Modulation of TMEM16B channel activity by the calcium-activated chloride channel regulator 4 (CLCA4) in human cells

Abstract

The Ca²⁺-activated Cl^- channel regulator CLCA1 potentiates the activity of the Ca²⁺-activated Cl^- channel (CaCC) TMEM16A by directly engaging the channel at the cell surface, inhibiting its reinternalization and increasing Ca²⁺-dependent Cl^- current (I_CaCC) density. We now present evidence of functional pairing between two other CLCA and TMEM16 protein family members, namely CLCA4 and the CaCC TMEM16B. Similar to CLCA1, (i) CLCA4 is a self-cleaving metalloprotease, and the N-terminal portion (N-CLCA4) is secreted; (ii) the von Willebrand factor type A (VWA) domain in N-CLCA4 is sufficient to potentiate I_CaCC in HEK293T cells; and (iii) this is mediated by the metal ion-dependent adhesion site motif within VWA. The results indicate that, despite the conserved regulatory mechanism and homology between CLCA1 and CLCA4, CLCA4-dependent I_CaCC are carried by TMEM16B, rather than TMEM16A. Our findings show specificity in CLCA/TMEM16 interactions and suggest broad physiological and pathophysiological links between these two protein families.

Keywords: CLCA family; TMEM16 family; calcium-activated chloride channel; calcium-activated chloride channel regulator; von Willebrand factor type A domain.

Figure 1

CLCA4 self-cleavage is required to potentiate I_CaCCin HEK293T cells.A, domain schematic representation of human CLCA4 constructs used in this study. The anti-CLCA1 antibody 8D3 is cross-reactive with CLCA4 and the region containing the epitope is highlighted. Domain abbreviations are as follows: CAT = MMP-like catalytic domain; SS = signal sequence; CYS = MMP-like cysteine-rich domain; VWA = von-Willebrand Type A domain; FnIII = fibronectin type-III domain; GPI = glycosylphosphatidylinositol anchor. B–D, CLCA4 is cleaved and secreted. B, Western blot (using 8D3) of cells transfected with CLCA4. Full-length CLCA4 is only detected in solubilized cells, whereas the cleaved N-terminal fragment (N-CLCA4) is found in both cell lysate and secreted into the medium. C, Western blot (using anti-FLAG M2) of cells transfected with CLCA4-FLAG. Both full-length and C-CLCA4 are only detected in solubilized cells. D, Western blot (using 8D3) of cells transfected with either WT CLCA4 or CLCA4 containing mutations to predicted active-site residues in CLCA4 MMP-like domain. These mutations (H155A and E156Q) prevent self-cleavage. E, anti-FLAG Western blot of cells transfected with CLCA-4-FLAG treated with PI-PLC to release GPI-anchored proteins. C-CLCA4-FLAG is released into media following PI-PLC treatment. F, CLCA4 self-cleavage is required to potentiate CaCC-like currents, i.e., I_CaCC. Cells were transfected with expression constructs or empty pHLsec plasmid (mock) and assayed by whole-cell patch clamp 48 h later. Representative whole-cell currents; pulse protocol on top. G, current density at +100 mV, measured at the end of the 600-ms voltage pulse. Symbols are data from individual patches (n = 10–25); bars are means ± S.E.M. of all experiments. Statistical differences between CLCA4 transfected and other conditions are indicated (Kruskal-Wallis one-way ANOVA on ranks, H (3) = 28.813, p < 0.001, followed by Dunn’s test).

Figure 2

CLCA4-dependent I_CaCCare carried by TMEM16B, and paracrine CLCA4-TMEM16B interactions are mediated by a MIDAS motif in the CLCA4 VWA domain.A, HEK293T cells transfected with scramble (siControl), TMEM16A siRNA, or TMEM16B siRNA were cultured in medium from mock-transfected cells or from cells expressing full-length CLCA4 or CLCA4 VWA domain (VWA), and assayed by whole-cell patch clamp. B and C, I_CaCC were activated in CLCA4- (B) and VWA-conditioned (C) cells transfected with siControl and TMEM16A siRNA, but current density remained at background levels in cells transfected with TMEM16B siRNA. D, homology model of the CLCA4 VWA domain, based on the crystal structure of CLCA1 VWA (PDB ID: 6PYO) (22). Residues composing the MIDAS site and the N-linked glycosylation site (N340) are shown as sticks. E, purified CLCA4 VWA expressed in the presence of kifunensine was treated with the deglycosylation enzyme EndoHf, resulting in a shift to lower molecular weight. F, mutation of the CLCA4 VWA glycosylation site (N340Q) results in a single band corresponding to the non-glycosylated protein. G, CLCA4-dependent I_CaCC potentiation was reduced in naïve HEK293T cells conditioned with MIDAS motif double mutant S316A/T383A VWA. Experiments were performed as in (A). B, C and G, current density at +100 mV, measured at the end of the 600-ms voltage pulse (protocol as in Fig. 1F). Symbols are data from individual patches (n = 10–25); bars are means ± S.E.M. of all experiments. Statistical differences were evaluated by Kruskal-Wallis one-way ANOVA on ranks, as follows: (B) H (5) = 26.647, p < 0.001; (C) H (3) = 15.780, p = 0.001; and (G) H (2) = 9.267, p = 0.010. Post hoc all-pairwise comparisons between groups were determined by Dunn’s method. Statistical differences are indicated in (B) between mock- and CLCA4-conditioned media for each siRNA transfection in CLCA4-conditioned versus scrambled siRNA control in mock-conditioned. Statistical differences are indicated in (C) between mock- and CLCA4-conditioned media for each siRNA transfection. Statistical differences are indicated in (G) between all treatments.

Figure 3

Working model for CLCA family self-activation and engagement of TMEM16 family proteins.A, soluble CLCA1 undergoes self-cleavage mediated by its MMP-like domain (CAT) to produce the N-CLCA1 and C-CLCA1 fragments. N-CLCA1 engages TMEM16A α9-α10 loop using VWA MIDAS motif. B, membrane-anchored CLCA4 undergoes self-cleavage mediated by its MMP-like domain (CAT) to produce the N-CLCA4 and C-CLCA4 fragments. N-CLCA4 engages TMEM16B using its VWA domain.