Calcium-induced conformational changes in C-terminal tail of polycystin-2 are necessary for channel gating

Abstract

Polycystin-2 (PC2) is a Ca(2+)-permeable transient receptor potential channel activated and regulated by changes in cytoplasmic Ca(2+). PC2 mutations are responsible for ∼15% of autosomal dominant polycystic kidney disease. Although the C-terminal cytoplasmic tail of PC2 has been shown to contain a Ca(2+)-binding EF-hand domain, the molecular basis of PC2 channel gating by Ca(2+) remains unknown. We propose that the PC2 EF-hand is a Ca(2+) sensor required for channel gating. Consistent with this, Ca(2+) binding causes a dramatic decrease in the radius of gyration (R(g)) of the PC2 EF-hand by small angle x-ray scattering and significant conformational changes by NMR. Furthermore, increasing Ca(2+) concentrations cause the C-terminal cytoplasmic tail to transition from a mixture of extended oligomers to a single compact dimer by analytical ultracentrifugation, coupled with a >30 Å decrease in maximum interatomic distance (D(max)) by small angle x-ray scattering. Mutant PC2 channels unable to bind Ca(2+) via the EF-hand are inactive in single-channel planar lipid bilayers and inhibit Ca(2+) release from ER stores upon overexpression in cells, suggesting dominant negative properties. Our results support a model where PC2 channels are gated by discrete conformational changes in the C-terminal cytoplasmic tail in response to changes in cytoplasmic Ca(2+) levels. These properties of PC2 are lost in autosomal dominant polycystic kidney disease, emphasizing the importance of PC2 to kidney cell function. We speculate that PC2 and the Ca(2+)-dependent transient receptor potential channels in general are regulated by similar conformational changes in their cytoplasmic domains that are propagated to the channel pore.

FIGURE 1.

SAXS analysis of Ca²⁺-induced global conformational changes in the PC2 EF-hand domain. A, SAXS patterns for PC2-EF in the apo (left panel) and Ca²⁺-bound (right panel) states. Fitted scattering data calculated from the DAMAVER PC2-EF without Ca²⁺ model and the PC2-EF NMR structure (Protein Data Bank code 2K6Q) (5) are shown in green (apo) and red (Ca²⁺-bound). B, P(r) curves for apo (green) and Ca²⁺-bound (red) PC2-EF show decreases in both the radius of gyration and maximum interparticle distance upon binding Ca²⁺. C, calculated dummy atom model of apo PC2-EF shows a broadly unstructured protein (left panel, green spheres). Calculated dummy atom model of Ca²⁺-bound PC2-EF (right, red mesh) shows high similarity to the NMR structure of PC2-EF (Protein Data Bank code 2K6Q) (5) (yellow spheres). The same scale is used. PyMOL was used to make structural figures.

FIGURE 2.

Ca²⁺ binding induces a folding transition in the PC2 EF-hand domain. A, difference in C_α chemical shifts from random coil values for apo (black bars) and Ca²⁺-bound (gray bars) PC2-EF. The positions of helices in Ca²⁺-bound PC2-EF are shown. B, regions of PC2-EF with increased ordered structure upon Ca²⁺ binding (residues colored orange have ΔCSI_Cα (holo-apo) >1 ppm) are mapped onto the NMR structure of Ca²⁺-bound PC2-EF (Protein Data Bank code 2K6Q) (5). Strikingly, the α1-α2 helix-loop-helix motif remains intact in the Ca²⁺-free state, and helix α2 and the first half of helix α3 remain unchanged by ligand binding. The helix-loop-helix motif and the α2-α3 turn may catalyze folding of the EF-hand motif in the presence of Ca²⁺. C, CSI plot for C_α atoms in apo PC2-EF. The positions of secondary structural elements determined by NMR for apo PC2-EF are shown. D, CSI plot for C_α atoms of Ca²⁺-bound PC2-EF. HLH, helix-loop-helix.

FIGURE 3.

AUC analysis of Ca²⁺-induced conformational and oligomerization state changes in PC2-C. A, AUC velocity sedimentation analysis of PC2-C in the presence of equimolar amounts of Ca²⁺ reveals a mixture of oligomerization and conformation states ranging from a compact dimer to an extended tetramer. f/f_o > 1 indicates an elongated shape. B, saturating concentrations of Ca²⁺ (50:1) convert PC2-C into a predominantly compact dimeric globular state.

FIGURE 4.

SAXS analysis of Ca²⁺-induced global changes in PC2-C. A, SAXS patterns for PC2-EF in the apo (left panel) and Ca²⁺-bound (right panel) states. Fitted scattering data calculated from the DAMAVER P4 models are shown in green (apo) and red (Ca²⁺-bound). B, P(r) curves for apo (green) and Ca²⁺-bound (red) PC2-C show a reduction in D_max and conformational changes. C, calculated dummy atom models (DAMAVER, P4) of apo (green) and Ca²⁺-bound (red) PC2-C compared with the structures of the EF-hand (Protein Data Bank code 2K6Q) (5) and coiled coil (Protein Data Bank code 3HRN) (7). Of 265 residues in the PC2-C construct, 78 correspond to the EF-hand domain, 63 correspond to the coiled coil domain, and 124 correspond to regions whose high resolution structure has not yet been determined. These 124 residues are predicted to be disordered (4). The lengths of the NMR and crystal structures are indicated. AUC and SAXS analyses of PC2-C suggest that conformational changes occur upon the addition of Ca²⁺ that result in a transition from an extended state to a compact state. D, schematic hypothesis for PC2 channel regulation. Based on our AUC, NMR, and SAXS experiments, we hypothesize that Ca²⁺ binding to the EF-hand domain of PC2 can alter the conformation of this domain and regulate channel activity.

FIGURE 5.

Single channel recordings of PC2-x-z. A, representative traces from single channel experiments for wild type PC2 (left panel) versus PC2-x-z (right panel) under increasing concentrations of free cytoplasmic (cis-side) Ca²⁺ (0.01, 0.1, and 1.0 μm free Ca²⁺). B, Ca²⁺ dependence of the activity of wild type PC2 (diamonds and black line) and PC2-x-z (squares and gray dashed line). The data obtained for one complete Ca²⁺ dependence experiments with either wild type or PC2-x-z are shown; five experiments with each PC2 were completed.

FIGURE 6.

Live cell Ca²⁺ imaging of PC2-x-z. SH-SY5Y cells overexpressing pcDNA 3.1 empty vector, wild type PC2, or PC2-x-z were excited with 200 μm carbachol in Ca²⁺-containing buffer to induce a transient release of Ca²⁺ from intracellular stores. Thapsigargin (TG, 5 μm) was added to confirm cell viability. Ca²⁺-induced fluorescence intensity ratios (F/F₀) are plotted as a function of time in seconds, with F₀ calculated as the average of the first 10 base-line points. The response duration was defined as the time the Ca²⁺ signal took to decrease to 50% of the peak value.