Molecular Determinants of the Sensitivity to Gq/11-Phospholipase C-dependent Gating, Gd3+ Potentiation, and Ca2+ Permeability in the Transient Receptor Potential Canonical Type 5 (TRPC5) Channel

Abstract

Transient receptor potential canonical type 5 (TRPC5) is a Ca²⁺-permeable cation channel that is highly expressed in the brain and is implicated in motor coordination, innate fear behavior, and seizure genesis. The channel is activated by a signal downstream of the G-protein-coupled receptor (GPCR)-G_q/11-phospholipase C (PLC) pathway. In this study we aimed to identify the molecular mechanisms involved in regulating TRPC5 activity. We report that Arg-593, a residue located in the E4 loop near the TRPC5 extracellular Gd³⁺ binding site, is critical for conferring the sensitivity to GPCR-G_q/11-PLC-dependent gating on TRPC5. Indeed, guanosine 5'-O-(thiotriphosphate) and GPCR agonists only weakly activate the TRPC5_R593A mutant, whereas the addition of Gd³⁺ rescues the mutant's sensitivity to GPCR-G_q/11-PLC-dependent gating. Computer modeling suggests that Arg-593 may cross-bridge the E3 and E4 loops, forming the "molecular fulcrum." While validating the model using site-directed mutagenesis, we found that the Tyr-542 residue is critical for establishing a functional Gd³⁺ binding site, the Tyr-541 residue participates in fine-tuning Gd³⁺-sensitivity, and that the Asn-584 residue determines Ca²⁺ permeability of the TRPC5 channel. This is the first report providing molecular insights into the molecular mechanisms regulating the sensitivity to GPCR-G_q/11-PLC-dependent gating of a receptor-operated channel.

Keywords: G-protein-coupled receptor (GPCR); calcium; ion channel; patch clamp; transient receptor potential channels (TRP channels).

FIGURE 1.

The putative membrane topology of mouse TRPC5 and effects of Gd³⁺ on GTPγS-activated currents in mouse TRPC5_R545A- and mouse TRPC5_R593A-expressing HEK cells. A, the TRPC5 subunit contains six transmembrane helices (S1-S6). The eCBS consists of the two acidic residues Glu-543 and Glu-595 (solid blue circles) located on E3 and E4 loops. The arginine residues Arg-545 and Arg-593 are shown with the solid red circles. B–D, sample traces of TRPC5 currents induced by a dialysis of GTPγS via the patch pipette. Gd³⁺ (100 μm) or NMDGCl was added at the times indicated by the horizontal bars. The broken lines indicate the level of the zero current. The upper and lower traces represent the outward and inward whole cell currents recorded at +100 mV (orange lines) and −100 mV (blue line). Insets show the current-voltage relations acquired during the voltage ramps from −100 mV to +100 mV in the absence (black lines) and presence (red lines) of Gd³⁺ at the time points indicated with a and b in the same experiment. E, comparison of the mean current densities of GTPγS-activated currents measured at p in (B–D). F, sample traces of TRPC5, TRPC5_R545A, and TRPC5_R593A currents activated by the depolarizing pulses to +150 mV from a holding potential of −60 mV. G, comparison of the densities of TRPC5, TRPC5_R545A, and TRPC5_R593A currents activated by the depolarizing pulses to +150 mV. The current density values were averaged within a 20-ms interval. H, Western blot analysis of cell-surface biotinylated proteins isolated from HEK cells overexpressing nothing, TRPC5, or TRPC5_R593A. The upper Western blot confirms the specificity of the TRPC5 antibody (NT = non-transfected HEK lysate). The middle and lower images show the results of Western blot analyses of cell-surface biotinylated proteins probed with the TRPC5 antibody or the Na⁺,K⁺-ATPase antibody, respectively (six independent biotinylation experiments). The exposure times were 10 s for the TRPC5 antibody and 30 s for the Na⁺,K⁺-ATPase antibody. 1, 2, and 3 indicate the lanes where biotinylated proteins from HEK-TRPC5 cells were separated, whereas 4, 5, and 6 indicate the lanes where biotinylated proteins from HEK-TRPC5_R593A cells were separated. I, densitometry analyses of the data shown in H. The upper panel shows a comparison of amounts of cell-surface biotinylated TRPC5_R593A proteins relative to that of cell-surface biotinylated TRPC5 proteins. The lower panel shows a comparison of biotinylated protein band densities of TRPC5 and TRPC5_R593A normalized to that of Na⁺,K⁺-ATPase. J and K, summary data of current ratios and densities measured at the time points of a and b”, before and after Gd³⁺ addition to the bath, at the holding potentials of −100 mV (blue dots), and +100 mV (orange dots) for the data sets shown in B–D. In all three plots of K, the y axis label is Current densities before and after Gd³⁺ application. The ratios quantify the relative current amplitude increases in the presence of Gd³⁺. One-way ANOVA on Rank followed by the all-pairwise comparison Dunn's test was used in E, G, and J, the unpaired t test was used in I, and the before and after treatment Wilcoxon Signed Rank Test was used in K to determine whether there is a significant difference between the tested groups (*, p < 0.05; **, p < 0.01). ns stands for not significant. The vertical error bars represent standard deviations, and the mean values are indicated with horizontal bars.

