Journal of the American Society of Nephrology
2007 JASN IMPACT FACTOR 7.111 HOME   AUTHOR INFO   EDITORIAL BOARD   SUBSCRIBE   FEEDBACK   ALERTS   HELP 
    advanced
CURRENT ISSUE ARCHIVES JASN Express ONLINE SUBMISSION


This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Venglarik, C. J.
Right arrow Articles by Lu, X.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Venglarik, C. J.
Right arrow Articles by Lu, X.
J Am Soc Nephrol 15:1168-1177, 2004
© 2004 American Society of Nephrology

BASIC SCIENCE

Evolutionary Conservation of Drosophila Polycystin-2 as a Calcium-Activated Cation Channel

Charles J. Venglarik, Zhiqian Gao and Xiangyi Lu

Department of Environmental Health Sciences, University of Alabama at Birmingham, Birmingham, Alabama.

Correspondence to Dr. Charles J. Venglarik, Department of Environmental Health Sciences, University of Alabama at Birmingham, RPHB-530, 1530 3rd AVE S, Birmingham, AL 35294. Phone: 205-934-7032; Fax: 205-975-6341; E-mail: cjv{at}uab.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
ABSTRACT. Mutations in the PKD2 gene cause autosomal dominant polycystic kidney disease (ADPKD) in humans. The protein encoded by PKD2 has similarity to voltage-sensitive cation channels and TRP channels and was named polycystin-2 (PC2). In agreement with this structural information, expression of PC2 in Xenopus oocytes or reconstitution of human PC2 in planar lipid bilayers produced Ca2+-activated cation channels. Although these studies provided a basic description of the biophysical, regulatory, and pharmacologic properties of the PC2-induced channels, it is still unknown how defective PC2 activity leads to cyst formation and expansion in ADPKD patients. To establish a genetic model for studying PC2 function and regulation, the authors identified and cloned a Drosophila PC2 (DmPC2). It is here shown that expression of DmPKD2 in Drosophila S2 cells produced a novel channel. On the basis of the similarity of this channel’s properties to mammalian PKD2-induced channels, this Drosophila channel is expected to provide a convenient genetic model for dissecting the mechanisms underlying ADPKD.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Polycystin-2 (PC2) is an approximately 110 kD transmembrane protein having sequence and structural similarities to voltage-dependent cation channels and TRP channels (1,2). Mutations in the PKD2 gene that encodes PC2 cause autosomal dominant polycystic kidney disease (ADPKD). This genetic disorder occurs at a frequency of 1:400 to 1:1000 (3) and is characterized by large fluid-filled cysts in the kidney and other target organs (4,5). The formation and expansion of cysts progressively destroys the normal parenchyma and often causes end-stage renal failure, requiring transplant or dialysis (3,5).

Heterologous expression of human PC2 after injection of Xenopus oocytes with PKD2 cDNA introduced novel nonselective cation channels in the plasma membrane and endoplasmic reticulum (6). These channels conducted both monovalent and divalent cations (Na+, K+, Ba2+, and Ca2+); however, Cl– or large monovalent cations (choline+ or NMDG+) did not serve as efficient charge carriers (6). Increasing calcium from 100 nM to 1 µM at the cytoplasmic surfaces of excised inside-out patches evoked a transient increase in open probability (Po), suggesting that channel opening is regulated by intracellular calcium (6). Similar effects of calcium were also demonstrated for a murine PC2 homologue, polycystin-L (PCL), expressed in Xenopus oocytes (7). The calcium dose-response relation has been quantified only for the PC2 channels reconsitituted in planar lipid bilayers, and the KM was approximately 7 µM. No specific blockers of PC2 or PCL have yet been identified, but La3+, Gd2+, or amiloride were shown to inhibit current (6,8,9).

Although previous studies demonstrate that PC2 likely functions as a calcium-activated cation channel and provide a basic description of its properties, many important questions remain unanswered regarding the upstream mediators that are responsible for regulating channel function and the downstream signaling pathways that normally serve to prevent cyst formation. In one study, PC2 was shown to localize to the endoplasmic reticulum of renal epithelial cells, and expression of PC2 in LLC-PK1 kidney cells enhanced vasopressin-induced intracellular Ca2+ release (10). In a subsequent study, PC2 was localized on the primary cilium of renal epithelia and appeared to mediate Ca2+ influx in response to changes in apical fluid flow (11,12). Additional information is needed to understand the relative importance of these divergent observations and to identify the mechanisms underlying ADPKD.

