibrium model illustrating various physical states (U, B₁, B_c, and B₂) of a CFTR channel bound to bivalent PDZ1–2 is described. The CFTR channel is represented by two PDZ binding motifs. See text for details. (B) Single-channel current traces of CFTR at various concentrations of PDZ1–2. Arrowheads indicate closed level. (C) A representative single-channel experiment showing the biphasic dependence of CFTR P_o on PDZ1–2 concentration. (D) Representative single-channel experiments showing inhibition of PDZ1–2-mediated effect on CFTR P_o by monovalent PDZ domains (PDZ1, filled circles; PDZ2, open circles). The P_o of CFTR before addition of 40 nM PDZ1–2 is indicated (lower dashed line).