Identification and Properties of TRPV4 Mutant Channels Present in Polycystic Kidney Disease Patients

Abstract

Polycystic kidney disease (PKD), a disease characterized by the enlargement of the kidney through cystic growth is the fourth leading cause of end-stage kidney disease world-wide. Transient receptor potential Vanilloid 4 (TRPV4), a calcium-permeable TRP, channel participates in kidney cell physiology and since TRPV4 forms complexes with another channel whose malfunction is associated to PKD, TRPP2 (or PKD2), we sought to determine whether patients with PKD, exhibit previously unknown mutations in TRPV4. Here, we report the presence of mutations in the TRPV4 gene in patients diagnosed with PKD and determine that they produce gain-of-function (GOF). Mutations in the sequence of the TRPV4 gene have been associated to a broad spectrum of neuropathies and skeletal dysplasias but not PKD, and their biophysical effects on channel function have not been elucidated. We identified and examined the functional behavior of a novel E6K mutant and of the previously known S94L and A217S mutant TRVP4 channels. The A217S mutation has been associated to mixed neuropathy and/or skeletal dysplasia phenotypes, however, the PKD carriers of these variants had not been diagnosed with these reported clinical manifestations. The presence of certain mutations in TRPV4 may influence the progression and severity of PKD through GOF mechanisms. PKD patients carrying TRVP4 mutations are putatively more likely to require dialysis or renal transplant as compared to those without these mutations.

Keywords: PKD; TRPV4; channelopathies; gain-of-function; ion channels; kidney disease; polycystic kidney disease; renal function.

Graphical Abstract

Figure 1.

Mutations of two residues in the N-terminal intrinsically disordered region (IDR) result in constitutive activity of TRPV4. (A) Structure of the human TRPV4 channel (PDB: 8FC8), where one subunit is highlighted for visual clarity. The N-terminal IDR was modelled with the SWISS-MODEL server (Guex and Peitsch, 1997; Guex et al., 2009; Waterhouse et al., 2018) for representative purposes. Residues E6 and S94 are depicted. (B-D) Representative currents were obtained at −120 mV and +120 mV for 100 ms from a holding potential of 0 mV in the inside-out configuration in wild-type (WT, B) and mutant E6K (C) and S94L (D) channels. Leak or initial currents and currents in the presence of 10 n m or 1 µm GSK1016790A (GSK) are shown, as indicated by labels. (E) Dose-response curves for activation by GSK measured at +120 mV in inside-out patches of HEK293 cells expressing WT or mutant E6K or S94L channels. The smooth curves are fits with the Hill equation [K_D = 95 ± 5 n m for WT (n = 8-17), 65 ± 5 n m for E6K (n = 4-8) and 93 ± 6 n m for S94L (n = 5-11)]. Hill coefficients were 1.9 for WT, 2.6 for E6K, and 2.4 for S94L. A single GSK concentration was tested per membrane patch and normalized to the current at saturating 1 µm GSK in the same patch. Group data are reported as the mean ± s.e.m. Statistical significance was of *P < 0.01 between WT versus E6K and *P < 0.01 versus S94L in the presence of 10 n m GSK; *P < 0.01 between WT versus E6K and *P < 0.01 versus S94L in the presence of 20 n m GSK; *P < 0.05 between WT versus S94L in the presence of 50 n m GSK and *P < 0.01 versus E6K in the presence of 100 n m GSK. No statistical differences were observed starting from 200 n m to higher GSK concentrations between activation of the WT and mutant channels. A parametric unpaired t-test was performed. (F) Representative traces of single-channel recordings from WT TRPV4 channels in the unliganded state (P_o = 0; i = 0 pA, n = 7) and with 10 n m (P_o = 0.34 ± 0.06; i = 6.3 ± 0.5 pA, n = 7) or 100 n m (P_o = 0.8 ± 0.07; i = 6.3 ± 0.3 pA, n = 6) GSK. (G) Representative single-channel recordings for the mutant TRPV4- E6K channel in the unliganded state (P_o = 0.18 ± 0.003; i = 6.7 ± 0.5 pA, n = 4) and with 10 n m (P_o = 0.94 ± 0.03; i = 5.8 ± 0.2 pA, n = 5) or 100 n m (P_o = 0.91 ± 0.08; i = 6.15 ± 0.2 pA, n = 2) GSK. (H) Representative traces for single-channel recordings from the mutant TRPV4-S94L channel in the unliganded state (P_o = 0.51 ± 0.08; i = 5.96 ± 0.4 pA, n = 11) and with 10 n m (P_o = 0.82 ± 0.08; i = 5.98 ± 0.4 pA, n = 6) or 100 n m (P_o = 0.94 ± 0.03; i = 5.97 ± 0.09 pA, n = 3) GSK. Closed and open states are marked with letters C and O, respectively. The histograms below each trace plot the average single-current amplitude elicited in the unliganded state or in the presence of different GSK concentrations. All single-channel recordings were performed in the inside-out configuration of the patch-clamp technique. Representative open probability (P_o) of WT (I), mutant E6K (J) and S94L (K) channels through time. Values for the P_o are shown at +60 mV from 0 (3 s) to up to 160 sweeps (480 s) in the unliganded state and in the presence of 10 and 100 n m GSK. P_o was determined for the 3 s duration for each sweep at +60 mV.