As many signaling mechanisms are conserved throughout phylogeny, one possible means of elucidating the PC2-mediated regulatory pathways is to use Drosophila as a model. Drosophila has shorter generation time compared with vertebrates, and sophisticated tools have been developed that greatly facilitate genetic and functional analyses. The aim of this study was to identify a Drosophila counterpart of PC2 and assay for channel activity. Using known PC2 proteins as query, we identified several putative PKD2 homologues in the Drosophila genome by BLAST (13). Of these, the one located at cytological position 33E3 (accession #CG6504) is most closely related to the mammalian PKD2 genes. We cloned cDNA (accession #AY283154) for the fly PKD2 at 33E3 (referred to as DmPKD2) and found that it encodes a 924–amino acid polypeptide (referred to as DmPC2) that is predicted to have a nearly identical structure to the PC2 family of cation channels. Stable transfection of this cDNA into cultured Drosophila cells produced a novel channel that was activated after addition of Ca2+ to the bath solutions of excised inside-out patches and conductive to Ba2+, Ca2+, and Na+ and relatively impermeable to Cl–. Thus the properties of the DmPKD2-induced channel appear similar to those previously reported for mammalian PC2 and PCL.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Identification and Cloning of Drosophila PKD2 cDNA
We identified Drosophila PC2 (DmPC2) (translation of CG6504) via a BLAST search (13) of Flybase (http://flybase.bio.indiana.edu:82/) by querying with human and mouse PC2 amino acid sequences (AAC16004 and NP032887). As the cDNA for this gene was unavailable from several Drosophila cDNA libraries, we initially cloned a partial cDNA fragment corresponding to a highly conserved region of the gene by RT-PCR. Analysis of this partial cDNA provided important information regarding intron-exon structure. We then cloned the full-length cDNA of 3245 bp by a combination of RT-PCR and RACE protocols using templates derived from reverse transcription of Drosophila embryonic and adult mRNA samples. The full-length cDNA was cloned as two overlapping 5' and 3' fragments that later joined together at the unique AccIII site in pGEM–T vector and sequenced. There are four individual base differences in the cloned cDNA (AY283154) comparing to the genomic sequence (CG6504). These base differences do not change amino acid sequence and are probably natural polymorphism associated with the yw fly strain from which mRNA was extracted and used for cDNA cloning.

Generation of Drosophila PKD2 cDNA Expression Construct
The approximately 3.3-kb cDNA insert was released by SalI and ApaI from the full-length clone in pGEM–T vector, blunted, and ligated into the pMK33/pMtHY Drosophila expression vector (14) at the EvoRV site. The resulting plasmid pMK33/pMtHY-DmPKD2 allows an inducible expression of DmPKD2 cDNA under the control of a metallothionein (Mt) promoter. The pMK33/pMtHY vector also contains a bacterial hygromycin-resistance gene driven by the Drosophila copia promoter, thus enabling selection of stably transformed cell lines. The correct orientation of DmPKD2 cDNA relative to the Mt promoter was verified by restriction enzymes.

Establishment of Drosophila PKD2-Expressing Cell Line
Drosophila S2 cells (15) originally from ATCC were used at indeterminate passage. These cells are well suited for the present studies, as they have been used successfully to express other Drosophila ion channels and do not appear to contain endogenous Ca2+-activated channels (16–18). S2 cells were adapted to grow in HyQ SFX medium (HyClone) at 25°C and were passaged every 3 d at 1:10 dilution to maintain exponential growth. Cells were transfected with either pMK33/pMtHy containing the Drosophila PKD2 cDNA (pMK33/pMtHY-DmPKD2) or the pMK33/pMtHY empty vector using Effectene (Qiagen Inc.) following the manufacturer instructions. The cells were grown for 3 d without drug selection after transfection. Thereafter cells were exposed to 600 µg/ml hygromycin B (CALBIOCHEM, San Diego, CA) until the cells returned to the normal doubling time of 24 h. The cells were cultured continuously under hygromycin B selection (400 µg/ml) except immediately before patch-clamp experiments. Similar treatment of non-transfected S2 cell cultures with hygromycin B rendered them nonviable.

Antibody Production, Purification, and Uses
Anti-DmPC2(29/30) was produced commercially (Zymed Laboratories, Inc) by immunizing rabbits with a GST-DmPC2 (317 to 417) fusion protein that was purified from BL21 Gold (Stratgen) transfected with a pGEX-SK1 clone. Residues 317 to 417 correspond to the 99–amino acid "insertion" located in the first extracellular loop of DmPC2. Anti-DmPC2(29/30) antiserum was partially purified to remove anti-GST antibody by preabsorption with equal volume of glutathione resin (Sigma) coupled with a unrelated GST fusion protein for 2 h at 23°C. The preabsorbed anti-DmPC2(29/30) serum was used at 1:5000 dilutions for Western blot and immunolocalization.

Western blots were performed on pMK33/pMtHY-DmPKD2 transfected Drosophila S2 cells seeded (1:5 vol/vol) in a T75 flask 1 d before treatment with 0.6 mM CuSO4. Cells were then counted, and samples of 107 cells were collected at 24-h intervals for 5 d. The cells were solubilized using a SDS buffer and separated by SDS-PAGE.

Patch-Clamp Recording and Analysis
This assay was implemented as described previously (19–21). Briefly, S2 cells were grown in media supplemented with 0.6 mM CuSO4 for 1 to 5 d to induce DmPC2 expression. Cover slips containing the cells were then placed in a chamber located on the stage of an inverted Nikon microscope. All experiments were performed at 23 to 25°C. Patch pipettes were fabricated from Kimax 51 glass using a Narashige PP-83 puller and had resistances of 1 to 3 M{Omega} when filled with solutions identical to the bath (114 mM BaCl2; 10 PIPES). We compensated for liquid junction potentials before experiments. All clamp potentials are reported as intracellular voltages with negative and positive current signifying inward and outward flow of cations, according to convention. Currents were monitored using an Axopatch 200A amplifier (Axon Instruments, Union City, CA) and Tektronix digital storage oscilloscope. The current output was also low pass filtered at 5 kHz and recorded to VHS videotape in PCM format at 20 kHz for subsequent analyses. Recordings were played back, refiltered using a low pass 8 pole Bessel filter (Frequency Devices), and acquired to a Pentium PC hard drive using a TL-1 A/D interface (Scientific Solutions Inc) and pClamp 5.5 (Axon Instruments). Single channel amplitudes were measured using BioPatch (BioLogic Calaix, France). Time-dependent decays of the Ca2+-induced currents were analyzed by first averaging the data using a custom program or filtering using a low fc and then fitting these data to exponential decay functions (y = ae–kt). SigmaPlot (SPSS) and CorelDraw (Corel Corp) were used for graphic presentation.

