External Ca2+ regulates polycystin-2 (TRPP2) cation currents in LLC-PK1 renal epithelial cells
Introduction
PC2 is a member of the superfamily of TRP cation channels [23] that is encoded by the PKD2 gene, whose mutations are responsible for ADPKD [21]. PC2 is a Ca2+-permeable nonselective cation channel [10], [36] that is implicated in a number of cellular functions [9]. PC2 has been detected in different cellular locations [7], including the plasma membrane [18], [25], the endoplasmic reticulum [15], [25], and the primary cilium [2], [14], [29]. Ciliary located PC2 in particular, is reported to be one of the key elements in the mechano-sensory response of renal epithelia to fluid flow [24], whose integrity may prevent cyst formation [32]. Previous studies from our laboratory determined a functional PC2 in the primary cilium of LLC-PK1 cells [29], which is regulated by a ciliary located type-2 vasopressin receptor and the production of local cAMP [30]. This PC2 function may have a relevant role in ciliary Ca2+ transport, and the regulation of ciliary length. PC2 activation is also associated with a rise in cell Ca2+ [24], which may not be mediated by the ciliary PC2 but instead functional channels located in the plasma membrane. Thus, Ca2+ influx through a functional plasma membrane PC2 seems to be essential in normal cell responses. Little is known, however, as to the expression and function of endogenous PC2 to the plasma membrane, and how Ca2+-dependent mechanisms may regulate this function. This is particularly evident from expected Ca2+ feedback signals entailing PC2-mediated Ca2+ transport that should depend on the availability of external Ca2+. Recent studies from our laboratory, for example, determined that the isolated protein is not affected by extracellular Ca2+, but instead to a feedback mechanism mediated by Ca2+ influx through the channel, and the regulatory binding of Ca2+ to putative intracellular cytoskeletal partners that are controlled by a local pool of Ca2+ [3]. PC2 regulatory mechanisms that may be associated with external Ca2+ concentrations still are heretofore largely unknown. The fact that PC2 itself does not respond to varying external Ca2+ concentrations [3], invokes the need for other sensing mechanisms of external Ca2+ regulation that may implicate putative “Ca2+ detectors” of extracellular Ca2+. One such mechanism is the Ca2+-sensing receptor (CaSR) [12], [20] that responds to extracellular Ca2+ concentrations in the range of 0.5–10 mM, and has been observed in the various nephron sections of the mammalian kidney [12], [31].
The aim of the present study was to assess whether changes in external Ca2+ concentration modify the whole cell conductance of wild type LLC-PK1 renal epithelial cells, and whether this contribution may be associated with endogenous PC2 function. Our data indicate that high external Ca2+ increased the whole cell conductance of LLC-PK1 cells particularly the stimulation of endogenous PC2-mediated currents, which were blocked either by silencing of the PKD2 gene, or intracellular dialysis with an active anti-PC2 antibody, raised against the carboxy-terminus of the channel protein. The stimulatory effect of high external Ca2+ on the PC2 currents, was mimicked by CaSR agonists, including spermine, and the calcimimetic R-568, in the absence of external Ca2+, and gentamicin in the presence of normal Ca2+. CaSR was identified by immunocytochemistry, and observed immuno-colocalized with the PC2 channel protein in a manner that depended on external Ca2+. The encompassed data are consistent with a regulatory pathway where external Ca2+ modulates a Ca2+ sensing mechanism that effects PC2 regulation in LLC-PK1 renal epithelial cells. This regulatory mechanism may help explain the connection between Ca2+ signals and the onset of ADPKD.
Section snippets
Cell culture
Wild-type LLC-PK1 cells were cultured as described [28], in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), without antibiotics. Cells were grown at 37 °C, in a humidified atmosphere with 5% CO2 to reach partial confluence in two-to-three days. Islands of confluent cells and attached single cells were used for immunocytochemical and electrical studies, respectively.
Immunocytochemistry
Cells were grown on glass coverslips, rinsed twice with phosphate-buffered saline (PBS),
Effect of external Ca2+ on the whole cell conductance of LLC-PK1 cells
To explore the effect of external Ca2+ on the whole cell conductance of cultured wild type LLC-PK1 cells, whole cell voltage clamping was applied to isolated cells by stepping voltages between ±100 mV from a holding potential of zero mV. In the presence of 1.2 Ca, the whole cell conductance was 158±4.0 S/F (n=24, Fig. 1a–c), with a reversal potential (Vrev) of −7.56±1.02 mV. The basal whole cell conductance measured over the linear voltage range (+60 to +100 mV) of LLC-PK1 cells in the absence of
Conclusions
The data in the present study demonstrate that PC2 is normally expressed in the plasma membrane of wild type LLC-PK1 renal epithelial cells, where it makes a significant contribution to the whole cell conductance. This background activity responds to the external Ca2+ concentration, such that it is almost absent under Ca2+-free conditions, and conversely, it is magnified by higher external Ca2+ concentrations. Previous studies determined that PC2 itself does not interact with external Ca2+,
Acknowledgements
This work was partially supported by NIDDK grant 1R01DK077079-01A2 (HFC), and PICT 2012 #1559 MinCyT, Argentina, to HFC. MRC, PLP, MS and HFC are members of the National Research Council of Argentina (CONICET). DCM and GS were visiting graduate students from the School of Medicine, Mendoza, Argentina. We are grateful to Drs. Fernando Pieckenstain for the kind gift of spermine-tetrahydrochloride, and Ana Clara Casadoumecq and Dr. Cecilia Villa Etchegoyen for excellent technical support. XQD and
References (44)
- et al.
