Noncanonical binding of calmodulin to aquaporin-0: implications for channel regulation

Abstract

Aquaporins (AQPs) are a family of ubiquitous membrane channels that conduct water across cell membranes. AQPs form homotetramers containing four functional and independent water pores. Aquaporin-0 (AQP0) is expressed in the eye lens, where its water permeability is regulated by calmodulin (CaM). Here we use a combination of biochemical methods and NMR spectroscopy to probe the interaction between AQP0 and CaM. We show that CaM binds the AQP0 C-terminal domain in a calcium-dependent manner. We demonstrate that only two CaM molecules bind a single AQP0 tetramer in a noncanonical fashion, suggesting a form of cooperativity between AQP0 monomers. Based on these results, we derive a structural model of the AQP0/CaM complex, which suggests CaM may be inhibitory to channel permeability by capping the vestibules of two monomers within the AQP0 tetramer. Finally, phosphorylation within AQP0's CaM binding domain inhibits the AQP0/CaM interaction, suggesting a temporal regulatory mechanism for complex formation.

Figure 1. Calmodulin binds AQP0 carboxyl terminus in a Ca²⁺ dependent manner

A, CaM pull-down assay with lens membranes. Molecular weight markers are indicated on the left in kDa. Lane 1: lens membranes incubated with calmodulin. Lanes 2, 5, 8 and 11: supernatants containing unbound CaM. Lanes 3, 6, 9 and 12: supernatants following 4 rounds of pellet washing. Lanes 4, 7, 10 and 13: pellets containing lens membranes. CaM is associated with lens membranes only when Ca²⁺ is present (Lanes 10 and 13). B, Western blot detection identifying AQP0 (left) as a 26kDa species and CaM (right) as a 17kDa species. C, Pull-down assay with proteolytically cleaved lens membrane proteins. Lanes 1, 2, and 3: CaM, lens membranes and cleaved lens membranes; respectively. “tAQP0” is C-terminally truncated AQP0 (Gonen et al., 2004b). Lanes 4 and 5: pellets containing untreated membranes and cleaved lens membrane proteins, respectively. CaM is found only in the untreated, un-cleaved preparation (Lane 4).

Figure 2. Identification of the AQP0-Calmodulin binding domain and effect of serine phosphorylation

A, Aquaporins form tetrames in which each monomer forms a functional and independent water pore (asterisk) (Shi et al., 1994). The AQP0 tetramer is viewed from the extracellular side of the cell membrane (Protein Data Bank Accession number 2B6O). B, Side view of an AQP0 monomer highlighting the cytoplasmic C-terminal α-helix as the calmodulin binding domain (AQP0^CBD, yellow). C, Native gel electrophoresis of calmodulin bound to AQP0^CBD. A clear shift in the calmodulin band is seen in the presence of Ca²⁺ (Lanes 1 versus 2) but not when Ser 235 is phosphorylated (Lane 3) or in the presence of EGTA even with excess peptide (Lanes 4 and 5, respectively). D, Sequence alignment of AQP0^CBD and 4 other 1-8-14 calmodulin binding motifs (highlighted in blue) from different proteins as indicated in the figure. The AQP0^CBD is missing the large hydrophobic residue at position 14. Ser 235 is highlighted in red and is the site of phosphorylation assayed in this study. The sequence of the non-cononical plant glutamate decarboxylase (PGD) CaM binding site is at the bottom.

Figure 3. Calmodulin binds two AQP0^CBD peptides in a step-wise manner

A, Overlay of ¹⁵N-calmodulin HSQC spectra of unbound (yellow), singly bound (green) and doubly bound (blue) species in the presence of Ca²⁺. B, Combined ¹H/¹⁵N chemical shift changes (Δδ) for several amino acids from the N- and C-lobes of calmodulin. Green and blue bars correspond to Δδ values from the 1:1 and 1:2 complexes, respectively. Red bars correspond to the overall Δδ values from the S235-P titration. Horizontal lines indicate the mean and standard deviation of the overall Δδ values from the AQP0^CBD titration. C, Close-up view of the boxed area from panel A, for AQP0^CBD. Residue assignments are identified in the figure. Arrows indicate different points in the titration experiment, corresponding to singly bound (Δδ₁) and doubly bound (Δδ₂) ¹⁵N-calmodulin species. D, Titration curves of normalized Δδ values for several ¹⁵N-calmodulin residues versus mole ratio of AQP0^CBD peptide (colored and labeled). The flat portion of the trace “Δδ₁” indicates slow-exchange. Theoretical fits to the fast exchange Δδ₂ used to obtain binding constants are overlaid in black hatch lines. E and F, are the same as C and D, but were obtained for the S235-P modified AQP0^CBD. In E, arrows indicate S235-P titration points that resulted in fast-exchange. In F, the titration data for both binding events were fit to a two-state binding isotherm.

Figure 4. Two calmodulin molecules bind to a single AQP0 tetramer

A, ¹H dimension of ¹⁵N-calmodulin HSQC titration series using full-length AQP0 tetramers in the presence of Ca²⁺. The molar stoichiometric ratio of CaM:AQP0 is indicated. B, Plot of signal intensity for several resonances from ¹⁵N-calmodulin during the titration. Standard deviations are indicated. The NMR signal for ¹⁵N-calmodulin is almost completely attenuated at a 1:2 molar ratio, indicating that two calmodulin molecules bind to a single AQP0 tetramer. C, As in panel A, but in the presence of EGTA. No AQP0/CaM interaction is detected in the absence of Ca²⁺.

Figure 5. Model of the AQP0/calmodulin complex

A, Structure of the petunia glutamate decarboxylase (PGD) calmodulin complex (Protein Data Bank Accession number 1NWD). Calmodulin wraps itself around two PGD α-helices oriented in an anti-parallel fashion. Ca²⁺ indicated by black speheres. B, AQP0 tetramer viewed from the cytoplasmic side of the cell membrane (bottom view). The carboxyl terminal tails are highlighted in yellow. Curved arrows indicate the proposed movement of the carboxyl tails. C, Rotation of the AQP0 carboxyl terminal tails places neighboring AQP0^CBD in close proximity in an orientation similar to the PGD calmodulin structure (Yap et al., 2003). D, Overlay of the PGD α-helices with AQP0^CBD. E and F, Side and bottom views of the modeled AQP0/CaM complex, respectively. In this model, calmodulin obstructs only two of the water pores out of the AQP0 tetramer (grey versus blue). Structure fitting and modeling was performed in UCSF chimera (Pettersen et al., 2004).