FIGURE 2.

Effects of Gd³⁺ or Ca²⁺ on histamine-induced currents in mouse TRPC5- and mouse TRPC5_R593A-expressing HEK cells. A–D, sample traces of time courses for currents via TRPC5 or TRPC5_R593A. Histamine (5 μm), Gd³⁺ (100 μm), Ca²⁺ (2 mm), and NMDGCl were added at the times indicated by the horizontal bars. The broken lines indicate the level of the zero current. The upper traces represent the outward whole cell currents recorded at +100 mV (orange lines), whereas the lower traces represent the inward currents recorded at −100 mV (blue lines). Insets show the current-voltage relationships acquired during the voltage ramps from −100 mV to +100 mV in the absence (black lines) and presence (red lines) of Gd³⁺ in the same experiment. E, comparison of current densities measured at the indicated points (b, e, i, l) at the holding potentials of −100 (blue dots) and +100 mV (orange dots). F, comparison of the current ratios of I_c/I_c′ or I_j/I_j′ characterizing Gd³⁺-induced potentiation at the holding potentials of −100 (blue dots) and +100 mV (orange dots). G, comparison of current densities measured before and after the addition of Gd³⁺ at the holding potentials of −100 (blue dots) and +100 mV (orange dots). H, comparison of current densities before and after re-addition of 2 mm Ca²⁺. In E and F, the red dotted lines show the positions where the y axis scale changes. The Mann-Whitney Rank Sum test was used in E and F, whereas the Wilcoxon Signed Rank Test was used in G and H to determine whether there is a significant difference between the tested groups (*, p < 0.05, **, p < 0.01, ***, p < 0.001). ns stands for not significant. The vertical error bars represent standard deviations, and the mean values are indicated with horizontal bars.

FIGURE 3.

Dose-response curves for histamine in mouse TRPC5- and mouse TRPC5_R593A-expressing HEK cells. A–D, sample traces of the currents induced by incremental increases in concentration of histamine in HEK cell expressing TRPC5 or TRPC5_R593A. Histamine (0.01, 0.1, 1, 10, 100, 300 μm), Gd³⁺ (100 μm), and NMDGCl were added at the time points indicated by the horizontal bars. The broken lines indicate the level of the zero current. The upper traces represent the outward whole cell currents recorded at +100 mV (orange lines), whereas the lower traces represent the inward currents recorded at −100 mV (blue lines). Insets show the current-voltage relations acquired during the voltage ramps from −100 mV to +100 mV at the time points indicated with a–e. E, the dose-response curves for histamine obtained in HEK-TRPC5 cells (n = 5) and HEK-TRPC5_R593A cells (n = 6) in the presence or absence of Gd³⁺ (100 μm). The inset shows the dose-response curve for histamine with an expended y axis scale obtained in HEK-TRPC5_R593A cells in the absence of Gd³⁺. F, comparison of current densities of histamine-activated currents recorded at −100 mV (blue dots) and +100 mV (orange dots) measured at different points in A–D. The red dotted lines show the positions where the y axis scale changes. The vertical error bars represent standard deviations, and the mean values are indicated with horizontal bars (*, p < 0.05, Mann-Whitney Rank Sum test).