Figure 2.

Mutations of two residues of ARD of the N-terminus of TRPV4 result in constitutively activated macroscopic currents and a shift in the response to the agonist. (A) Depiction of two subunits of the cryo-EM structure (PDB: 8FC8) [82] of the human TRPV4 channel. Residues A217 and R315W are depicted. Representative currents at −120 mV and +120 from a holding potential of 0 mV of wild-type (WT, B) and mutant A217S (C) or R315W (D) channels. Inside-out membrane patches were exposed to bath solution without agonist (initial), 10 n m and 1 µm GSK1016790A (GSK). (E) Dose-response curves for activation by GSK measured at +120 mV in inside-out patches of HEK293 cells expressing WT or mutant A217S or R315W channels. The smooth curves are fits with the Hill equation (K_D = 95 ± 5 n m for WT [n = 8-17, same data as in Figure 1E), 66 ± 6 n m for A217S (n = 5-11) and 83 ± 4 n m for R315W (n = 4-11)]. Hill coefficients were 1.9 for WT, 2.1 for A217S, and 2.7 for R315W. A single GSK concentration was tested per membrane patch and normalized to the current at saturating 1 µm GSK in the same patch. Group data are reported as the mean ± s.e.m. Statistical significance was of *P < 0.01 between WT versus A217S and *P < 0.001 versus R315W in the presence of 10 n m GSK; *P < 0.01 between WT versus A217S and *P < 0.0001 versus R315W in the presence of 20 n m GSK; *P < 0.01 between WT versus A217S and *P < 0.01 versus R315W in the presence of 50 n m GSK and *P < 0.001 versus A217S and *P < 0.01 versus R315W in the presence of 100 n m GSK. No statistical differences started from 200 n m GSK between the WT and mutant channels. A parametric unpaired t-test was performed. Representative single-channel recordings for the A217S TRPV4 mutant in the (F) unliganded state and in the presence of (G)10 n m or (H) 100 n m GSK at +60 mV. The average single-current amplitude elicited in the unliganded state or in the presence of different GSK concentrations is plotted in the histograms below each trace. (I) Changes in open probability (P_o) through time in the unliganded state and in the presence of 10 and 100 n m GSK, as indicated in the figure. Values for the P_o are shown at +60 mV from 0 (3 s) to up to 150 sweeps (450 s). P_o was determined for the 3 s duration for each sweep at +60 mV. The Hill equation was fitted to the data to obtain the Hill coefficient (n) and the apparent dissociation constant (K_D) All-point histograms were constructed, and P_o was calculated and graphed as described in Figure 1.