Patch-Clamp Solutions
The bath and pipette solutions both initially contained: 114 mM BaCl2 and 10 mM PIPES – NMDG+ (pH, 6.2). This pH is physiologic for Drosophila. Barium was initially selected for study because it does not appear to interfere with known Ca2+ channels, and human PC2 channels were previously shown to conduct Ba2+ (6,10). In later experiments, we varied the ionic composition of the pipette solutions to determine ion selectivity. The Ba2+ versus Cl– selectivity was assayed using pipette solutions containing: 11.4 mM BaCl2, 46 mM HCl, 46 mM NMDG+, 215 mM mannitol, and 10 mM PIPES-NMDG+ (pH = 6.2). Using the Nernst equation, the calculated reversal potentials (Vrev) for Ba2+ and Cl– are –30 and +30 mV, respectively. The Cl– to Ba2+ permeability ratio (PCl/PBa) can be calculated from the observed reversal potential (Vrev) using a form of the equation derived by Lewis (22):Go


The subscripts signify bath (B) and pipette (P) concentrations and R, T, z, and F all have the usual meanings. Provided Cl– permeability is low, the Ca2+ versus Ba2+ selectivity ratio (PCa/PBa) can be determined using a pipette solution containing 114 mM CaCl2 plus 10 mM PIPES-NMDG+ (pH, 6.2). Noting that the cation concentrations are identical on both sides, PCa/PBa can be calculated using a form of the simple bionic equation (23):Go


Similarly, the Ba2+ versus Na+ selectivity ratio (PBa/PNa) can be determined using a pipette solution containing 114 mM NaCl, 114 mM mannitol, and 10 mM PIPES-NMDG+ (pH, 6.2). Noting that the cation concentrations are identical on both sides, PBa/PNa can be derived by solving the following equation for species that differ in valence (24):Go



   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Analyses of DmPC2 Amino Acid Sequence
Conceptual translation of Drosophila PKD2 cDNA yields a DmPC2 protein of 924 amino acids. The TMHMM (25) and Coils programs (26) predict that DmPC2 has six transmembrane helices (TM1-TM6) and a C-terminal coiled-coil domain (residues 882 to 922). Overall, this predicted topology is nearly identical to mammalian and C. elegans PC2 family of cation channels and is similar to the TRP channels (1,2,27). Comparison of DmPC2 with human PC2 using LAlign (28) showed that the region of greatest homology (75% similarity and 40.4% identity) occurs over 396 amino acids (Dm 424–814/Hs 304–698) as illustrated in Figure 1A. This includes the "polycystin motif," the five putative transmembrane segments (TM2 to TM6), along with the putative pore-forming "P-loop" region (2). The extracellular "polycystin motif" is highly conserved among all PC1, PC2, and polycystin-L proteins and is of unknown function. The sequence spanning TM1 (Dm 231–323/Hs 214–304) is partially conserved (18.3% identity), while both human PC2 and DmPC2 possess proline-rich N-termini. The tree shown in Figure 1B presents a more global comparison of DmPC2 and related proteins using ClustalW (29). These proteins include PC2, polycystin-1 (PC1), receptor for egg jelly (REJ), and voltage-activated Ca2+ channels. Importantly DmPC2 is more closely related to polycystin-2-like-2 (PC2L2) and human PC2 than other proteins exemplified by PC1/REJ3 or N- and T-type Ca2+ channels. Overall, this comparison suggests that DmPC2 functions as a cation channel.



View larger version (35K):
[in this window]
[in a new window]
[as a PowerPoint slide]
  		Figure 1. Analyses of the primary structure of Drosophila polycystin-2 (DmPC2). (A) Comparison of the region with highest sequence conservation between human PC2 (AAC16004) and DmPC2 (AY283154). This region includes the polycystin motif in the extracellular loop between TM1 and TM2, TM2-TM6, and the putative P-loop sequence between TM5 and TM6. There is 40.4% identity (:) and 75% similarity (.) between the two sequences. (B) The overall sequence relatedness of DmPC2 to PC2, PC1, receptor for egg jelly (REJ), alpha subunits of N- and T-type calcium channels, and other similar proteins as calculated by the ClustalW program (29). The tree was constructed by importing the numeric output of ClustalW into PhyloDraw (Graphics Application Lab, Pusan National University). Accession numbers are as follows: MmPC2L1 (XP283565), MmPC2L (AF271381), PC2L1 (NP057196), DrPC2 (CAD52124), MmPC2 (O35245), PC2 (NP000288), PC2L2 (Q9NZM6), MmPC2L2 (NP058623), CePC2 (NP502838), CeLOV1 (NP496184), REJ3 (AF4221531), PC1 (NP006062), MmPC1 (NP035235), RnTRP6C (BAB43812), TrPC1 (NP648970), DdPC2 (AAM08926), mucolipin2 (AAM08926), Gg N-type {alpha} Ca+2 (AF1730161), N-type {alpha} 1B Ca+2 (NP000709), N-type {alpha}1 Ca+2 (T45115), Mm T-type {alpha} Ca+2 (NP033913), T-type {alpha} Ca+2 (AF1349851).

