The Cytoplasmic C-terminal Fragment of Polycystin-1 Regulates a Ca2+-permeable Cation Channel*

David H. VandorpeDagger, Marina N. Chernova, Lianwei Jiang, Lorenz K. Sellin§, Sabine Wilhelm, Alan K. Stuart-Tilley, Gerd Walz§, and Seth L. Alper

From the Molecular Medicine and Renal Units, Beth Israel Deaconess Medical Center and the Departments of Medicine and Cell Biology, Harvard Medical School, Boston, Massachusetts 02215

Received for publication, July 14, 2000, and in revised form, October 3, 2000



    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The cytoplasmic C-terminal portion of the polycystin-1 polypeptide (PKD1(1-226)) regulates several important cell signaling pathways, and its deletion suffices to cause autosomal dominant polycystic kidney disease. However, a functional link between PKD1 and the ion transport processes required to drive renal cyst enlargement has remained elusive. We report here that expression at the Xenopus oocyte surface of a transmembrane fusion protein encoding the C-terminal portion of the PKD1 cytoplasmic tail, PKD1(115-226), but not the N-terminal portion, induced a large, Ca2+-permeable cation current, which shifted oocyte reversal potential (Erev) by +33 mV. Whole cell currents were sensitive to inhibition by La3+, Gd3+, and Zn2+, and partially inhibited by SKF96365 and amiloride. Currents were not activated by bath hypertonicity, but were inhibited by acid pH. Outside-out patches pulled from PKD1(115-226)-expressing oocytes exhibited a 5.1-fold increased NPo of endogenous 20-picosiemens cation channels of linear conductance. PKD1(115-226)-injected oocytes also exhibited elevated NPo of unitary calcium currents in outside-out and cell-attached patches, and elevated calcium permeability documented by fluorescence ratio and 45Ca2+ flux experiments. Both Ca2+ conductance and influx were inhibited by La3+. Mutation of candidate phosphorylation sites within PKD1(115-226) abolished the cation current. We conclude that the C-terminal cytoplasmic tail of PKD1 up-regulates inward current that includes a major contribution from Ca2+-permeable nonspecific cation channels. Dysregulation of these or similar channels in autosomal dominant polycystic kidney disease may contribute to cyst formation or expansion.



    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

~85% of autosomal dominant polycystic kidney disease (ADPKD)1 is secondary to mutations in the polycystin-1 (PKD1) gene (1). The 4303-aa PKD1 polypeptide has a large N-terminal extracellular domain region encompassing leucine-rich repeats, a C-type lectin domain, a low density lipoprotein A-like domain (2), 16 PKD domain repeats, and a single sperm receptor for egg jelly domain (3). Following a complex polytopic transmembrane domain is a 226-aa C-terminal cytoplasmic tail.

The hallmark pathology of ADPKD is the two-hit generation and gradual expansion of renal cysts. These cysts compress and, ultimately, render nonfunctional adjacent, apparently normal renal tissue (4). Cyst expansion is associated with the conversion of tubular epithelial cells from the normal phenotype of net solute reabsorption to the cystic phenotype of net secretion. This secretory phenotype may involve the function of the apically localized chloride channel, CFTR (5), but a direct link between the ADPKD disease genes for PKD1 and PKD2 and the ion transport processes required to mediate cyst expansion has remained elusive, despite the creation of mouse lines genetically deficient in PKD1 (6) or PKD2 (7). PKD disease mutations have been found throughout the PKD1 coding region (8-10), but absence of the C-terminal cytoplamic tail suffices to cause ADPKD (11).

Multiple potential signaling and binding functions of the 226-aa PKD1 C-terminal cytoplasmic tail have been defined through study of overexpressed transmembrane fusion proteins encompassing all or portions of this C-terminal region (12-14). In this fusion protein context, the PKD1 C-terminal cytoplasmic tail activates PKCalpha and (in a process requiring active Rac or Cdc-42) c-Jun N-terminal kinase. Both pathways are required for downstream activation of AP-1-dependent transcriptional events (12, 13).

Overexpression of the extreme C-terminal part of the PKD1 cytoplasmic tail, PKD1(115-226), activates signaling by Wnt polypeptides via frizzled family receptors to inhibit glycogen synthase kinase 3beta and stabilize beta -catenin, leading to activation of the TCF/LEF family of transcription factors (13). PKD1(115-226) also binds and stabilizes the G protein regulator RGS7 (15) and, through RGS7, 14-3-3 (16). PKD1(115-226) also binds, stabilizes (14, 17), and may target to the plasma membrane (18, 19) the ADPKD2 gene, PKD2, a structural homolog of Ca2+ and cation channels (20). Overexpressed PKD2 or its C-terminal cytoplasmic tail also up-regulate AP-1-mediated transcription through pathways synergistic with those activated by the PKD1 C-terminal cytoplasmic tail (21). The signaling activities associated with overexpression of C-terminal fragments of PKD1 have been noted independent of type of membrane-anchored fusion protein (12-16). Additional reported activities of the C-terminal cytoplasmic tail of PKD1 include activation of Galpha o and Galpha i proteins in vitro (22), and phosphorylation on serine and tyrosine residues in vitro and in transfected cells (22-24). Endogenous PKD1 can be precipitated in a complex with E-cadherin and the alpha /beta /gamma -catenin complex and colocalizes with these components in human fetal kidney (25, 26).

