Contribution of Coiled-Coil Assembly to Ca2+/Calmodulin-Dependent Inactivation of TRPC6 Channel and its Impacts on FSGS-Associated Phenotypes

Abstract

Background: TRPC6 is a nonselective cation channel, and mutations of this gene are associated with FSGS. These mutations are associated with TRPC6 current amplitude amplification and/or delay of the channel inactivation (gain-of-function phenotype). However, the mechanism of the gain-of-function in TRPC6 activity has not yet been clearly solved.

Methods: We performed electrophysiologic, biochemical, and biophysical experiments to elucidate the molecular mechanism underlying calmodulin (CaM)-mediated Ca²⁺-dependent inactivation (CDI) of TRPC6. To address the pathophysiologic contribution of CDI, we assessed the actin filament organization in cultured mouse podocytes.

Results: Both lobes of CaM helped induce CDI. Moreover, CaM binding to the TRPC6 CaM-binding domain (CBD) was Ca²⁺-dependent and exhibited a 1:2 (CaM/CBD) stoichiometry. The TRPC6 coiled-coil assembly, which brought two CBDs into adequate proximity, was essential for CDI. Deletion of the coiled-coil slowed CDI of TRPC6, indicating that the coiled-coil assembly configures both lobes of CaM binding on two CBDs to induce normal CDI. The FSGS-associated TRPC6 mutations within the coiled-coil severely delayed CDI and often increased TRPC6 current amplitudes. In cultured mouse podocytes, FSGS-associated channels and CaM mutations led to sustained Ca²⁺ elevations and a disorganized cytoskeleton.

Conclusions: The gain-of-function mechanism found in FSGS-causing mutations in TRPC6 can be explained by impairments of the CDI, caused by disruptions of TRPC's coiled-coil assembly which is essential for CaM binding. The resulting excess Ca²⁺ may contribute to structural damage in the podocytes.

Keywords: Calcium signal; TRPC channel; chronic kidney disease; electrophysiology; focal segmental glomerulosclerosis; podocyte.

Graphical abstract

Figure 1.

Functional analysis of CaM mutants and Ca²⁺-dependent binding. (A) Representative currents recorded from HEK293 cells coexpressing with TRPC6 and CaM_WT or a CaM mutant (CaM₁₂, CaM₃₄, or CaM₁₂₃₄). Muscarinic receptor (M₁R) is also coexpressed for the electrophysiologic experiments in HEK293 cells. The inset shows typical I–V curve of TRPC6. (B) Fractions of residual currents plotted as a function of time after the peak. Black circles indicate control data from TRPC6 with M₁R-transfected cells only (n=14, CaM_endo), here and throughout. (C) Representative profiles of Ca²⁺-dependent binding of CaMs to the TRPC6 CBD. FRET measurement is explained in the Methods section. (D) Quantification of the affinity of binding of CBD_WT to CaM mutants by plotting donor free ([D]_free) concentrations against FRET signal (FR). Data from various cells were collected at their maximum FR values in (C). Lines represent fitting of CBD_WT versus CaM_MUT. FRET data are summarized in Supplemental Table 1.

Figure 2.

NMR and ITC titrations of CBDp binding to Ca²⁺CaM. (A) Overlay of 2D ¹H-¹⁵N SOFAST-HMQC spectra of ¹³C, ¹⁵N–labeled CaM with increasing amounts of nonlabeled CBDp. The right panels show zoomed views of crosspeaks (NMR signals). T5, I27, T29, N42, and T44, which are residues within the N-lobe of CaM, showed nonlinear direction changes during the titration. (B) Zoomed view of 2D ¹H-¹⁵N SOFAST-HMQC spectra of ¹³C, ¹⁵N–labeled CaM_ΔC and CaM_ΔN with the increasing amounts of nonlabeled CBDp. The color code is the same as in (A). In contrast to CaM_WT, signals of all residues shifted in a linear manner. (C) Saturation of NMR signal shift changes as a function of the equivalent molar ratio of added CBDp. Each value (Δδ_ij) was divided by Δδ_total (=Δδ₀₁+Δδ₁₂+Δδ₂₃+Δδ₃₄) to yield the saturation ratio. All residues reached 80%–90% of saturation at 2 eq. (D) Combined CSPs upon addition of CBD were plotted as a function of the residue number of CaM. In stacked bar graph, Δδ₀₁ (blue) and Δδ₁₂ (red) represent CSPs from 0 to 1 eq and from 1 to 2 eq, respectively. (E) ITC thermograms for CBD binding of CaM_WT in the presence of Ca²⁺. Upper panel represents the raw data for sequential injections of CBDp into CaM. In the lower panel, the data points are obtained by integration of the peaks in the upper panel and plotted against the molar ratios. Fitting was performed with two-site binding model to determine thermodynamic parameters and binding affinity. ITC data for CaM_ΔC, CaM_ΔN are shown in Supplemental Figure 3 and summarized in Supplemental Table 2.

Figure 3.