FIGURE 4.

Homology modeling and molecular dynamics simulations. A, superimposed mouse TRPC5 homology model (green) and the cryo-EM structure of the rat TRPV1 channel (wheat) in the open state. Glu-543 and Glu-595 residues in TRPC5 are colored red. B–D, The interatomic interactions are depicted in the selected conformations predicted from molecular dynamics simulations. The S5 domain and the E3 loop are colored in green. The S6 domain, E4 loop, and the pore helix are colored in sky blue. Interatomic distances in angstroms are shown as black numbers next to the yellow broken lines. E and F, the interatomic Gd³⁺ (the orange ball) and side chain interactions within the eCBS predicted from molecular dynamics simulations. The color coding is as in B–D.

FIGURE 5.

Effects of Gd³⁺ on GTPγS-activated currents mediated by mouse TRPC5, mouse TRPC5_Y541A, mouse TRPC5_E543Q, mouse TRPC5_Y542A, and mouse TRPC5_F540A. A, the sequence alignment for mouse TRPC1, TRPC3, TRPC4, TRPC5, TRPC6, and TRPC7. The TRPC5's Tyr-541 and Tyr-542 residues are highlighted in sky blue. The glutamate residues Glu-543 and Glu-595 are highlighted in yellow. The Arg-593 residue in TRPC5 is highlighted in red. B–E, time courses of the whole-cell currents induced by a dialysis of 500 μm GTPγS in HEK cells overexpressing TRPC5 or one of the mutants listed above. Gd³⁺ (100 μm) and NMDGCl were added at the times indicated by the horizontal bars. The broken lines indicate the level of the zero current. The upper and lower traces represent the outward and inward whole cell currents recorded at +100 mV (orange lines) and −100 mV (blue lines). Insets show the current-voltage relations recorded in the absence (black lines) and presence (red lines) of Gd³⁺. F, comparison of the mean current densities of GTPγS-activated currents recorded at −100 mV (blue dots) and +100 mV (orange dots) in HEK cells transfected with TRPC5-WT, TRPC5_Y541A, TRPC5_Y542A, TRPC5_F540A, or TRPC5_E543Q. The red dotted lines show the positions where the y axis scale changes. G and H, summary data of current ratios and current densities measured at the time points of a and b at holding potentials of −100 mV (blue dots) and +100 mV (orange dots) for the data sets shown in B–E. The ratios quantify the relative current amplitude increases in the presence of Gd³⁺. In all five plots of H, the y axis label is GTPγS-induced current density measured at a and b (pA/pF). The vertical error bars represent standard deviation, and the mean values are indicated with horizontal bars (*, p < 0.05; ***, p < 0.001; ns, stands for not significant; one-way ANOVA on Rank followed by the all pairwise comparison Dunn's test in F and G, unpaired t test (black line in F), paired t test or Wilcoxon Signed Rank Test in H).

FIGURE 6.