The varied signaling and binding functions of the PKD1 C-terminal cytoplasmic tail led us to hypothesize that it might regulate ion transport processes that mediate or regulate cyst expansion. We now report that the distal fragment of the cytoplasmic C-terminal tail of PKD1 (PKD1(115-226)), expressed as a transmembrane fusion protein, up-regulates inward current and a Ca2+-permeable nonspecific cation channel of Xenopus oocytes.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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CDNAs-- cDNAs encoded tripartite fusion proteins in which the N-terminal extracellular domain of human CD16 was fused to the human CD7 transmembrane region (27), whose cytoplasmic terminus was fused in turn to portions of the 226-amino acid (aa) C-terminal cytoplasmic domain of the 4303-aa human PKD1 polypeptide. These fusion proteins were subcloned in the oocyte expression vector, pXT7. The longest construct encoded the complete C-terminal cytoplasmic fragment of PKD1 (PKD1 aa 4078-4303), and was termed "CD16.7-PKD1-(1-226)" (12, 13, 15). "CD16.7-PKD1(115-226)" encoded only the C-terminal 112 aa of the 226-aa PKD1 C-terminal tail (PKD1 aa 4192-4303). "CD16.7-PKD1(1-92)" encoded the N-terminal 92 aa of the PKD1 C-terminal tail (PKD1 aa 4078-4169).2 Other PKD1 constructs studied included "CD16.7-PKD1(1-155)" encoding PKD1 aa 4078-4232, "CD16.7-PKD1(115-189)" encoding PKD1 aa 4192-4266, and "CD16.7-PKD1(115-203)" encoding PKD1 aa 4192-4280. "CD16.7 control" encoded a cytoplasmic tail polypeptide of the same length as PKD1(1-92), but of unrelated, novel sequence.3

These and other CD16.7-PKD1 wild-type constructs have been described (15). PKD1 C-terminal cytoplasmic domain fragments fused to the CD16.7 transmembrane anchor display intracellular protein binding, signaling, and 293T cell surface expression properties indistinguishable from the same PKD1 constructs linked to immunoglobulin ectodomain membrane anchors and to various epitope tags (12-16). Point mutations in CD16.7-PKD1(115-226) were constructed by four primer polymerase chain reaction techniques. Primer sequences are available upon request. Integrity of the polymerase chain reaction products and of ligation junctions was confirmed by DNA sequencing.

Preparation of cRNA-- SalI- or NotI-linearized recombinant pXT7 templates were transcribed with T7 RNA polymerase. Xenopus oocyte isolation, culture, and microinjection were performed using standard techniques (28, 29). Oocytes injected with 12-25 ng of cRNA in a volume of 50 nl maintained integrity up to 18 days after injection. Generally, however, oocytes were subjected to electrical recording studies 2-3 days after microinjection.

Immunocytochemistry-- Oocytes injected 3, 5, or 10 days previously with cRNA were incubated with purified 3G8 mouse anti-human CD16 monoclonal antibody (gift of O. Mandelstam, J. Strominger, and J. Unkeless; or purchased from Meditech) at a concentration of 2 µg/ml in ND96 at room temperature for 3-4 h, then rinsed several times in ND96. Oocytes were fixed either in absolute methanol or in 3% paraformaldehyde in 140 mM NaCl, 20 mM sodium phosphate, pH 7.4 (PBS). Paraformaldehyde-fixed oocytes were rinsed in PBS, quenched with several washes in PBS plus 50 mM glycine, then in PBS. Methanol-fixed oocytes were rehydrated into PBS. Oocytes were then incubated in secondary Cy3-conjugated donkey anti-mouse IgG (Jackson Immunoresearch Laboratories, West Grove, PA) diluted 1/500 in PBS, rinsed several times in PBS, then dehydrated in methanol, and cleared in benzyl benzoate/benzyl alcohol (2:1) prior to imaging with a Bio-Rad MRC 1024 confocal microscope. Some oocytes were incubated with both primary and secondary antibody prior to fixation, with indistinguishable results.

Two-electrode Voltage Clamp-- Oocytes were placed in a 1-ml chamber (model RC-11, Warner Instruments, Hamden, CT) on the stage of a dissecting microscope and impaled with microelectrodes under direct view. Electrodes were pulled from borosilicate glass with a Narashige puller, filled with 3 M KCl, and had resistances of 2-3 megohms. Currents were measured with a Geneclamp 500 amplifier (Axon Instruments, Burlingame, CA) interfaced to a Hewlett Packard computer with a Digidata 1200 interface (Axon Instruments). Data acquisition and analysis utilized pCLAMP 6.0.3 software (Axon Instruments). Voltage pulse protocols consisting of 720-ms 20-mV steps between -100 and +20 mV were generated by the Clampex subroutine. Bath resistance was minimized by the use of agar bridges filled with 3 M KCl, and a virtual ground circuit clamped bath potential to zero.

Standard bath solution was 96 mM NaCl, 2 mM KCl, 5 mM HEPES, 1.8 mM CaCl2, and 1 mM MgCl2, and holding potential was -30 mV. All solutions were of pH 7.40. Cation/anion permeability determinations were by equimolar replacement of bath Na+ with N-methyl-D-glucamine (NMDG), and of bath Cl- with gluconate. Determination of relative monovalent cation permeabilities was by equimolar substitution of bath Na+ with K+, Li+, and NH4-.

Patch Clamp Recording of Cation Currents-- Patch pipettes were pulled to a resistance of 5-8 megohms, and oocytes were devitellinized as described previously (29). Gigaseals were first attained in the cell-attached mode. Outside-out patches were formed by breaking the oocyte plasma membrane with a pulse and withdrawing the pipette slowly. Currents were measured with an Axopatch 1D amplifier (Axon Instruments) interfaced via a Digidata 1200 AD/DA board to a HP Vectra computer. Data was acquired at 1 kHz and digitized at 5 kHz. For outside-out experiments, pipette solutions contained 128 mM cesium aspartate, 12 mM cesium EGTA, and 10 mM HEPES and the bath solution was 140 mM sodium methanesulfonate, 10 mM HEDTA, 10 mM HEPES. In cell-attached experiments, the pipette contained 100 mM CaCl2, 10 mM HEPES, and 10 mM HEDTA.