 
One notable divergence is present in the first putative extracellular loop between TM1 and TM2, where DmPC2 contains a 99–amino acid sequence "insertion" that is not present in the corresponding regions of C. elegans or mammalian PC2 proteins. We produced an antibody, anti-DmPC2(29/30), against this sequence. This antibody detected a major protein band at 122 kD on Western blots of Drosophila male adult homogenate (Figure 2A) as did another antisera (anti–DmPC2-C) raised against a peptide near the C-terminus of DmPC2 (not shown). These data demonstrate that the sequence addition in the first extracellular loop of DmPC2 is translated in vivo.



View larger version (35K):
[in this window]
[in a new window]
[as a PowerPoint slide]
  		Figure 2. Detection of DmPC2 expression in Drosophila S2 cultured cells by Western blot and immunolocalization. (A) Comparison of DmPC2 expression in parental S2 cells versus CuSO4-induced expression in the transfected S2 cells by Western blot analysis. The band appears at the same molecular weight (122 kD) as the endogenous DmPC2 in adult male Drosophila. The slowly migrating bands may be due to protein oligomers. Nuclear lamin (NL) was used as a control for loading. (B–D) Comparison of representative photomicrographs from three DmPKD2-transfected S2 cells doubly incubated with preimmune serum plus anti-NL (B) or anti-DmPC2 (29/30) serum plus anti-NL (C and D). Cells in panels B and C were first incubated with the sera (4°C for 1 h) before fixation, whereas the cell in panel D was first fixed and thereby permeablized before sera incubation. The first photomicrograph (B) shows no NL antibody penetrated into cell interior under this staining protocol and no staining with preimmune serum. The dotted pattern shown in the second photomicrograph (C) is consistent with the hypothesis that anti-DmPC2 (29/30) serum recognizes the first extracellular loop of DmPC2 and provides strong evidence for DmPC2 localization at the external surface of the plasma membrane. The third micrograph (D) shows the presence of both DmPC2 and NL in the cell interior.

 
Expression of DmPC2 Yields Novel Ca2+-Activated Current
Drosophila S2 cells do not express detectable amounts of DmPC2 from the endogenous locus as shown by Western blot (Figure 2A). We established stable S2 cells after transfection with DmPKD2 cDNA under the metallothionein (Mt) promoter. In the absence of CuSO4 in the culture media, the transfected S2 cells expressed a approximately 122-kD protein band that was recognized by the anti-PKD2(29/30) antibody. Expression of this protein increased progressively over 5 d after addition of 0.6 mM CuSO4 to the culture media (Figure 2A). Immunostaining of nonpermeablized DmPKD2-transfected S2 cells with anti-DmPC2(29/30) that recognizes the first extracellular loop of DmPC2 revealed that DmPC2 is present on the exterior surface of the plasma membrane as indicated by punctate staining (Figure 2C). An antibody against the intracellular protein nuclear lamin (NL) was used to monitor cellular permeability during the staining procedure. Upon permeabilization of the DmPC2-transfected cells, a large amount of DmPC2 and NL detected intracellularly (Figure 2D). We then proceeded to test the conductance of the transfected S2 cell membranes using the patch clamp technique.

Previous studies suggest that Drosophila S2 cells do not have an endogenous Ca2+-activated conductance (17,18). As shown in the representative current trace in Figure 3A, DmPC2 expression produced a novel population of Ca2+-activated channels in excised inside-out patches. Figure 3A compares the current recorded before and after addition of 300 µM Ca2+ to the bath solution. These experiments were performed in the presence of symmetric 114 mM BaCl2 solutions in the bath and pipette. There was no spontaneous channel activity under basal conditions. After the addition of CaCl2, a population of channels activated rapidly and subsequently "ran down" or inactivated over the next 5 to 10 min. An identical biphasic response after CaCl2 addition was observed in 71 out of 230 patches from DmPKD2-transfected cells. However, CaCl2 addition had no effect on > 30 patches from CuSO4-treated S2 cells that were transfected with the empty pMK33/pMtHy vector (P < 0.05 by {chi}2 analysis). Figure 3B replots the data shown in Figure 3A as the average current per 10-s interval (±SD), and Figure 3C illustrates a long duration record obtained from another experiment. The decays can be described by single exponential functions with time constants ({tau}) of 5.4 and 5.6 min as illustrated by the curves in Figure 3, B and C. Consistent with this estimate, channel activity ceased 10 to 15 min (i.e., 2 to 3 times {tau}) after CaCl2 addition in all experiments (not shown).



View larger version (27K):
[in this window]
[in a new window]
[as a PowerPoint slide]
  		Figure 3. Calcium activates a novel population of channels in DmPKD2-transfected S2 cells. (A) Representative current trace before and after addition of 300 µM CaCl2. The large bidirectional deflections immediately after the arrow are artifacts caused by the addition and subsequent stirring. This record was obtained from an excised inside-out membrane patch in the presence of symmetric 114 mM BaCl2 solutions. The holding potential was +30 mV, which accounts for the positive (outward) currents. The inset in panel A shows an expanded 15-s trace, and the y-axis scaling is identical to the original. These data were low passed filtered at 100 Hz and sampled at 333.3 Hz. All data points are plotted. The Ca2+-stimulated current declined over time, and the averaged current per 10-s interval was replotted as shown in panel B. The solid line shows the best fit of these data to a single exponential function by nonlinear regression and the time constant ({tau}) for the decay was estimated to be 5.4 min (R2 = 0.95). (C) Another representative record whereby the current was activated by addition of bath calcium (100 µM) and subsequently decayed. This record contained >20 channels and was filtered at 5 Hz to reduce the open-channel noise and number of data points (10 Hz). The dashed line shows the best fit of these data to a single exponential decay function, and the time constant ({tau}) was 5.6 min (R2 = 0.89). (D) Dose-response curve for calcium activation. These results were obtained by first adding calcium at 1, 3, 10, or 30 µM (n = 3 for each) and normalizing the data on the basis of the subsequent response to 100 µM Ca+2. The response to 300 µM (n = 3) was quantified in reverse order. The error bars illustrate ± SD and these points were consistent with a Hill plot (R2 = 0.99) as shown by the dashed line. The KM was 8.6 µM and n = 1.6.