Identification and characterization of polycystin-2, the PKD2 gene product
J. Biol. Chem.
(1999) - et al.
Calcium transport and local pool regulate polycystin-2 (TRPP2) function in human syncytiotrophoblast
Biophys. J.
(2013) - et al.
The calcium sensing receptor modulates fluid reabsorption and acid secretion in the proximal tubule
Kidney Int.
(2013) - et al.
Calcium-sensing receptor decreases cell surface expression of the inwardly rectifying K+ channel Kir4.1
J. Biol. Chem.
(2011) - et al.
Extracellular polyamines regulate fluid secretion in rat colonic crypts via the extracellular calcium-sensing receptor
Gastroenterology
(2004) - et al.
Constitutive activity of TRP channels: methods for measuring the activity and its outcome
Methods Enzymol.
(2010) - et al.
Coordinate expression of the autosomal dominant polycystic kidney disease proteins, polycystin-2 and polycystin-1, in normal and cystic tissue
Am. J. Pathol.
(1999) - et al.
Morphological and electrical properties of human trophoblast choriocarcinoma, BeWo cells
Placenta
(2008) - et al.
Characterization of Na+-permeable cation channels in LLC-PK1 renal epithelial cells
J. Biol. Chem.
(2004) - et al.
Characterization of single channel currents from primary cilia of renal epithelial cells
J. Biol. Chem.
(2005)
Ketamine – more mechanisms of action than just NMDA blockade
Trends Anaesth. Crit. Care
Polycystin-2 is a novel cation channel implicated in defective intracellular Ca2+ homeostasis in polycystic kidney disease
Biochem. Biophys. Res. Commun.
cDNA cloning of porcine PKD2 gene and RNA interference in LLC-PK1 cells
Gene
Calcimimetic R-568 and its enantiomer S-568 increase nitric oxide release in human endothelial cells
PLoS ONE
Cellular and subcellular distribution of polycystin-2, the protein product of the PKD2 gene
J. Am. Soc. Nephrol.
Termination of cAMP signals by Ca2+ and Gαi via extracellular Ca2+ sensors: a link to intracellular Ca2+ oscillations
J. Cell Biol.
The versatile nature of the calcium-permeable cation channel TRPP2
EMBO Rep.
Polycystin-2, the protein mutated in autosomal dominant polycystic kidney disease (ADPKD), is a Ca2+-permeable nonselective cation channel
Proc. Natl. Acad. Sci. USA
SUMOylation protects against IL-1β-induced apoptosis in INS-1 832/13 cells and human islets
Am. J. Physiol. Endocrinol. Metab.
Extracellular calcium sensing and signalling
Nat. Rev. Mol. Cell Biol.
Novel Ca receptor signaling pathways for control of renal ion transport
Curr. Opin. Nephrol. Hypertens.
Subcellular localization and trafficking of polycystins
Pflu¨g. Arch.
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2018, Life SciencesCitation Excerpt :The combination of protein kinase with the corresponding protein kinase phosphorylation sites can induce phosphorylation of CaSR, and result in the activation of downstream signaling pathways to transmit CaSR message [2,3]. CaSR, as a membrane receptor, it cannot directly permeate ions into cells, sometimes it complexes with the TRP family induce cations such as Ca2+, Na+, and Mg2+ enter into cells viz., CaSR-TRPV4 [4], CaSR-TRPP2 [5], CaSR-TRPA1 [6], CaSR-TRPC3 [7] and CaSR-TRPC6 [8]. CaSR is activated by numerous stimuli including cations (Ca2+, Be2+, Sr2+, Mg2+, Gd3+, Ba2+), amino acids (phenylalanine, tyrosine), polyamines (spermine, spermidine), polypeptides (poly‑l‑arginine, poly‑l‑lysine), and aminoglycoside antibiotics (neomycin, gentamicin C), which can bind at the orthostatic site of ECD [9–11].
Role of the microtubules in the electrical activity of the primary cilium of renal epithelial cells
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Xiao Qing Dai and Paula L. Perez made equal contributions to the present study.