Calcium Fluorescence Measurements-- Oocytes from both groups were injected with 70-kDa Calcium Green dextran (Molecular Probes) to a final concentration of 3.5-7 µM, then subjected to bath [Ca2+] change from 0 to 10 mM. Relative change in intracellular calcium concentration ([Ca2+]i) was determined by exciting the fluorophore at 490 nm and imaging at 530 nm as described (29). Additional oocytes were loaded for 45 min with 8 µM Fura2-AM, then subjected to bath [Ca2+] changes from 1.8 mM to nominal 0 mM to 10 mM. In these oocytes [Ca2+]i was determined by alternately exciting the fluorophore at 340 and 380 nm. 510-nm emission images were acquired and analyzed as described (29), using IMAGE1 and IMAGE1-FL software (Universal Imaging).

45Ca2+ Influx Studies-- The uptake procedure was as reported (30), with modifications. Oocytes were incubated 1 h in ND-96 modified to contain 0.13 mM CaCl2 with 2.5 µCi/µl 45Ca2+, washed in ND-96, then counted in a liquid scintillation counter (Packard 2200CA).


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

CD16.7-PKD1(115-226) Expression Induces a Large, Inward Current-- Expression in Xenopus oocytes of CD16.7-PKD1(115-226) was associated with an inward current not observed in oocytes expressing CD16.7 control (Fig. 1, A and B). The current measured at -100 mV in the CD16.7-PKD1(115-226) group was -910 ± 68 nA (n = 97 oocytes from 18 frogs) compared with -131 ± 11 nA in the CD16.7 control group (n = 79 oocytes from 15 frogs, t = 10.2, p < 0.0001 by unpaired two-tailed t test, Fig. 1C). The average conductances of 2.1 and 8.7 µS and the whole cell reversal potentials (Erev) of -31 mV and +2 mV in oocytes expressing CD16.7 control and CD16.7-PKD1(115-226), respectively (Fig. 1C), each differed significantly by unpaired t tests (p < 0.0001). The currents in CD16.7 control-expressing oocytes were indistinguishable from those measured in oocytes previously injected with CD16.7-PKD1(1-92) cRNA or with water (Fig. 1D).



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Fig. 1.   Expression in oocytes of CD16.7-PKD1(115-226) induces a large current not observed in oocytes expressing CD16.7 control or CD16.7-PKD1(1-92). A, current traces in normal bath (ND96) generated in a two-microelectrode voltage clamp experiment on a CD16.7 control-injected oocyte. The voltage pulse protocol generated 20-mV voltage steps from -100 mV to +20 mV, from a holding potential of -30 mV. B, representative current traces from a PKD1(115-226)-injected oocyte. C, composite current-voltage relationship of CD16.7 control-injected oocytes and CD16.7-PKD1(115-226)-injected oocytes. Slope conductances were 2.1 and 8.7 µS, and reversal potentials were -31 and +2 mV in the control and PKD1(115-226) groups, respectively. D, analysis of the indicated deletion constructs implicates a region of the cytoplasmic tail encompassing residues 115-189 as required for expression of increased current. La3+-sensitive current (2 mM La3+) was assayed at -100 mV during two microelectrode studies. *, p < 0.05; **, p < 0.01, by post-test versus control. Oocytes expressing CD16.7-PKD1(115-226) (shaded bar) were studied in a separate experiment (inset, I-V relationship of La3+-sensitive difference current).

Several subdomains of the PKD1 cytoplasmic C-terminal tail as CD16.7-PKD1 fusion proteins were expressed in oocytes further to delimit the regions required for activation of cation currents (Fig. 1D), which were sensitive to block by La3+ (inset). La3+-sensitive inward currents elicited at -100 mV in oocytes expressing CD16.7-PKD1(115-203) or -(115-189) were of magnitude comparable to those observed with CD16.7-PKD1(115-226) (Fig. 1D and data not shown) with CD16.7-PKD1(1-226). In contrast, expression of CD16.7 control, or of CD16.7-PKD1(1-92), (), or () did not induce such currents. Expression of the constructs shown in Fig. 1D was not associated with evident oocyte morbidity over at least 4 days after cRNA injection.

The increased inward current and positive voltage-shifted Erev elicited by CD16.7-PKD1(115-226) but not by CD16.7 control was not explained by differences in oocyte surface expression of the different fusion proteins. As shown in Fig. 2, immunostaining of intact, unfixed oocytes with anti-CD16 antibody revealed comparable intensities of CD16 signal at the oocyte surface whether the epitope was borne on CD16.7-PKD1(115-226) or by CD16.7 control. Surface staining intensity increased at longer times after cRNA injection and in oocytes coexpressing both cRNAs (data not shown). The inactive CD16.7-PKD1(1-92) constructs were also expressed at the surface of 57% of injected oocytes at fluorescence intensities comparable to that of the active construct CD16.7-PKD1(115-226). Surface staining was always absent in water-injected oocytes (data not shown) and in oocytes expressing CFTR, which lacks the CD16 epitope (Fig. 2d).