 
We quantified the calcium dose-dependence of the DmPKD2-induced channel activity; the data are summarized by the data plotted in Figure 3D. The curve shows the nonlinear fit to a Hill plot, which yielded a KM of 8.6 µM and a Hill coefficient of 1.6 (R2 = 0.99). Figure 4 shows that a similar type of channel activity was also evoked in cell-attached patches from DmPKD2-transfected S2 cells after addition of the Ca2+ ionophore, ionomycin. This trace is representative of three experiments. Conversely, no channel activity was observed in cell-attached patches from empty-vector transfected S2 cells (n = 6) (P < 0.05 by {chi}2 analysis). Thus, like mammalian PC2 proteins, heterologous expression of DmPC2 produced channels in the plasma membranes that were transiently activated by µM levels of intracellular Ca2+.



View larger version (29K):
[in this window]
[in a new window]
[as a PowerPoint slide]
  		Figure 4. The calcium ionophore, ionomycin, evoked transient channel openings in cell-attached patches from DmPKD2-transfected cells. This is a representative current record from a cell that was treated with Cu2+ for 1 d. This record was also obtained in the presence of symmetric 114 mM BaCl2 solutions, and the holding potential was –40 mV, which accounts for the inward current deflections. This is a continuous recording, except for the exclusion of a brief approximately 15-s interval during the addition of ionomycin (1 µM). These data were low passed filtered at 100 Hz and sampled at 333.3 Hz.

 
Conductance and Ion Selectivity of Drosophila Polycystin-2
Figure 5A shows a current-voltage (I-V) relation for the Ca2+-activated currents in DmPC2-expressing cells, which was recorded in excised inside-out patches in the presence of symmetric 114 mM BaCl2 in the bath and pipette. The dashed line illustrates the best fit of the I-V data by linear regression (R = 0.99), which yielded a conductance of 41 pS. This value underestimates the actual open channel conductance due to the fast, flickering bursting behavior shown in the current traces plotted below the I-V relations (30). These events could not be resolved by increasing the fc of the low-pass filter due to limitations in the signal-to-noise ratio.



View larger version (35K):
[in this window]
[in a new window]
[as a PowerPoint slide]
  		Figure 5. Current-voltage (I-V) relations and representative single channel records from excised inside-out membrane patches performed under conditions that define ion selectivity. (A) Symmetric 114 mM BaCl2 bath and pipette (Erev = 0 mV). (B) 11.4 mM Ba2+ in the pipette and 114 mM Ba2+ in the bath (Erev = –30 mV). (C) Bionic conditions with 114 mM Ca2+ in the pipette and 114 mM Ba2+ in the bath (Erev = 9.6 mV). (D) Bionic conditions with 114 mM Na+ in the pipette and 114 mM Ba2+ in the bath (Erev = 12.3 mV). In addition to 114 mM BaCl2 all bath solutions contained 10 mM PIPES-NMDG (pH = 6.2). 300 µM CaCl2 was added to the bath to activate channels before collection of these data. The membrane potential was varied, and the resulting unitary deflections recorded and analyzed as amplitude histograms using BioPatch software. All data were low passed filtered at 100 Hz and sampled at 333.3 Hz. The dashed lines show the best fits by linear regression (R2 > 0.96 for all).

 
Figures 5B to 5D show I-V relations and representative current tracings from excised inside-out patches under asymmetric ionic conditions. These data were obtained from DmPKD2-transfected cells after addition of calcium (100 to 300 µM) to the bath solutions. The results shown in Figure 5B were obtained in the presence of 114 mM BaCl2 in the bath and reduced concentrations of Ba+2 and Cl– in the pipette as described in the Materials and Methods section. The dashed line illustrates the best fit of the I-V data by linear regression (R2 = 0.99), which yielded a Vrev of –29 mV. PBa/PCl can be calculated using this value and the ratio (340:1) suggests that Cl– permeability is limited. The results plotted in Figure 5C were obtained with 114 mM CaCl2 in the pipette and 114 mM BaCl2 in the bath. The dashed line illustrates the best fit of these I-V data by linear regression (R2 = 0.96). The Vrev was 9.6 mV, which yields a relatively modest PCa/PBa of 2.0. Finally the data presented in Figure 5D were obtained with 114 mM NaCl in the pipette and 114 mM BaCl2 in the bath. The dashed line illustrates the best fit of these I-V data by linear regression (R2 = 0.99). The Vrev was 12 mV, which yields a PNa/PBa of approximately 10.

Lanthanum Blockade of Drosophila Polycystin-2
La3+ blocks mammalian PC2 and polycystin-L channel activity (6–8). Figure 6 illustrates the effect of LaCl3 on a representative record of Ca2+-activated multichannel currents in DmPC2-expressing cells. Addition of 10 mM LaCl3 to the bath solution abolished the channel activity, consistent with its effects on PC2 and polycystin-L (6–8).



