CLC anion channel regulatory phosphorylation and conserved signal transduction domains

Abstract

The signaling mechanisms that regulate CLC anion channels are poorly understood. Caenorhabditis elegans CLH-3b is a member of the CLC-1/2/Ka/Kb channel subfamily. CLH-3b is activated by meiotic cell-cycle progression and cell swelling. Inhibition is brought about by GCK-3 kinase-mediated phosphorylation of S742 and S747 located on a ∼176 amino acid disordered domain linking CBS1 and CBS2. Much of the inter-CBS linker is dispensable for channel regulation. However, deletion of a 14 amino acid activation domain encompassing S742 and S747 inhibits channel activity to the same extent as GCK-3. The crystal structure of CmCLC demonstrated that CBS2 interfaces extensively with an intracellular loop connecting membrane helices H and I, the C-terminus of helix D, and a short linker connecting helix R to CBS1. Point mutagenesis of this interface identified two highly conserved aromatic amino acid residues located in the H-I loop and the first α-helix (α1) of CBS2. Mutation of either residue to alanine rendered CLH-3b insensitive to GCK-3 inhibition. We suggest that the dephosphorylated activation domain normally interacts with CBS1 and/or CBS2, and that conformational information associated with this interaction is transduced through a conserved signal transduction module comprising the H-I loop and CBS2 α1.

Figure 1

CLH-3b structural features. (A) Schematic diagram showing organization of the membrane-associated domain of a CLC protein monomer and cytoplasmic C-terminus of CLH-3b. Rectangles indicate α-helices. Regions of helices highlighted in red function in channel gating and to coordinate Cl⁻ within the ion-conducting pore. R-helix is connected to CBS1 via a short cytoplasmic linker (green). (B) Model showing putative interface of CLH-3b intracellular domains with membrane-associated domains. Interface features are based on the crystal structure of CmCLC (5). CBS1 and CBS2 dimerize to form a Bateman module. Helices D, F, N, and R form the Cl⁻ (red circles) conducting pore of a CLC monomer. CBS2 interacts with the R-helix linker (green), the C-terminus of the D-helix, and an intracellular loop (black) connecting helices H and I, which form part of the interface between two CLC monomers. The inter-CBS linker is shown in purple. (C) Diagram of inter-CBS linker domain. Linker is ∼176 amino acids long (amino acids 615–790) and contains a highly charged motif, a GCK-3-binding motif (RFLI), and two regulatory phosphorylation sites, S742 and S747.

Figure 2

Effect of deletion of the distal C-terminus (Δ846-1001) on CLH-3b regulation. WT or Δ846-1001 mutant channels were coexpressed with kinase dead or WT GCK-3. Current properties were measured before and 1 min after cell swelling in 250 mOsm bath medium. Changes in WT and Δ846-1001 current amplitude (A), activation voltage (B), and 50% rise time (C) induced by GCK-3 and swelling were not significantly (P > 0.3) different. Representative whole cell current traces for WT and Δ846-1001 channels coexpressed with GCK-3 before and 1 min after induction of cell swelling are shown in the right panel of A. Values are mean ± SE (n = 4–5).

Figure 3

Biophysical properties of recombinant inter-CBS linker protein. (A) Prediction of disorder in the inter-CBS linker domain using PONDR (25). Predictions were generated for WT and S742E/S747E linkers. PONDR scores were identical for the two proteins except for amino acids 728–760, which are shown on the plot and contain the two regulatory phosphorylation sites (S742 and S747 shown in red). In the WT linker (black line), amino acids 734–753 have near-zero PONDR scores suggesting a high degree of order. Replacement of S742 and S747 with glutamate (red line) to mimic phosphorylation increases the PONDR scores in this region suggesting an increase in disorder. The GCK-3-binding motif (RFLI; amino acids 668–671) is shown. (B) SDS-PAGE gel of 1 μg of purified inter-CBS linker protein (amino acids 619–793). (C) Near ultraviolet CD spectra of WT and S742E/S747E mutant linkers. Spectra indicate that the WT and S742E/S747E mutant linkers contain little secondary structure.

Figure 4

Effect of inter-CBS linker deletions Δ728-748 and Δ748-768 on channel functional properties. Mutant and WT channels were coexpressed with kinase dead or WT GCK-3. When coexpressed with kinase dead GCK-3, Δ728-748 and Δ748-768 current amplitudes (A and B), activation voltages (C) and 50% rise times (D) were similar to those of GCK-3 inhibited WT channels. Coexpression of the deletion mutants with GCK-3 had no effect on functional properties. Values are mean ± SE (n = 4–8).

Figure 5

Effect of deletions in inter-CBS linker amino acids 728–768 on CLH-3b functional properties. (A) Sequence of amino acids 728–768 and schematic diagram showing location of 8–14 amino acid deletions. (B) Current amplitudes, (C) activation voltages, and (D) 50% rise times for WT CLH-3b coexpressed with functional GCK-3 and the Δ738-751 mutant coexpressed with kinase dead GCK-3. Functional properties of the WT and mutant channels were not significantly (P > 0.1) different. Values are mean ± SE (n = 6–11).

Figure 6

Structural and sequence features of CLC intracellular domains. (A) Ribbon diagram showing interface of CmCLC membrane and intracellular domains with CBS2. Intracellular loop connecting membrane helices H and I (yellow), membrane helix D (green), N-terminus of R-helix linker (purple), and α1 of CBS2 (red) are shown. Numbers indicate CmCLC amino acids that were used to guide point mutation studies in CLH-3b. The specific CmCLC amino acids are identified in the lower left corner and the equivalent residues mutated in CLH-3b are shown in parentheses. Figure was generated using UCSF Chimera software. (B) Alignments of CLH-3b membrane helices H and I and intracellular loop connecting them with various CLC proteins including CmCLC. Red shows Y232 in CLH-3b and F273 in CmCLC.

Figure 7

Effect of the H-I loop mutation Y232A and the CBS2 α1 H805A on CLH-3b regulation. Y232A and H805A mutant channels were expressed in the presence of kinase dead or functional GCK-3. Current amplitudes (A and B), (C) activation voltages, and (D) 50% rise times of Y232A and H805A mutants. Activation voltage and 50% rise times are plotted on the same scale as data in Fig. 2, B and C, to facilitate comparison with WT CLH-3b activity. Values are mean ± SE (n = 5–7). ^∗P < 0.02 compared to H805A mutant expressed with functional GCK-3.