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Fig. 2.   Surface immunostaining of CD16 epitope in unfixed oocytes injected 3 days (a-c) or 10 days (d-f) previously with cRNA encoding CD16.7 (a; 2 of 3 unfixed oocytes were CD16-positive), CD16.7 control (b and e; 11 of 11 unfixed oocytes were CD16-positive), CD16.7-PKD1(115-226) (c and f; 40 of 43 unfixed oocytes were CD16-positive), or CFTR (d; 0 of 4 unfixed oocytes were CD16-positive). Unfixed oocytes were incubated anti-CD16 monoclonal antibody 3G8, rinsed, post-fixed with methanol (a-c) or paraformaldehyde (d-f), then labeled with Cy3-conjugated secondary anti-Ig, and again fixed. Relative intensities in panels a-c are directly comparable, as are panels d-f. Water-injected oocytes (data not shown; 0 of 10 unfixed oocytes were CD16-positive) were indistinguishable from those expressing CFTR (d). 13 of 23 unfixed oocytes expressing the inactive CD16.7-PKD1(1-92) were detectably CD16-positive (data not shown). Each panel represents 1 mm2.

CD16.7-PKD1(115-226)-induced Current Exhibits Preferential Specificity for Cations-- The ion selectivity of the CD16.7-PKD1(115-226)-associated current was tested by NaCl bath substitution with either sodium gluconate or N-methyl-D-glucamine chloride. Bath change from NaCl to sodium gluconate produced little reduction in current and no change in Erev (-1 ± 1 mV, n = 15, Fig. 3A). In contrast, bath change from NaCl to N-methyl-D-glucamine chloride significantly decreased inward current at -100 mV from -573 ± 58 nA to -327 ± 52 nA, p < 0.001), and hyperpolarized Erev -19 ± 5 mV (n = 15), each significantly greater than the -5 ± 1 mV hyperpolarization (p < 0.001, n = 5, unpaired t test), and -99 ± 13 nA inward current at -100 mV (p = 0.0019, n = 5) observed in oocytes expressing CD16.7(control). In the presence of the impermeant cation NMDG, the depolarized Erev of CD16.7-PKD1(115-226)-expressing oocytes shifts toward the equilibrium potentials for Cl- and K+. These results are most simply explained by preferential activation of a cation conductance.



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Fig. 3.   CD16.7-PKD1(115-226)-stimulated currents preferentially pass cations. A, PKD1(115-226)-associated currents were significantly reduced and the reversal potential was significantly hyperpolarized when bath sodium was replaced by NMDG. Bath chloride subsitution with gluconate had no effect on reversal potential. B, significant La3+-sensitive currents persisted after 16 h chloride depletion and subsequent recording in a sodium methanesulfonate bath after injection of EGTA. Current at -100 mV was -421 ± 52 nA and -270 ± 34 nA in control and La3+-containing bath, respectively; t = 5.7, p = 0.0001, by paired t test.

The relative anion/cation permeability of oocytes expressing CD16.7-PKD1(115-226) was further tested by a mannitol dilution test. Shifting from 96 mM NaCl to a bath containing 9.6 mM NaCl, with mannitol added to maintain constant osmotic strength, decreased inward current at -100 mV from -634 ± 155 to -464 ± 130 nA (t = 5.5, p = 0.0009, by two-tailed t test), and hyperpolarized Erev from -11 ± 4 mV to -19 ± 6 mV (t = 4.1, p = 0.005, by paired t test). The direction of this shift in Erev indicates that CD16.7-PKD1(115-226)-stimulated current was more selective for cations than for anions. Oocytes were then ~90% depleted of intracellular chloride by overnight incubation in a Cl-free sodium methanesulfonate medium (31). Following subsequent acute injection with EGTA to minimize residual Ca2+-activated Cl- currents, oocytes expressing CD16.7-PKD1(115-226) continued to exhibit La3+-sensitive current (Fig. 3B) significantly greater than that observed in PKD1(1-92)fs-expressing oocytes.

Monovalent cation selectivity of the induced current was tested by equimolar substitution of bath NaCl with KCl, LiCl, and NH4Cl. Bath substitution with KCl or LiCl depolarized Erev by +15 ± 3 mV and +7 ± 3 mV, respectively (n = 5), in oocytes expressing CD16.7 control, as expected for the moderate K+ conductance and small cation conductance native to oocytes (32). In contrast, these bath changes did not alter Erev in oocytes expressing CD16.7-PKD1(115-226). Whereas bath substitution with NH4Cl depolarized Erev in CD16.7 control-expressing oocytes by +41 ± 4 mV and significantly increased conductance (n = 5, data not shown) as in native oocytes (32), CD16.7-PKD1(115-226)-expressing oocytes shifted the (extrapolated) Erev only slightly, +6 ± 3 mV (n = 6; F = 5.7, p = 0.02, data not shown). Thus, CD16.7-PKD1(115-226)-associated currents exhibited little specificity among monovalent cations, and approximated in magnitude the native NH4+ conductance of oocytes (33).

The CD16.7-PKD1(115-226)-induced Current Is Associated with Elevated Ca2+ Permeability-- The hypothesis that the PKD1(115-226)-associated current might conduct Ca2+ was tested by subjecting oocytes to voltage ramps first in the absence and then in the presence of 10 mM bath Ca2+ (Fig. 4A). The difference current at -100 mV attributable to Ca2+ was -436 ± 95 nA in CD16.7-PKD1(115-226)-expressing oocytes compared with +21 ± 27 nA in oocytes previously injected with water (p = 0.004, two-tailed t test). Thus, the nonspecific cation current activated in oocytes by expression of PKD1(115-226) also exhibited substantial permeability to Ca2+. The time course of holding currents was studied with calcium bath substitution (see Fig. 4B). The holding potential chosen was -30 mV, close to the chloride equilibrium potential, to minimize potential contribution of endogenous chloride currents. The upper trace from an oocyte expressing CD16.7-PKD1(115-226) reveals decreased inward current upon bath Na+ substitution with NMDG, which increased upon subsequent addition to the NMDG of 10 mM Ca2+ (representative of four experiments). The lower trace from an oocyte expressing CD16.7 control shows that current remained unaltered during the same bath change protocol (representative of four experiments), as expected from the dominant endogenous chloride conductance. Fig. 4C shows current voltage relationships of oocytes subjected to the protocol of Fig. 4B. The calcium difference current (solid line) was significant, and the Erev was consistent with that expected of a cation current.