View larger version (21K):
[in this window]
[in a new window]
[as a PowerPoint slide]
  		Figure 6. Lanthanum blocks DmPC2 channel activity. The record was derived from an excised inside-out patch. The bath and pipette solutions both contained BaCl2, and the holding potential was +30 mV throughout the experiment. Channels were activated by addition of CaCl2 to the bath solution, approximately 1 min before the start of this record. The addition of 10 mM LaCl3 to the bath solution caused an immediate decrease in current. The sample rate was 333.3 Hz, and fc was 100 Hz.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have cloned a Drosophila PKD2 cDNA (AY283154) that corresponds to a gene CG6504 located at cytological position 33E3 on the left arm of the second chromosome. This protein’s sequence, predicted membrane topology, and structure are similar to human PC2. Using this information, it is possible to construct a simple model for DmPC2 as depicted in Figure 6A. Like human PC2 and PCL, (1,2,31) DmPC2 consists of six membrane-spanning helices, a large extracellular domain containing a well-conserved "polycystin motif" between the first two helices, and a P-loop between membrane-spanning domains 5 and 6 that presumably forms a conductive pore.

Transfection of DmPKD2 cDNA into S2 cells produced a novel population of channels with similar properties as mammalian PC2. (1) The channel activity had a biphasic response after addition of Ca2+ to the cytosolic side, consisting of a transient activation followed by inactivation. The time constant for inactivation (5.2 ± 1.0 min, n = 8) was 3.5-fold to 5-fold longer than human PC2 (6) or polycystin-L, (7) which presents an advantage regarding data collection. (2) The calcium dose-response for channel activation (KM = 8.6 µM) was similar to the dose-response for human PC2-induced channels in planar lipid bilayers (KM = 6.7 µM). (3) The Drosophila channel had a unitary conductance of 41 pS in the presence of symmetric 114 mM BaCl2. This compares favorably with the 46 pS conductance recently reported for endogenous human PC2 in symmetric 100 mM BaCl2 solutions (32). (4) Cations (Ba++, Ca++, and Na+) were preferred as charge carriers over anion such as Cl–, as is the case for human PC2 (6). Specifically, the permeability ratio of Ca2+ to Cl– under our experimental condition is 340:1. (5) The channel showed a slightly enhanced (2:1) permeability of Ca2+ versus Ba2+ and it had a 10:1 preference for Na+ over Ba2+ (6,8). (6) Finally lanthanum can block the channel-mediated currents for both DmPC2 and PC2 (6). These data indicate that DmPC2 has generally similar structural and functional properties as mammalian PC2.

Mammalian PC2 channels can demonstrate voltage-dependent gating depending in part on experimental conditions and following the application of high voltages (8,9). However we observed little effect of voltage on DmPC2 currents at physiologic membrane potentials. In this regard, the voltage-dependent gating of heterologously expressed polycystin-L was recently shown to be caused by a blocking effect of Mg2+ (33).

Based on the known structures of homologous cation channels such as voltage dependent Na+ and Ca2+ channels, functional PC2 channels are expected to require an assembly of four channel subunits (2,34). Furthermore, induction of channels with similar properties after the transfection of PKD2 in a variety of expression systems (6,32), including cell free systems (8), suggests that PC2 alone may function as a channel. This study provides further credence to this notion using Drosophila. Figure 7B depicts one way that four homologous PC2 subunits may align hypothetically to form a central conductive element or pore with their P-loops. The six membrane-spanning domains and C-termini are depicted near the central part of the channel complex due to their proximity to the P-loop. The N- and C-termini of adjacent subunits are drawn near each other as PC2 channels are evolutionarily related to voltage-activated N- and T-type Ca2+ channel sequences (refer to Figure 1B) that are covalently linked in sequence. This description is not inconsistent with evidence the C-termini are important in the biochemical and subcellular localization of PC2 (6,35); importantly this proposed structure accounts for a conductive pore.



View larger version (22K):
[in this window]
[in a new window]
[as a PowerPoint slide]
  		Figure 7. Simple models illustrate DmPC2. (A) is derived from the known structures of other homologous cation channels plus the sequence analysis of DmPC2 shown in Figure 1. The homotetramer structure shown in panel B is suggested by the observation that heterologous expression of PC2 in a variety of system yields channels with generally similar properties. This model extends panel A based on the hypothesis that four P-loops are required for pore formation as has been described previously for other cation channels

 
DmPC2 can be used to test this model and investigate structure-function relationships for the conserved P-loop and polycystin motif. The pore region presumably defines the ionic conductance, selectivity, and blocker sensitivity. In the central portion of this sequence, there is a highly conserved, negatively charged Asp residue (Figure 1A) that has been hypothesized to confer Ca2+ selectivity to TRP and other calcium channels (27,34). Similarly the extracellular polycystin motif is conserved as shown by the comparison in Figure 1A, suggesting that it has an important function. Finally, in addition to the similarities between DmPC2 and mammalian PC2 at levels of protein sequence and channel properties presented here, we recently published evidence of functional similarity (36). Mammalian PC2 has been found to localize to primary cilia of renal epithelia where it is involved in calcium entry in response to mechanical signals such as fluid flow (11,12). Interestingly, DmPC2 is localized to fly sperm flagella, a special type of motile cilium (36,37). We generated loss-of-function mutations and found that DmPKD2 mutant males were sterile because the mutant sperm did not response to directional cues (chemical or mechanical signals) in the female reproductive tract (36). Thus it appears that both Drosophila and mammalian PC2 functions on ciliated structures. We anticipate that the DmPC2 Drosophila model, due to its powerful genetic tools, will facilitate the identification of upstream signaling modulators that regulate PC2 channel activity, such as possible PC1 homologues, as well as the specific mediators and pathways stimulated by Ca+2 entry through PC2.


