Acid-induced off-response of PKD2L1 channel in Xenopus oocytes and its regulation by Ca(2.).

Shaimaa Hussein,Ying Cao,Xing-Zhen Chen,Qian Wang,Chris Dyte,Wang Zheng,Jungwoo Yang,Fan Zhang,Jingfeng Tang

doi:10.1038/srep15752

Abstract

Polycystic kidney disease (PKD) protein 2 Like 1 (PKD2L1), also called transient receptor potential polycystin-3 (TRPP3), regulates Ca2+-dependent hedgehog signalling in primary cilia, intestinal development and sour tasting but with an unclear mechanism. PKD2L1 is a Ca2+-permeable cation channel that is activated by extracellular Ca2+ (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 PKD2L1 plasma membrane localization but did not alter the observed properties of the off-response. PKD2L1 off-response was inhibited by an increase in intracellular Ca2+. 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.

Highlights

Polycystic kidney disease (PKD) protein 2 Like 1 (PKD2L1), called transient receptor potential polycystin-3 (TRPP3), regulates Ca2+-dependent hedgehog signalling in primary cilia, intestinal development and sour tasting but with an unclear mechanism
This large current induced in an off-response manner resembled those associated with PKD2L1/PKD1L3 co-expressed in HEK cells, oocytes and TRCs15,16,18,25
In the present study we have examined acid-dependent properties of human PKD2L1 expressed in Xenopus oocytes using the two-microelectrode voltage clamp electrophysiology, together with molecular biology and mutagenesis techniques

Summary

Introduction

Polycystic kidney disease (PKD) protein 2 Like 1 (PKD2L1), called transient receptor potential polycystin-3 (TRPP3), regulates Ca2+-dependent hedgehog signalling in primary cilia, intestinal development and sour tasting but with an unclear mechanism. In mammalian cells and oocytes, PKD2L1, in complex with PKD protein 1 Like 3 (PKD1L3, another PKD1 homologue), was activated by acids in an off-response manner, i.e., the PKD2L1/PKD1L3 channel complex mediated a large current only after the removal of the extracellular acid[15,16]. It was reported that weak acids, such as citric acid, induce larger off-response currents in HEK cells expressing the PKD2L1/ PKD1L3 complex than strong acids, such as HCl, at the same pH All these acids possessed similar activation threshold values of around pH 3.018. Two negatively charged residues in the PKD2L1 pore loop, D52326 and D52516, have been identified to affect Ca2+ permeation and cation selectivity of the PKD2L1/PKD1L3 channel complex, and may be involved in channel gating and selectivity

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Results

Conclusion