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Fig. 4.   Oocytes expressing CD16.7-PKD1(115-226) exhibit Ca2+ currents. A, difference currents were constructed from 2600 msec voltage ramps (-150 to +50 mV, upper panel), executed in baths containing first 0 and then 10 mM CaCl2. The difference currents (lower panel) in PKD1(115-226)-expressing oocytes (black trace) were significantly larger than those of water-injected oocytes (gray trace). B, representative experiment illustrating time course of holding currents during bath change from ND-96 with NMDG (0 Ca2+) to NMDG with 10 Ca2+. For an oocyte expressing CD16.7-PKD1(115-226) (upper panel), inward current was reduced at the holding potential when NMDG (0 Ca2+) replaced the control bath, and the inward current reappeared with the substitution of a Ca2+-containing bath. In contrast, for an oocyte expressing CD16.7 control (lower panel), inward current was unchanged during these maneuvers. C, current-voltage relation of CD16.7-PKD1(115-226)-expressing oocytes recorded in NMDG baths containing first 0 and then 10 mM Ca2+. The Ca2+ difference current exhibited a 6.8-µS slope conductance and Erev of +15 mV (r2 = 0.994). D, 45Ca2+ uptake into CD16.7-PKD1(115-226)-expressing oocytes greatly exceeded uptake into CD16.7-PKD1(1-92)-expressing oocytes, and was blocked by La3+. E, Calcium Green fluorescence intensity (495/530 nm) indicates a greater Ca2+ permeability in oocytes expressing CD16.7-PKD1(115-226) than in those expressing CD16.7 control. At the arrow, 10 mM Ca2+ was added to the Ca2+-free NMDG bath. A single oocyte trace is shown for each group. Similar results were obtained with Fura-2 fluorescence ratio imaging.

45Ca2+ influx into oocytes expressing PKD1(115-226) was 3.94 ± 0.14 pmol/oocyte·h (Fig. 4D, n = 24), and greatly exceeded the 0.85 ± 0.26 pmol/oocyte·h in oocytes expressing CD16.7-PKD1(1-92) (n = 9, p < 0.00001). 1 mM La3+ inhibited 45Ca2+ influx into oocytes expressing PKD1(115-226) by 69% to 1.24 ± 0.17 pmol/oocyte·h (n = 26, p < 0.00001), whereas La3+ was without effect on 45Ca2+ influx into oocytes expressing PKD1(1-92) (n = 4, Fig. 4D). Thus, 1 mM La3+ inhibited 88% of the 45Ca2+ influx attributable to expression of CD16.7-PKD1(115-226).

Calcium Green fluorescence estimates of oocyte [Ca2+] indicated that the elevated Ca2+ influx into oocytes expressing PKD1(115-226) led to elevated free [Ca2+]i (Fig. 4E). Upon bath shift from 0 to 10 mM Ca2+, global oocyte [Ca2+]i in oocytes expressing CD16.7-PKD1(115-226) exhibited a sustained increase of 38 ± 7 fluorescence intensity units, significantly higher than the 9.5 ± 1.4 units in oocytes expressing CD16.7 control (t = 4.0, p = 0.007, by unpaired t test, n = 4). Since the nonratiometric Calcium Green did not allow comparison of absolute [Ca2+]i values among oocytes, we also measured [Ca2+]i in Fura2-AM-loaded oocytes. Basal [Ca2+]i in oocytes expressing CD16.7-PKD1(1-92) and CD16.7-PKD1(115-226) was indistinguishable, 87 ± 2 nM and 91 ± 6 nM, respectively (n = 4, p > 0.1). These values did not change detectably upon shift to nominally Ca2+-free bath. However, subsequent bath shift to 10 mM Ca2+ increased [Ca2+]i 34 ± 9 nM in PKD1(115-226)-expressing oocytes, but only 5 ± 1 nM in PKD1(1-92)-expressing oocytes (n = 4, p < 0.01). The elevated [Ca2+]i persisted without decay for at least 45 min.

Single-channel Properties of CD16.7-PKD1(115-226)-activated Na+ Current-- Fig. 5A shows unitary currents recorded at a holding potential of -50 mV from a representative outside-out patch with cesium aspartate in the pipette and sodium methanesulfonate in the bath. Addition to the bath of 1 mM La3+ suppressed nearly completely the inward Na+ currents. The unitary Na+ current exhibited an amplitude of -1.1 pA at -50 mV (Fig. 5B), and a linear conductance of 20 pS (r2 = 0.99) with Erev ~0 mV (PNa/PCs ~1; Fig. 5C). The mean NPo of 0.16 ± 0.04 was 5.1-fold higher than the NPo (0.03 ± 0.03) of indistinguishable channels detected in patches pulled from oocytes expressing CD16.7 control (Fig. 5D, n = 5, p = 0.03). Bath addition of 1 mM La3+ to patches pulled from oocytes expressing CD16.7-PKD1(115-226) reduced mean NPo by 96% (Fig. 5A), from 0.47 ± 0.19 to 0.02 ± 0.01 (n = 5, p = 0.04, one-tailed t test). Bath addition of 10 µM SKF96365 reduced NPo 82% to 0.03 ± 0.02 (n = 5, p = 0.03). Both inhibitors were considerably more potent as antagonists of outside-out patch currents than of whole oocyte currents (Table I).



