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Sci Rep
2015 Jan 12;5:15752. doi: 10.1038/srep15752.
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Acid-induced off-response of PKD2L1 channel in Xenopus oocytes and its regulation by Ca(2.).
Hussein S
,
Zheng W
,
Dyte C
,
Wang Q
,
Yang J
,
Zhang F
,
Tang J
,
Chen XZ
.
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Polycystic kidney disease (PKD) protein 2 Like 1 (PKD2L1), also called transient receptor potential polycystin-3 (TRPP3), regulates Ca(2+)-dependent hedgehog signalling in primary cilia, intestinal development and sour tasting but with an unclear mechanism. PKD2L1 is a Ca(2+)-permeable cation channel that is activated by extracellular Ca(2+) (on-response) in Xenopus oocytes. PKD2L1 co-expressed with PKD protein 1 Like 3 (PKD1L3) exhibits extracellular acid-induced activation (off-response, i.e., activation following acid removal) but whether PKD1L3 participates in acid sensing remains unclear. Here we used the two-microelectrode voltage-clamp, site directed mutagenesis, Western blotting, reverse transcriptase-polymerase chain reaction (RT-PCR) and immunofluorescence, and showed that PKD2L1 expressed in oocytes exhibits sustained off-response currents in the absence of PKD1L3. PKD1L3 co-expression augmented the PKD2L1plasma membrane localization but did not alter the observed properties of the off-response. PKD2L1 off-response was inhibited by an increase in intracellular Ca(2+). We also identified two intra-membrane residues aspartic acid 349 (D349) and glutamic acid 356 (E356) in the third transmembrane domain that are critical for PKD2L1 channel function. Our study suggests that PKD2L1 may itself sense acids and defines off-response properties in the absence of PKD1L3.
Figure 1. Acid-induced off-response currents in Xenopus laevis oocytes over-expressing human PKD2L1.(A) Representative currents recorded by the two-microelectrode voltage clamp at â50âmV from an oocyte expressing human PKD2L1 or a water-injected oocyte (as a control). An off-response current is defined as the difference between the current amplitudes at pH 7.5 and pH 3.0. The pH values of the standard extracellular solutions are indicated. (B) Representative currentâvoltage relationship curves obtained using a voltage ramp protocol at the time points indicated by âaâ and âbâ in the panel A tracings. (C) RT-PCR using Xenopus oocytes detecting the RNA signals of PKD2L1 (2L1), PKD1L3 (1L3) and β-actin. The predicted sizes are 658âbp, 706âbp and 718âbp for β-actin, PKD1L3 and PKD2L1, respectively. (D) Second (nested) PCR detecting PKD1L3 and PKD2L1 signals using Xenopus oocytes (O), tongue (T), kidney (K) and brain (B). β-actin signals served as positive controls.
Figure 2. Off-response currents in oocytes expressing PKD2L1 with or without PKD1L3.(A) Averaged and normalized off-response currents elicited by extracellular pH 3.0 from oocytes injected with 4 or 25âng of PKD2L1 (2L1) mRNA alone or co-injected with 25âng of PKD1L3 (1L3) mRNA, and voltage clamped at â50âmV. Shown are off-response currents averaged from different numbers of oocyte, as indicated. *pâ=â0.05 and **pâ=â0.004, unpaired t-test. (B) Representative off-response I-V curves obtained using a ramp protocol under the same conditions as in panel A. (C) Representative immunofluorescence data showing the PM intensity of PKD2L1 protein in oocytes injected with different amounts of mRNAs, as indicated.
Figure 3. Dose dependence of the acid-induced off-response.(A) representative current recorded at â50âmV and different extracellular pH, as indicated, from a PKD2L1-expressing oocyte. (B) pH dose dependence of the off-response currents for oocytes injected with PKD2L1 mRNA or water. [H+] indicates protons concentration. Data points represent average values from Nâ=â5â9 with the standard error of the mean (SEM) and were fitted to the Michaelis Menton Equation, which produced a Km value of pH 3.5â±â0.5.
