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Gating of the polycystin ion channel signaling complex in neurons and kidney cells¹

PATRICK DELMAS^*,2, SURYA M. NAULI, XIAOGANG LI, BERTRAND COSTE^*, NANCY OSORIO^*, MARCEL CREST^*, DAVID A. BROWN and JING ZHOU

^* Intégration des Informations Sensorielles, CNRS, Faculté de Médecine, IFR Jean Roche, Marseille, France;
Wellcome Laboratory for Molecular Pharmacology, Department of Pharmacology, University College London, London, UK; and
Renal Division, Department of Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston, Massachusetts, USA

² Correspondence: Intégration des Informations Sensorielles (UMR 6150), Faculté de Médecine, IFR Jean Roche, Bd. P. Dramard, 13916 Marseille, Cedex 20, France. E-mail: delmas.p{at}jean-roche.univ-mrs.fr

SPECIFIC AIMS

It is speculated that polycystin-1 (PC1) and polycystin-2 (PC2) are part of a regulatory signaling complex involved in the control of membrane ion transport in target tissues that are defective in autosomal dominant polycystic kidney disease (ADPKD), the most common monogenetic cause of kidney failure in man. We studied gating mechanisms and functional properties of polycystin complexes in living cells and demonstrated for the first time that PC1 and PC2 co-assemble and form a novel macromolecular complex that functions either as a Ca²⁺-permeable cation channel or as a G protein-coupled receptor. Within these complexes, polycystin-1 acts as a receptor that gates polycystin-2 channel via a structural rearrangement of its cytosolic tail.

PRINCIPAL FINDINGS

1. Reconstitution of polycystin ion channel complexes in sympathetic cells
Polycystin complexes were reconstituted in sympathetic cells by intranuclear microinjection of PKD2 and PKD1 cDNAs. PC2 staining was restricted to the endoplasmic reticulum (ER) when expressed alone or when co-expressed with PC1_C193, a C-terminally truncated form of PC1. In contrast, PC2 co-localized with PC1 in the plasma membrane when co-expressed with full-length PC1.

2. Properties of channels formed by polycystin-1/polycystin-2 complexes
Whole-cell perforated patch-clamp recordings made 48 h after cDNA delivery revealed that cells expressing PC1/PC2 complexes displayed a standing cation current that was absent in uninjected cells, mock cells or cells expressing PC1 or PC2 alone. PC1/PC2 current had a reversal potential of –2 ± 3 mV and was voltage-independent. Relative permeability of Na⁺ to K⁺ to Ca²⁺ was 1:0.98:0.57. PC2 formed the channel pore in the PC1/PC2 complex because the current was depressed by amiloride (IC₅₀ 42±8 µM) and La³⁺ (IC₅₀ 62±9 µM), two known blocking agents of PC2 channel homomers, and was also suppressed by cytoplasmic microinjection of an antibody raised against an intracellular epitope (amino acids 44–62) of PC2. The PC2 mutant R742X lacking the C-terminal 226 amino acids, which includes the PC1 interaction domain and the ER retention signal, produced a larger cation current, though it had a similar reversal potential and pharmacology as the current generated by PC1/PC2 channels. Single channel recordings (using Na⁺ as charge carrier) from cells expressing PC1/PC2 and PC2 R742X revealed channels with main chord conductances of 110 and 90 pS, respectively. The open probability (p_o) of PC2 R742X channels was however significantly higher (P<0.001) than that of PC1/PC2 channels (mean p_o at –40 mV: 0.37 for PC2 R742X and 0.11 for PC1/PC2).

3. Activation of polycystin ion channel complex by antibodies raised against extracellular domains of polycystin-1
Local application of MR3, an antibody that binds to amino-acids 2938-2956 on the N-terminal domain of (h)PC1, to cells co-expressing PC1/PC2 induced a robust cation current (–8.6±0.8 pA/pF) (Fig. 1 ). This again was carried by PC2 since it was voltage-independent, had the same reversal potential as the PC2 current, was blocked by amiloride, and was suppressed by cytosolic microinjection of the PC2 antibody (Fig. 1C, D ). No currents were seen in mock cells or cells expressing either PC1 or PC2 (Fig. 1A, B ). In addition, MR3 had no effect on membrane currents of cells co-expressing PC1_C193/PC2, indicating that integrity of the C-terminal tail of PC1 is required for the functionality of the polycystin complex.

View larger version (32K): [in this window] [in a new window]   Figure 1. Activation of polycystin ion channel complex by the MR3 anti-hPC1 antibody. Representative inward currents evoked by 10 s-application of antibody MR3 (1/100) in sympathetic cells expressing hPC1 (A), mPC2 (B) and hPC1/mPC2 (C–D). Note that MR3 activated an inward current only in hPC1/mPC2-expressing cells (C–D), which was blocked by bath application of amiloride (80 µM, C) or cytoplasmic microinjection of the anti-mPC2 antibody (44-62; 1/200, D). Dashes indicate the null-current baseline at –60 mV. Inset in (D): left, schematic diagram of intracellular microinjection of the anti-mPC2 antibody. Right, detergent lysates of HEK293 cells transfected (+) or not (–) with myc-tagged mPC2 were immunoprecipitated with anti-myc antibody and blotted with the anti-mPC2 antibody used to block PC2 channel activity. The arrowhead indicates PC2. Note that lower bands represent reduced heavy chains of precipitated antibody.