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Fig. 5.   Outside-out patches from oocytes expressing CD16.7-PKD1(115-226) display increased activity of La3+-sensitive cation channels. A, representative traces from an outside-out patch pulled from a CD16.7-PKD1(115-226)-expressing oocyte into a 96 mM sodium methanesulfonate bath with cesium aspartate in the pipette, before and after addition of 1 mM La2+ (Vm was -50 mV). B, amplitude histogram of single-channel events recorded from a representative experiment during control conditions (Vm was -50 mV). C, current-voltage relation of the cation channel, with slope conductance of 20 pS. Erev near 0 mV indicates PNa/PCs ~1. D, NPo of single-channel events in outside-out patches from CD16.7-PKD1(115-226)-expressing oocytes was significantly higher than in CD16.7 control-expressing oocytes (Vm = -50 mV, n = 5, p < 0.04).


                              
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Table I
Pharmacology of PKD1(115-226) currents (in Xenopus oocytes, measured at -100 mV holding potential)

Single-channel Properties of CD16.7-PKD1(115-226)-activated Ca2+ Currents-- Fig. 6A shows an outside-out patch from an oocyte expressing CD16.7-PKD1(115-226). Ca2+ currents were recorded at -50 mV in a 100 mM Ca2+ bath with EGTA in the pipette. Unitary Ca2+ current in this patch was -1.1 pA at -50 mV (Fig. 6B). This current exhibited a linear I-V relationship (Fig. 6C, n = 10-12), and was undetectable in oocytes expressing CD16.7 control (Fig. 6D). Unitary Ca2+ current was inhibited 88% by addition of 1 mM La3+, reducing NPo from 1.13 ± 0.39 to 0.14 ± 0.10 (n = 7, p < 0.02 by paired t test, Fig. 6E).



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Fig. 6.   Oocytes expressing CD16.7-PKD1(115-226) exhibit increased La3+-sensitive Ca2+ currents in outside-out patches. A, representative trace from outside-out patch of a CD16.7-PKD1(115-226)-expressing oocyte, recorded in a calcium bath with EGTA-buffered cesium aspartate in the pipette (Vm = -50 mV). B, representative amplitude histogram from recordings such as in panel A (fitted by gaussian, Vm = -50 mV). C, single-channel current-voltage relationship from recordings such as in panel A, with slope conductance of 11 pS (n = 7, gray line). D, NPo of single-channel currents from outside-out patches at -50 mV in Ca2+ bath was significantly greater in oocytes expressing CD16.7-PKD1(115-226) (0.775 ± 0.232) than in those expressing CD16.7 control (0.006 ± 0.004; t = 2.23, p = 0.04, two-tailed, unpaired t test). E, NPo of single-channel currents in outside-out patches from oocytes expressing CD16.7-PKD1(115-226) is inhibited 88% by 1 mM La3+.

Induced Ca2+ channel activity was also detected in cell-attached patches on oocytes expressing CD16.7-PKD1(115-226). Fig. 7A shows representative traces from a cell-attached patch with 100 mM Ca2+ as the main charge-carrying ion in the pipette. Oocytes were preinjected with EGTA (estimated final concentration 5 mM) and then preincubated 2 h in sodium methanesulfonate to deplete intracellular chloride and minimize endogenous Ca2+-activated chloride currents. Unitary current was again 1.1 pA at -50 mV (Fig. 7B), with a linear conductance of 18 pS (Fig. 7C). NPo in cell-attached patches from oocytes expressing CD16.7-PKD1(115-226) was 0.53 ± 0.09, significantly higher than in oocytes expressing CD16.7 control (0.01 ± 0.005, t = 3.5, p = 0.005, by unpaired t test, Fig. 7D). Thus, cation channels activated by CD16.7-PKD1(115-226) were also permeable to Ca2+ in outside-out and cell-attached patch configurations.



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Fig. 7.   Oocytes expressing CD16.7-PKD1(115-226) exhibit increased unitary Ca2+ currents in cell-attached patches. A, representative traces from cell-attached patches on CD16.7-PKD1(115-226)-expressing oocytes with CaCl2 in the pipette. Oocytes were preincubated in sodium methanesulfonate and preinjected with EGTA to reduce activity of endogenous Ca2+-activated chloride currents. B, amplitude histogram of -50 mV trace shown in A. C, single-channel current-voltage relationship of experiment in panel A with slope conductance 18 pS (gray line). D, NPo of cell-attached patches recorded on CD16.7-PKD1(115-226)-expressing oocytes (0.525 ± 0.093) significantly exceeded that in CD16.7 control-expressing oocytes (0.01 ± 0.005; t = 3.5, p = 0.005, unpaired two-tailed t test).

Pharmacology and Regulation of CD16.7-PKD1(115-226)-associated Cation Currents-- Na+ current measured at -100 mV holding potential in CD16.7-PKD1(115-226)-expressing oocytes was reduced by bath addition of 2 mM LaCl3 from -694 ± 110 nA to -365 ± 72 nA (n = 10, p < 0.01, two-tailed t test). Water-injected oocytes exhibited much lower currents at the same holding potential (-112 ± 13 nA) that were reduced minimally by La3+ to -89 ± 12 nA, n = 4, data not shown). The current was insensitive to 500 µM DIDS and to 100 µM niflumic acid, but exhibited moderate block by 50 µM SKF96365 (13%, p = 0.001), 1 mM amiloride (13%, p = 0.02), and 10 µM Zn2+ (19%, p = 0.008), with increased inhibition at higher Zn2+ concentrations. The degree of inhibition by Gd3+ was similar to that of La3+ (Table I). Because cation channels of comparable unitary conductance are activated by hypertonicity (34), oocytes expressing CD16.7-PKD1(115-226) or CD16.7 control were subjected to a bath change from isotonic ND-96 (212 mosM) to ND-96 rendered hypertonic by addition of mannitol (final 280 mosM). Although this degree of hypertonicity suffices to activate endogenous oocyte Na+/H+ exchange (35), currents recorded in the hypertonic and isotonic baths did not differ in either group of oocytes (data not shown).