   Acknowledgments
 
We thank Dr. Yedvobnick and Anumeha Kumar (Emory University) for generously providing the Drosophila S2 cells and Dr. Ronald L. Johnson for his kind gift of the pMK33/pMtHY Drosophila expression vector. We are also grateful to Drs. Douglas Ruden and Eric J. Sorscher for sharing equipment and providing various assistance and support. This work is supported by NIDDK grants (DK57301, DK60821) and a supplemental grant from the Polycystic Kidney Disease Research Foundation to X.L.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Mochizuki T, Wu G, Hayashi T, Xenophontos SL, Veldhuisen B, Saris JJ, Reynolds DM, Cai Y, Gabow PA, Pierides A, Kimberling WJ, Breuning MH, Deltas CC, Peters DJ, Somlo S: PKD2, a gene for polycystic kidney disease that encodes an integral membrane protein. Science 272: 1339–1342, 1996[Abstract]
  2. Stayner C, Zhou J: Polycystin channels and kidney disease. Trends Pharmacol Sci 22: 543–546, 2001[CrossRef][Medline]
  3. Gabow PA: Autosomal dominant polycystic kidney disease. N Engl J Med 329: 332–342, 1993[Free Full Text]
  4. Sullivan LP, Wallace DP, Grantham JJ: Epithelial transport in polycystic kidney disease. Physiol Rev 78: 1165–1191, 1998[Abstract/Free Full Text]
  5. Igarashi P, Somlo S: Genetics and pathogenesis of polycystic kidney disease. J Am Soc Nephrol 13: 2384–2398, 2002[Free Full Text]
  6. Vassilev PM, Guo L, Chen XZ, Segal Y, Peng JB, Basora N, Babakhanlou H, Cruger G, Kanazirska M, Ye C, Brown EM, Hediger MA, Zhou J: Polycystin-2 is a novel cation channel implicated in defective intracellular Ca2+ homeostasis in polycystic kidney disease. Biochem Biophys Res Comm 282: 341–350, 2001[CrossRef][Medline]
  7. Chen XZ, Vassilev PM, Basora N, Peng JB, Nomura H, Segal Y, Brown EM, Reeders ST, Hediger MA, Zhou J: Polycystin-L is a calcium-regulated cation channel permeable to calcium ions. Nature 401: 383–386, 1999[CrossRef][Medline]
  8. González-Perrett S, Kim K, Ibarra C, Damiano AE, Zotta E, Batelli M, Harris PC, Reisin IL, Arnaout MA, Cantiello HF: Polycystin-2, the protein mutated in autosomal dominant polycystic kidney disease (ADPKD), is a Ca2+-permeable nonselective cation channel. Proc Natl Acad Sci U S A 98: 1182–1187, 2001[Abstract/Free Full Text]
  9. Gonzalez-Perrett S, Batelli M, Kim K, Essafi M, Timpanaro G, Moltabetti N, Reisin IL, Arnaout MA, Cantiello HF: Voltage dependence and pH regulation of human polycystin-2-mediated cation channel activity. J Biol Chem 277: 24959–24966, 2002[Abstract/Free Full Text]
  10. Koulen P, Cai Y, Geng L, Maeda Y, Nishimura S, Witzgall R, Ehrlich BE, Somlo S: Polycystin-2 is an intracellular calcium release channel. Nat Cell Biol 4: 191–197, 2002[CrossRef][Medline]
  11. Yoder BK, Hou X, Guay-Woodford LM: The polycystic kidney disease proteins, polycystin-1, polycystin-2, polaris, and cystin, are co-localized in renal cilia. J Am Soc Nephrol 13: 2508–2516, 2002[Abstract/Free Full Text]
  12. Nauli SM, Alenghat FJ, Luo Y, Williams E, Vassilev P, Li X, Elia AE, Lu W, Brown EM, Quinn SJ, Ingber DE, Zhou J: Polycystins 1 and 2 mediate mechanosensation in the primary cilium of kidney cells. Nat Genet 33: 129–137, 2003[CrossRef][Medline]
  13. Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ: Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25: 3389–3402, 1997[Abstract/Free Full Text]
  14. Koelle MR, Talbot WS, Segraves WA, Bender MT, Cherbas P, Hogness DS: The Drosophila EcR gene encodes an ecdysone receptor, a new member of the steroid receptor superfamily. Cell 67: 59–77, 1991[CrossRef][Medline]
  15. Schneider I: Cell lines derived from late embryonic stages of Drosophila melanogaster. J Embryol Exp Morphol 27: 353–365, 1972[Medline]
  16. Millar NS, Buckingham SD, Sattelle DB: Stable expression of a functional homo-oligomeric Drosophila GABA receptor in a Drosophila cell line. Proc R Soc Lond B Biol Sci 258: 307–314, 1994[Medline]
  17. Hardie RC, Reuss H, Lansdell SJ, Millar NS: Functional equivalence of native light-sensitive channels in the Drosophila trp301 mutant and TRPL cation channels expressed in a stably transfected Drosophila cell line. Cell Calcium 21: 431–440, 1997[CrossRef][Medline]
  18. Asmild M, Willumsen NJ: Chloride channels in the plasma membrane of a foetal Drosophila cell line, S2. Pflugers Arch 439: 759–764, 2000[CrossRef][Medline]