4. Concordant activation of polycystin ion channel complex and G_i/o-type G-proteins by polycystin-1 antibodies
We tested whether enhancement of PC2 activity was accompanied by G-protein activation using endogenous N-type Ca²⁺ channels (I_Ca) as sensors for activated G-proteins. We have previously shown that co-expression of PC2 represses tonic activation of G-proteins by PC1, so voltage-dependent inhibition of I_Ca was virtually absent in cells expressing PC1/PC2. MR3 application onto PC1/PC2 cells removed this repression and produced a gradual inhibition of Ca²⁺ currents that presented all the characteristic features of Gß modulation (i.e., slowing in activation kinetics of I_Ca and relief of inhibition by large depolarizing voltages). MR3 no longer inhibit Ca²⁺ currents in the presence G-transducin, a potent Gß sequestering agent or following treatment with pertussis toxin (PTX), demonstrating that MR3 activates G_i/o-type G-proteins and releases their associated ß dimers.

5. Activation of polycystin ion channel complex is independent of G-proteins
Our data suggest that MR3 causes the rearrangement of the C-terminal domain of PC1, which unmasks the PC1 G-protein binding site and leads to G-protein activation. However, G-protein activation was not responsible for MR3-induced PC2 activity because such activity could still be induced in PC1/PC2-expressing cells after pre-treatment with PTX, intracellular injection of the G_q/11 antibody CQ1 (1/200), bath application of N-ethylmaleimide, or intracellular dialysis of GDP-ß-S. In addition, application of U73122 and intracellular dialysis of heparin or 8-NH₂-cADPR had no significant effect on the MR3-evoked PC1/PC2 current. These data suggest that PC1 gates PC2 channel by a mechanism that concomitantly activates G-proteins but that does not require either G-protein or PLC/Ca²⁺ signaling to occur.

6. Gating of polycystin ion channel complexes in kidney epithelial cells derived from wild-type or Pkd1 mutant mice
We applied MR3 antibody on kidney epithelia of distal tubule origin (E15.5) that express PC1 and PC2 and examined Ca²⁺ mobilization. We found that MR3 antibody (1/50), but not normal rabbit serum, evoked a large increase in cytosolic Ca²⁺ concentration in wild-type cells (Fig. 2 A). MR3-induced Ca²⁺ mobilization was prevented in Ca²⁺ free external solution (0 mM Ca²⁺ + 1 mM EGTA) and by the application of the blocking antibody p57 (1/50) directed against the external residues 278-428 of (m)PC2. MR3 response was mediated by PC1 because MR3 failed to generate a Ca²⁺ response in kidney epithelial cells isolated from Pkd1^del34/del34 homozygous mutant embryos (Fig. 2B ). The del34 mutation is predicted to truncate PC1 by 836 amino acids, and mimics many mutations found in human ADPKD patients. Likewise, MR3 was inactive in kidney epithelial cells isolated from Pkd1^null/null mutants that lack PC1.

View larger version (13K): [in this window] [in a new window]   Figure 2. MR3-induced Ca2+ mobilization in mouse embryonic kidney cells requires integral PC1/PC2 complexes. Responses to MR3 antibody (1/100 for 10 s) in increases of cytosolic Ca2+ are shown for both wild-type (A) and PKD1del34/del34 (B) cells. The average (thick lines) and standard errors (thin lines) in response to application of MR3 antibody are shown. At least 2 populations of respective cell lines were used at passages 15 and 16 for each genotype.

We confirmed that plasmalemmal localization of PC2 was severely reduced in cells isolated from Pkd1^del34/del34 mutants, consistent with the inability for this PC1 mutant to physically bind to PC2, and was virtually lost in cells derived from Pkd1^null/null mutants.

CONCLUSIONS AND SIGNIFICANCE

Collectively, our data suggest that PC1 and PC2 form functionally associated "subunits" of a heteromultimeric receptor-channel signaling complex, which is capable of triggering Ca²⁺ responses upon structural rearrangement. Binding of antibodies to the large extracellular domain of PC1 activated bidirectional signaling events, simultaneously enhancing PC2 gating and stimulating G_i/o-proteins. The concordant activation of PC2 and G-proteins appear to proceed through a structural rearrangement of the carboxy terminal tail of PC1 (Fig. 3 ). We propose that genetic alteration of polycystins such as those occurring in ADPKD impede the Ca²⁺-dependent signaling pathways of polycystin complexes thereby providing a likely mechanistic explanation to the pathogenesis of ADPKD. Our proposed mechanism represents a novel form of receptor function that may be paradigmatic for the function of other polycystin orthologs and related proteins in a variety of nonrenal cell types, including sperm, muscle cells and neurons.

View larger version (29K): [in this window] [in a new window]   Figure 3. Model for polycystin ion channel signaling complex. Upper: PC1 and PC2 co-assemble in the plasma membrane via their COOH termini to form a stable polycystin complex that has little constitutive activity. Lower: ligand binding to the N-terminal extracellular domain (arrow) of PC1 causes a structural rearrangement of the polycystin complex that unmasks the G-protein binding site located on the C terminus of PC1 (gray boxes) and opens PC2. This leads to bi-directional signaling events via Gi/o G-proteins and Ca2+.

A challenging momentum remains to identify the putative ligands and molecular cues that activate the polycystin complexes and to define how these different factors adapt polycystin functions to the unique requirements of each tissue. It is hoped that detailed explorations of polycystin complexes will lead to a better understanding of cellular and biochemical mechanisms of cyst development in ADPKD. matic) in order to meet the guidelines. Make all necessary revisions on proof itself; please do not submit a revised disk.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0319fje; doi: 10.1096/fj.03-0319fje

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