Assembly of coiled-coil segments and their functional role. (A) FRET measurement of binding strength comparison between CBDs (left panel) and CBD-CC segments (right panel) in live cells. The binding affinity was evaluated by the same analysis style as in Figure 1D. A negative control (CFP and YFP only) for FRET measurements is shown in the panel for CBD only (gray circles). (B) Crosslinking of CBD-CC fragments. Purified CBD-CC (200 μM) was treated with 0–3 mM EGS crosslinker. Arrows indicate monomer (M, 11.6 kDa), dimer (D), tetramer (T), and oligomer (O) formations of CBD-CC. (C) Size exclusion chromatography results for CBD-CC with and without CaM are shown as red and black traces, respectively. The inset shows the standard curve estimation of the complex sizes of CBD-CC with and without CaM. (D) Crosslinkings of CaM to CBD-CC by EDC/NHS (1 mM) in the presence (1 mM) or absence of Ca²⁺ were separated in SDS-PAGE and were imaged by CBB stain (left), or immunoblotting for CaM (right). The positions of the expected mol wt for single CaM (16.7 kDa) and single or multiple CBD-CC complexes (1:1, 1:2, 1:3, 1:4) are indicated by arrows. (E) Currents from TRPC6 with a truncated coiled-coil (TRPC6_ΔCC), evoked by CCh. Inactivation plots constructed by plotting residual currents against time for TRPC6_WT and TRPC6_ΔCC are shown as black and red circles, respectively. (F) Representative TRPC6 currents trace from the cell cotransfected with the N-lobe (CaM_ΔC) and C-lobe (CaM_ΔN). Residual current plots of CaM_ΔC/CaM_ΔN (red circles) versus TRPC6WT without overexpression of CaM (CaMendo) (black circles). Data from CaM_ΔC or CaM_ΔN only are shown in Supplemental Figure 5.

Figure 4.

FSGS mutations in the coiled-coil segment lead to impairment of CDI. (A) The sequence of the locus of the CBD (black) and coiled-coil segment (gray), and FSGS mutations within the TRPC6 channel. Positions of the coiled-coil heptad repeat (abcdefg) are indicated above the sequence. Helical-wheel diagrams showing the location of residues in the heptad repeat (right). (B) Receptor-activated whole-cell currents from FSGS mutations (TRPC6_K874×, TRPC6_Q889K, TRPC6_R895C, TRPC6_R895L, TRPC6_E897K) coexpressed with wild-type TRPC6 in HEK293 cells (upper panels). Scale bars depict the same time course and current amplitude throughout. The coexpression of wild-type and K874× mutation showed no difference compared with K874× alone (Supplemental Figure 6). Residual current plots are plotted against time after the peak (data from FSGS-associated TRPC6 channels are shown in gray circles, lower panels). (C) Summary of the peak current densities (pA/pF) induced by the receptor stimulation. (D) Inside-out recordings of TRPC6_WT (upper), TRPC6_K874× (lower) with step-wise changing of intracellular Ca²⁺ concentrations (μM). (E) Summary of the inside-out recordings. TRPC6_WT (black circles) and TRPC6_K874× (gray circles) are represented as a plot of normalized NPo versus [Ca²⁺]_i. Normalized NPo was obtained by dividing NPo at nearly zero [Ca²⁺]_i. (F) Binding affinities between CBD-CC mutants in cells were measured by FRET, where individual data points represent FRET strength from single cells (black circles), and fitted by using 1:1 binding isotherm equations (black curve). The strength of interaction of CBD-CC_WT is shown as a gray line (control). FRET data are summarized in Supplemental Table 1.

Figure 5.

Impairment of CDI in cultured podocytes. (A) Gray lines represent individual [Ca²⁺]_i of nontransfected cells (left) and cells transfected with TRPC6_WT/M₁R (middle left) (n=23), TRPC6_WT/TRPC6_K874×/M₁R (center) (n=17), TRPC6 _WT/CaM₁₂/M₁R (middle right) (n=10), and TRPC6_WT/CaM₃₄/M₁R (right) (n=10). Cells are evoked by CCh (100 μM) at approximately 60 seconds. Average traces are indicated on the panel in black, red, pink, and light-blue lines, respectively. Note that the scales for [Ca²⁺]_i are identical. (B) Representative Ca²⁺-imaging of CCh-evoked TRPC6_WT and TRPC6_WT/TRPC6_K874×. In the TRPC6_K874× panels, a sustained [Ca²⁺]_i elevation is observed (200 seconds). (C) Decay time course of normalized to peak [Ca²⁺]_i from TRPC6_WT (black line) and TRPC6_K874× (red), CaM₁₂ (pink) and CaM₃₄ (light blue). The horizontal axis represents time after transient Ca²⁺ peak. (D) TRPC6 antagonist (Pyr-4, 10 μM) suppresses the [Ca²⁺]_i responses in podocytes expressing TRPC6_WT/M₁R (left) and TRPC6_WT/TRPC6_K874×/M₁R (right). (E) Whole-cell recordings from podocytes overexpressing TRPC6_WT, TRPC6_WT/TRPC6_K874×, and TRPC6_WT/CaM₁₂ with M₁R (V_h=−50 mV). (F) I–V relationship curve recorded from the representative traces (a–c). (G) Residual current plots of TRPC6_WT, TRPC6_WT/TRPC6_K874×, and TRPC6_WT/CaM₁₂ are shown as black circles, red squares, and pink triangles, respectively (n=5).

Figure 6.

Distribution of TRPC6 channels and pathologic F-actin organization in podocytes. (A) Confocal images of podocytes overexpressing GFP-TRPC6_WT/M₁R (left panels; BF, bright field) and GFP-TRPC6_K874×/M₁R (right panels). Expanded boxes represent GFP fluorescence in foot process–like structure. (B) The frequency of occurrence of the four types of F-actin manifestations in cultured podocytes stained by Alexa Fluor 546 phalloidin (parallel fiber: shown in black bar; rim: white bar; ARC: gray bar; and others: dark gray bar). No stimulation (−) and CCh (100 μM) stimulation for 10 minutes (+). Numbers of cells displaying each F-actin pattern are labeled on each stacked bar. (C) Summary figure. Disruption of the CaM-bridge due to the abnormal assembly between coiled-coil segments disrupts CDI of TRPC6, thereby prolonging channel opening and enhancing ion influx. Sustained Ca²⁺ elevation stimulates downstream signaling cascades and F-actin rearrangements in podocytes. DAG, diacylglycerol; PLC, phospholipase C.