Figure 4. Effects of acid application time and type on the off-response.(A) Representative current recorded at â50âmV and with the application of the standard solution at pH 3.0 for different time intervals in an oocyte expressing PKD2L1. (B) Averaged and normalized off-response currents elicited by HCl or citric acid at pH 3.0 were compared from the same oocytes. (C) Averaged and normalized off-response currents elicited by HCl or acetic acid at pH 3.0 were compared from the same oocytes (***pâ=â0.0004, paired t-test).
Figure 5. Acid-induced vs Ca2+-induced channel activation of PKD2L1.(A) Representative current recorded at â50âmV in a PKD2L1-expressing oocyte to show the on-response induced by 5 mM extracellular Ca2+ (at pH 7.5) and the subsequent off-response induced by extracellular pH 3.0 (no extracellular Ca2+). (B) Representative current recorded at â50âmV in a PKD2L1-expressing oocyte to show the on-response induced by 5 mM extracellular Ca2+ (at pH 3.0) and the subsequent off-response induced by extracellular pH 3.0 (no extracellular Ca2+). (C) IâV curves generated at the time points (âaâââdâ) indicated in panels A and B.
Figure 6. Roles of Ca2+ ions in the PKD2L1 off-response.(A) Representative tracing recorded at â50âmV, showing the off-response activation and ensuing inactivation in the presence of extracellular Ca2+ (5âmM). (B) Representative current recorded at â50âmV in a PKD2L1-expressing oocyte, showing the inhibition of the off-response current by extracellular Ca2+ (5âmM). (C) Representative current recorded at â50âmV in a PKD2L1-expressing oocyte 1âhr after injection of 50ânL of 50âmM EGTA, showing the effect of extracellular Ca2+ (5âmM). (D) Averaged and normalized off-response currents with EGTA or water (Ctrl) pre-injection. (E) Averaged percentage of the off-response current inhibition by extracellular Ca2+ with EGTA or water pre-injection. ***pâ=â0.0007, unpaired t-test.
Figure 7. Role of extracellular Ca2+ ions in the PKD2L1 off-response.(A) Representative current recorded at â50âmV in a PKD2L1-expressing oocyte under various extracellular conditions, as indicated. (B) Based on experiments described in panel A, averaged data to show the effect of extracellular EGTA (1âmM). *pâ=â0.04, unpaired t-test. (C) Dose-dependence of the relative Ca2+ inhibition of the off-response current. Data were averaged from 11 oocytes and the curve is a fit of the data to Equation 1, with Ki value of 167â±â33âμM.
Figure 8. Effects of mutation D523N on the PKD2L1 on- and off-responses.(A) Averaged and normalized on- and off-response currents elicited by extracellular 5âmM Ca2+ and pH 3.0, respectively, from oocytes expressing WT or mutant PKD2L1 voltage clamped at â50âmV. Shown are on- (***pâ=â3.0e-6, unpaired t-test) and off-response currents (**pâ=â0.002, unpaired t-test) averaged from different numbers of oocyte, as indicated. (B) Representative off-response I-V curves obtained by a ramp protocol under the same condition as in panel A. (C) Western blotting data showing the protein expression of WT and mutant PKD2L1 in expressing or water-injected (Ctrl) oocytes. (D) Representative immunofluorescence data showing the PM localization of WT and mutant PKD2L1 in oocytes.
Figure 9. Effects of negatively charged intramembrane residues on the PKD2L1 on- and off-responses.(A) Representative data showing mRNA and protein bands that were revealed in 1% agarose gel and 8% SDS-PAGE, respectively. (B) Averaged and normalized on- and off-response currents elicited by extracellular 5âmM Ca2+ and pH 3.0, respectively. Oocytes expressing WT, mutant or water-injected oocytes (Ctrl) were voltage clamped at â50âmV. The number of each group is indicated in a bracket. On-response currents were significantly reduced, with *pâ=â0.03 and 0.05 (unpaired t-test) for D349N and E356N, respectively. Off-response currents were also significantly reduced, with *pâ=â0.05 and **pâ=â0.01 (unpaired t-test) for D349N and E356N, respectively. (C) Representative immunofluorescence data showing the PM localization of PKD2L1 WT, D349N and E356N expressed in oocytes.
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