The effect of bath pH on the induced cation current was tested. Bath pH change from 7.4 to 6.0 decreased inward current measured at -100 mV by 33%, from -1041 ± 71 nA to -697 ± 69 nA (n = 5, p = 0.0002). In contrast, bath pH change to 8.0 was without effect on inward current.

Importance of Selected Serine and Tyrosine Residues for Cation Current Activation by CD16.7-PKD1(115-226)-- Several amino acid residues have been shown to be phosphorylated within PKD1(115-226) fusion proteins in vitro by exogenously added kinases and in transiently transfected cells (23). Within this region, serine 175 (Ser-4252 of holo-PKD1) is a preferred substrate of cAMP-dependent protein kinase, and tyrosine 160 (Tyr-4237 of holo-PKD1) is a preferred substrate of c-Src. The adjacent serine residues 174 and 175 (4251/4252) were mutated in tandem either to alanine or to aspartate, since each is a candidate cAMP-dependent protein kinase site. Each double mutation abolished CD16.7-PKD1(115-226)-associated inward currents (Fig. 8A), despite unimpaired expression of the doubly mutant polypeptides at the oocyte surface (Fig. 8B). Similarly, mutation of tyrosine 160 to phenylalanine (Y4237F) abolished CD16.7-PKD1(115-226)-associated current (Fig. 8A) despite unimpaired surface expression of this mutant polypeptide (Fig. 8B).



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Fig. 8.   PKD1(115-226)-associated La3+-sensitive current is abolished by mutation of Ser-174/175 (4251/4252 of holo-PKD1) to Ala or to Asp, and by mutation of Tyr-160 (Tyr-4237 of holo-PKD1) to Phe. A, oocytes injected 48 h earlier with cRNA encoding wild-type CD16.7-PKD1(115-226) (n = 19), CD16.7-PKD1(115-226) with double Ser mutations to Ala (n = 12) or Asp (n = 11), or with a Tyr mutation to Phe (n = 7) were analyzed for La3+-sensitive current measured at -100 mV. B, CD16 immunolocalization in unfixed oocytes expressing wild-type CD16.7-PKD1(115-226) (left panel), or the double mutants S174A/S175A (18 of 21 oocytes were CD16-positive) and S174D/S175D (32 of 33 oocytes were CD16-positive; middle panels) and the mutant Y160F (right panel, 9 of 10 oocytes were CD16-positive).



    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We report that expression of the CD16.7-PKD1(115-226) fusion protein, encoding the 112 C-terminal residues of the PKD1 cytoplasmic C-terminal tail fused to the CD16.7 transmembrane chimera, up-regulates a Ca2+-permeable cation conductance in Xenopus oocytes. The cation conductance is nonselective among monovalent cations, is inhibited by La3+, Gd3+, SKF96365, amiloride, and Zn2+, but not by DIDS or niflumate, and is not activated by hypertonicity. Expression of CD16.7 control, or of CD16.7-PKD1(1-92), CD16.7-PKD1(1-155), or of CD16.7 without any intracellular tail failed to activate this cation conductance, despite normal surface expression of these inactive polypeptides. Activation of whole cell currents was accompanied by increased open probability (NPo) of cation channels of 20-22 pS linear conductance, which likely mediated or substantially contributed to the recorded whole cell currents.

The CD16.7-PKD1(115-226) fusion protein spans the lipid bilayer only once. The transmembrane domain derived from CD7 within the CD16.7 fusion proteins is not expected itself to form an ion channel. Indeed, similar cation channel activity appeared present at 5-8-fold lower activity in oocytes previously injected with water or with cRNA encoding either CD16.7 control or CD16.7-PKD1(1-92). Taken together, the data suggest that the PKD1(115-226) domain activates a cation channel native to oocytes. This hypothesis suggests that CD16.7-PKD1(115-226) either triggers a signaling pathway that tonically activates cation channel activity, or that the PKD1 fragment can serve as a chaperonin or a functional subunit of an endogenous pore-forming polypeptide or polypeptide complex.

Which signaling pathways might mediate cation channel activation by CD16.7-PKD1(115-226)? This portion of the PKD1 C-terminal cytoplasmic tail activates the siamois promoter via inhibition of GKS-3beta , beta -catenin stabilization, and up-regulation of TCF-mediated transcriptional signaling (13). Xwnt8-mediated activation of the siamois promoter was also enhanced by PKD1(115-226). Although itself incapable of activating transcriptional signaling mediated by AP-1 elements, this PKD2-binding fragment of PKD1 enhanced activation by PKD2 of AP-1-mediated transcription (21). PKD1(115-226) also dorsalized and prevented posterior development in zebrafish embryos (13), and bound and stabilized RGS7 (15).

PKD1(115-226) also harbors a major in vitro cAMP-dependent protein kinase phosphorylation site (23, 24). We tested by mutagenesis the functional importance of this site for cation channel activation. Mutation of the tandem serine residues 4251/4252 to either alanine or to aspartate nearly abolished CD16.7-PKD1(115-226)-associated cation currents, despite undiminished expression of the mutant polypeptides at the oocyte surface. Mutation of the putative tyrosine kinase site Tyr-4237 (23) to Phe also reduced cation current without decreasing surface expression of polypeptide. Therefore, these residues play important roles in cation channel activation. Whether this occurs by direct phosphorylation or by another mechanism remains to be determined.

