Polycystin-2L1 (PKD2L1) belongs to the family of transient receptor potential (TRP) ion channels and bears 76% protein sequence similarity to polycystin-2 (PKD2) [1]. Mutations of the latter cause autosomal dominant polycystic kidney disease (ADPKD). PKD2L1 is characterized by a large permeability for Ca²⁺ [2], which has been attributed to the structure of its selectivity filter [3]. Interestingly, structural information of PKD2L1 suggests that a selectivity filter residue D523 forms a salt bridge interaction with a pore loop residue R518 [4]. Thus, R518 might stabilize the selectivity filter thereby affecting PKD2L1 ion channel properties. Here we investigate the relevance of R518 for the ion channel function of PKD2L1.

To eliminate its potential salt bridge interaction with D523, R518 was substituted with a cysteine residue using site-directed mutagenesis. Mutant PKD2L1_R518C and wildtype PKD2L1 constructs were heterologously expressed in Xenopus laevis oocytes for functional analysis using the two-electrode voltage clamp (TEVC) technique. To assess calcium entry into oocytes in a qualitative manner, a fluorescent assay was established using the calcium-sensitive dye Fluo-4 and 50mM CaCl₂ bath solution. Values are presented as mean +/- SEM and ANOVA with Bonferroni post-hoc-test was used for statistical analysis.

In control experiments, application of calcium ionophore ionomycin in 50 mM CaCl₂ bath solution resulted in a ~4-fold increase of fluorescence intensity in Fluo-4-injected oocytes [n=47]. Injecting the Ca²⁺ chelator EGTA into oocytes prevented the ionomycin-induced fluorescence increase, confirming that this was due to Ca²⁺ influx [n=48]. Importantly, PKD2L1 expressing oocytes placed in 50 mM CaCl₂ bath solution [n=42] demonstrated a similar increase of fluorescence as ionomycin-treated oocytes [n=40]. In contrast, the PKD2L1_R518C failed to produce an increase in fluorescence, indicating reduced Ca²⁺ permeability of the mutant channel [n=29]. In parallel TEVC experiments, application of 50 mM CaCl₂ resulted in large inward currents due to activation of calcium-activated chloride channels (CaCC) in PKD2L1 expressing oocytes [n=32, -2.49 µA +/- 0.18 µA]. This was not observed in oocytes pre-injected with EGTA [n=31, -0.33 µA +/- 0,02 µA] or when 50 mM MgCl₂ was applied instead of CaCl₂ [n=25, -0.22 µA +/- 0.03 µA]. Similarly, oocytes expressing PKD2L1_R518C  did not reveal a measurable activation of CaCC currents in 50 mM CaCl₂ [n=38, – 0.23 µA +/- 0.01 µA, n.s.], further supporting reduced calcium permeability of the mutant channel. Interestingly, in a solution containing 96 mM Na⁺ PKD2L1_R518C exhibited significantly larger Na⁺-inward currents than wildtype PKD2L1 [-1.00 µA +/- 0.08 µA, n=22 versus -0.53 µA +/- 0.05 µA, n=23; p<0.001]. This suggests that R518C selectively reduces PKD2L1 permeability for divalent but not for monovalent cations.

Taken together, our data indicate that the pore loop residue R518 is essential for Ca²⁺ permeability of PKD2L1, probably due to its role in stabilizing the selectivity filter. Interestingly, this arginine residue is conserved in PKD2 (R638) and the corresponding mutation R638C has been reported to be associated with the ADPKD [5]. Therefore, our findings regarding PKD2L1 may help to understand functional consequences of disease causing PKD2 mutations which remain to be explored in future studies.