  19. Hamill O, Marty A, Neher E, Sakmann B, Sigworth FJ: Improved patch-clamp techniques for high resolution current recording from cells and cell-free membrane patches. Pflugers Arch 391: 85–100, 1981[CrossRef][Medline]
  20. Venglarik CJ, Schultz BD, Frizzell RA, Bridges RJ: ATP alters current fluctuations of cystic fibrosis transmembrane conductance regulator: evidence for a three-state activation mechanism. J Gen Physiol 104: 123–146, 1994[Abstract/Free Full Text]
  21. Venglarik CJ, Schultz BD, DeRoos AD, Singh AK, Bridges RJ: Tolbutamide causes open channel blockade of cystic fibrosis transmembrane conductance regulator Cl- channels. Biophys J 70: 2696–2703, 1996[Abstract/Free Full Text]
  22. Lewis CA: Ion-concentration dependence of the reversal potential and the single channel conductance of ion channels at the frog neuromuscualr junction. J Physiol (London) 286: 417–445, 1979[Abstract/Free Full Text]
  23. Hille B. Ionic Channels of Excitable Membranes. Sunderland, MA, Sinauer Associates Inc., 1992.
  24. Fatt P, Ginsborg BL: The ionic requirements for the production of action potentials in crustacean muscle fibers. J Physiol (London) 115: 516–543, 1958
  25. Krogh A, Larsson B, von Heijne G, Sonnhammer EL: Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. J Mol Biol 305: 567–580, 2001[CrossRef][Medline]
  26. Lupas A, Van Dyke M, Stock J: Predicting coiled coils from protein sequences. Science 252: 1162–1164, 1991[CrossRef][Medline]
  27. Tsiokas L, Kim E, Arnould T, Sukhatme VP, Walz G: Homo- and heterodimeric interactions between the gene products of PKD1 and PKD2. Proc Natl Acad Sci U S A 94: 6965–6970, 1997[Abstract/Free Full Text]
  28. Huang X, Miller W: A time efficient, linear space local similarity algorithm. Adv Appl Mathemat 12: 337–357, 1991[CrossRef]
  29. Higgins DG: CLUSTAL V: multiple alignment of DNA and protein sequences. Methods Mol Biol 25: 307–318, 1994[Medline]
  30. Yellen G: Ionic permeation and blockade in Ca2+-activated K+ channels of bovine chromaffin cells. J Gen Physiol 84: 157–186, 1984[Abstract/Free Full Text]
  31. Guo L, Schreiber TH, Weremowicz S, Morton CC, Lee C, Zhou J: Identification and characterization of a novel polycystin family member, polycystin-L2, in mouse and human: Sequence, expression, alternative splicing, and chromosomal localization. Genomics 64: 241–251, 2000[CrossRef][Medline]
  32. Luo Y, Vassilev PM, Li X, Kawanabe Y, Zhou Z: Native polycystin 2 functions as a plasma membrane Ca2+-permeable cation channel in renal epithelia. Mol Cell Biol 23: 2600–2607, 2003[Abstract/Free Full Text]
  33. Liu Y, Li Q, Tan M, Zhang YY, Karpinski E, Zhou J, Chen XZ: Modulation of the human polycystin-L channel by voltage and divalent cations. FEBS Lett 525: 71–76, 2002[CrossRef][Medline]
  34. Varadi G, Mori Y, Mikala G, Schwartz A: Molecular determinants of Ca2+ channel function and drug action. Trends Pharmacol Sci 16: 43–49, 1995[CrossRef][Medline]
  35. Hanaoka K, Qian F, Boletta A, Bhunia AK, Piontek K, Tsiokas L, Sukhatme VP, Guggino WB, Germino GG: Co-assembly of polycystin-1 and –2 produces unique cation-permeable currents. Nature 408: 990–994, 2000[CrossRef][Medline]
  36. Gao Z, Yu Z, Wang L, Ruden D, Lu X: PKD2 cation channel is required for directions sperm movement and male fertility. Current Biology 13: 2175–2178, 2003[CrossRef][Medline]
  37. Watnick TJ, Jin J, Matunis E, Kernan MJ, Montell C: A flagellar polycystin-2 homolog required for male fertility in Drosophila. Current Biology 13: 2179–2184, 2003[CrossRef][Medline]
Received for publication June 18, 2003. Accepted for publication February 26, 2004.




This article has been cited by other articles:


Home page
 JCBHome page
K. Huang, D. R. Diener, A. Mitchell, G. J. Pazour, G. B. Witman, and J. L. Rosenbaum
Function and dynamics of PKD2 in Chlamydomonas reinhardtii flagella
J. Cell Biol., November 5, 2007; 179(3): 501 - 514.
[Abstract] [Full Text] [PDF]

Home page
 J. Am. Soc. Nephrol.Home page
T. Obara, S. Mangos, Y. Liu, J. Zhao, S. Wiessner, A. G. Kramer-Zucker, F. Olale, A. F. Schier, and I. A. Drummond
Polycystin-2 Immunolocalization and Function in Zebrafish
J. Am. Soc. Nephrol., October 1, 2006; 17(10): 2706 - 2718.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Venglarik, C. J.
Right arrow Articles by Lu, X.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Venglarik, C. J.
Right arrow Articles by Lu, X.

HOME CURRENT ISSUE ARCHIVES JASN Express ONLINE SUBMISSION AUTHOR INFO
EDITORIAL BOARD SUBSCRIBE FEEDBACK ALERTS HELP