With which cation channel might CD16.7-PKD1(115-226) directly or indirectly interact? Qian et al. and Tsiokas et al. showed that this region of PKD1 can complex with the C-terminal tail of PKD2 (14, 17). Although PKD2 and PKD1 colocalization within cells remains uncertain (19), PKD2 and PKD1 can be coimmunoprecipitated (14, 17). The PKD2 homolog PKDL, although not a cause of heritable human polycystic kidney disease (36), does produce Ca2+-permeable cation channels in Xenopus oocytes (30) different in character from those we have measured in oocytes expressing CD16.7-PKD1(115-226).

Tsiokas et al. (18) showed that PKD2 through distinct binding domains complexes with the Ca2+-permeable cation channel hTRPC1, but not with hTRPC3. Akaike and colleagues (37) showed that degenerate hTRP-1 oligonucleotides reduced thapsigargin-stimulated currents in oocytes injected with a rat TRP4 homolog. More recently, Berridge and colleagues have cloned Xtrp1 (38), but its function remains unreported. Our preliminary experiments failed to demonstrate reduction in CD16.7-PKD1(115-226)-associated cation channel activity after coinjection of Xtrp1 antisense oligonucleotides (data not shown).

Xenopus oocytes exhibit an Icrac-like store-operated Ca2+ current (39, 40). Preliminary experiments indicate that thapsigargin, although reproducibly activating a cation current in oocytes, did not activate current attributable to CD16.7-PKD1-(115-226). CD16.7-PKD1(115-226)-elicited cation current also differed from oocyte Icrac in its more modest La3+ sensitivity in intact oocytes, its moderate amiloride sensitivity, and its lack of inward rectification (40).

Nonselective cation channels and mechanosensitive cation channels have been reported in Xenopus oocytes (reviewed in Ref. 41). The cation conductances activated in oocytes by maitotoxin (42) and by palytoxin (43) each differed from CD16.7-PKD1(115-226)-activated cation conductance. Unlike the widely distributed hypertonicity-activated cation channel (33), CD16.7-PKD1(115-226)-activated cation conductance was insensitive to moderate increase in bath tonicity.

Unlike depolarization-activated hemi-gap junction-mediated cation currents (44), CD16.7-PKD1(115-226)-activated cation conductance was voltage-independent. Unlike the external divalent-blocked cation channel (45, 46), CD16.7-PKD1(115-226)-activated cation conductance was not blocked by DIDS or by niflumate. Endogenous oocyte cation conductance has been activated by simple overexpression of heterologous K+ channels (47), but, unlike these currents, CD16.7-PKD1(115-226)-activated cation current was insensitive to DIDS and not gated by hyperpolarization.

In summary, expression in Xenopus oocytes of a CD16.7 transmembrane fusion protein containing the PKD1 C-terminal cytoplasmic tail aa 115-226 activated Ca2+-permeable nonspecific cation currents in Xenopus oocytes likely mediated by channels of 20-pS linear conductance. Preliminary experiments (data not shown) indicate that similar channels were activated in 293T cells transiently transfected with CD16.7-PKD1(115-226). In oocytes, the elevated cation conductance correlated with increased Ca2+ entry and elevation of [Ca2+]i. These changes in ion channel activity are the first directly linked to the expression of any portion of the PKD1 polypeptide. We propose that changes in intracellular Ca2+ signaling resulting from mutations in PKD1 contribute to the creation and/or maintenance of the secretory and/or proliferative phenotype of renal cysts in ADPKD1, and to the resultant cyst expansion that ultimately leads to renal failure.


    ACKNOWLEDGEMENTS

We thank Leonidas Tsiokas and Joe Mindell for helpful discussions.


    FOOTNOTES

* This work was supported in part by National Institutes of Health Grants DK51059, DK34854 (Harvard Digestive Diseases Center), and HL15157 (Boston Sickle Cell Center) (to S. L. A.) and DK52897 (to G. W.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Supported by a Joseph E. Murray award from the Massachusetts/Rhode Island affiliate of the National Kidney Foundation.

§ Current address: University Hospital Freiburg, 79106 Freiburg, Germany.

To whom correspondence should be addressed: Molecular Medicine Unit, RW763, Beth Israel Deaconess Medical Center, 330 Brookline Ave., Boston, MA 02215. Tel.: 617-667-2930; Fax: 617-667-2913; E-mail: salper@caregroup.harvard.edu.

Published, JBC Papers in Press, October 23, 2000, DOI 10.1074/jbc.M006252200

2 In most experiments, CD16.7-PKD1(1-92) was expressed as either of two variants which included C-terminal extensions beyond PKD1 cytoplasmic domain residue 92, originating from polylinker sequence. One form added a single C-terminal Ser residue. The other form added the 7-residue C-terminal sequence Ser-Ala-Ala-Ala-Arg-Glu-Ile. Both of these fusion proteins were expressed at the oocyte surface, but neither increased oocyte currents. The experiment shown in Fig. 6D utilized a form of CD16.7-PKD(1-92) devoid of any C-terminal extension.

3 The construct CD16.7 control provides a novel, PKD1(1-92)-unrelated cytoplasmic C-terminal amino acid sequence of the same length as PKD1(1-92). The sequence is: ESWHLSPLLCVGLWALRLWGALRLGAVILRWRYHALRGELYRPAWEPQDYEMVELFLRRLRLWMGLSKVKES. This sequence is encoded by a frameshifted PKD1(1-92) cDNA (due to a single nucleotide deletion, GenBankTM NM000296 nucleotide 12446).


    ABBREVIATIONS

The abbreviations used are: ADPKD, autosomal dominant polycystic kidney disease; PKD, polycystic kidney disease; DIDS, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid; S, siemen(s); aa, amino acid(s); CFTR, cystic fibrosis transmembrane regulator; PBS, phosphate-buffered saline.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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