Effects of Gd³⁺ on histamine-activated currents mediated by mouse TRPC5, TRPC5_Y541A, TRPC5_E543Q, TRPC5_Y542A, TRPC5_F540A, and TRPC5_R545A. A–E, time courses of the whole-cell currents induced by 5 μm histamine in HEK cells overexpressing one of the mutants listed above. Gd³⁺ (100 μm) and NMDGCl were added at the times indicated by the horizontal bars. Insets show the current-voltage relations. F, comparison of the mean current densities of histamine-induced currents recorded at −100 mV (blue dots) and +100 mV (orange dots) in HEK cells transfected with TRPC5-WT, TRPC5_Y541A, TRPC5_Y542A, TRPC5_F540A, TRPC5_E543Q, or TRPC5_R545A. G and H, summary data of current ratios and current densities measured at the time points of a and b at holding potentials of −100 mV (blue dots) and +100 mV (orange dots) for the data sets shown in A–E. The ratios represent the relative current amplitude increases in the presence of Gd³⁺. In all six plots of H, the y axis label is Histamine-induced current density measured at a and b (pA/pF). The error bars represent standard deviation, and the mean values are indicated with horizontal bars (*, p < 0.05; **, p < 0.01, ns stands for not significant; one-way ANOVA on Rank followed by the all pairwise comparison Dunn's test in F and G, paired t test or Wilcoxon Signed Rank Test in H).

FIGURE 7.

The Asn-584 residue determines Ca²⁺ permeability in TRPC5. A, amino acid sequence alignment of TRPC5 (Leu-538 through Glu-598) and TRPV1. The epitope of the T5E3 antibody is indicated in the sequence with a purple box. The Glu-543 and Glu-595 residues in TRPC5 are highlighted in yellow. The Arg-593 residue in TRPC5 is highlighted in red. The Asn-584 residue in TRPC5 and the Asp-646 residue in TRPV1 are highlighted in green. The Tyr-541 and Tyr-542 residues in TRPC5 are highlighted in sky blue. B–D, sample traces of time courses of the histamine-induced currents through human TRPC5, human TRPC5_N584D, and human TRPC5_N584G mutants. Histamine (5 μm), Gd³⁺ (100 μm), and NMDGCl were added at the times indicated by the horizontal bars. The broken black lines indicate the level of the zero current. The upper and lower traces represent the outward and inward whole cell currents recorded at +100 mV (orange lines) and −100 mV (blue lines). The inset in D shows the summary data of current ratios measured at the time points of a and b at the holding potentials of −100 mV. E, F, H, and I, current-voltage relations of TRPC5_N584D and TRPC5_N584G currents acquired at the time points with 10 mm Ca²⁺ (sky blue lines) and with 150 mm NaCl (black lines) in the solution. The currents are activated by GTPγS (E and F) or histamine (H and I), G and J, summary data showing the Na⁺ and Ca²⁺ permeability ratio for TRPC5_N584D and TRPC5_N584G currents activated by GTPγS and histamine as compared with that for TRPC5. The vertical error bars represent standard deviation, and the mean values are indicated with horizontal bars (*, p < 0.05; **, p < 0.01; one-way ANOVA followed by the all pairwise comparison Student-Newman-Keuls test).

FIGURE 8.

Heating to 42 °C inhibits Gd³⁺-potentiated mouse TRPC5 and mouse TRPC5_R593A currents. A, B, D, and E, sample traces of histamine and GTPγS-induced TRPC5 and TRPC5_R593A currents. Gd³⁺ (100 μm) and the extracellular solution heated to 42 °C was added at the times indicated by the horizontal bars. The broken black lines indicate the level of the zero current. C and F, summary data for current inhibition by the extracellular solution heated to 42 °C (V_holding = −100 mV, the vertical error bars represent standard deviation, and the mean values are indicated with horizontal bars; ns stands for not significant, Mann-Whitney Rank Sum Test).

FIGURE 9.

A conformation of the open R593A mutant of mouse TRPC5. The model predicts that in the mutant, the Glu-543 to Glu-595 cross-distance is ∼11 Å. It is likely that Arg-545 may form a π-interaction with the Tyr-542 aromatic ring and a dipole-dipole interaction with the main-chain carbonyl moiety of Glu-543, whereas Lys-591 is too far (∼8 Å) from Glu-543 to form a salt bridge.