Cloning and Functional Expression of a Swelling-Induced Chloride Conductance Regulatory Protein, plCln, from Rabbit Ocular Ciliary Epithelium

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.

239, 692–696 (1997)

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Cloning and Functional Expression of a Swelling-Induced Chloride Conductance Regulatory Protein, plCln , from Rabbit Ocular Ciliary Epithelium1 Xiao Lin Wan, Shan Chen, and Marvin Sears2 Department of Ophthalmology and Visual Science, Yale University School of Medicine, P.O. Box 208061, New Haven, Connecticut 06520-8061

Received September 15, 1997

The cDNA encoding a swelling-induced chloride conductance regulatory protein, plcln , was cloned from rabbit ciliary epithelium by using a polymerase chain reaction (PCR)-based approach. The open reading frame encoding 236 amino acids possesses high amino acid identity (93/%) with the previously cloned plcln from human ciliary epithelium. Outwardly rectifying currents were recorded in Xenopus oocytes injected with plcln cRNA, a result consistent with plcln expression in ciliary epithelium. A widespread distribution and marked expression of plcln mRNA in both nonpigmented ciliary epithelial (NPE) cells and pigmented ciliary epithelial (PE) cells was found for the first time. In situ hybridization analysis showed that plcln expression is more abundant in NPE than PE. These findings are consistent with the idea that plcln may be an important regulatory element in these secretory cells. q 1997 Academic Press

The aqueous humor that nourishes the avascular interior of the eye is secreted by the ocular ciliary epithelium, a bilayer of nonpigmented ciliary epithelial cells (NPE) and pigmented ciliary cells (PE) whose apical surfaces are apposed (1, 2). Chloride channels in the ciliary epithelium are considered to play a critical role in the aqueous humor formation, and can regulate cell volume in response to swelling induced by hypotonicity (3-5). In fact, 4 chloride channels/channel regulatory, plcln (6), P-glycoprotein (7), CIC-2 (8), and CIC-3 (9), thus far have been reported to be volume sensitive, and are considered to be involved in the formation of aqueous humor (10-14). These swelling-induced CI0 1 This work was supported by Grants NIH EY 08879-07 and EY00785-25. The nucleotide sequence(s) reported in this paper has been submitted to the GenBank database under Accession No. AF003907. 2 To whom correspondence should be addressed. Fax: 203-7374227. E-mail: [email protected].

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were too similar to be distinguished only by electrophysiology. Recently, plcln , a swelling-induced chloride conductance regulatory protein, was cloned from Madin Darby canine kidney (MDCK) epithelial cells (6), and rat atria (15, 16), and Xenopus oocytes (15). plcln has been cloned from human NPE cell lines (17), but in situ hybridization is required to learn whether there is a difference in plcln localization between NPE and PE. Further, functional expression of these regulatory CI0 channels/ channel can be characterized physiologically using injected oocytes. Finally, it is from the rabbit eye about which much of our knowledge of aqueous humor dynamics comes and therefore we used this species. In this report, plcln was cloned from rabbit ciliary epithelium by polymerase chain reaction (PCR), and its molecular and physiological properties were characterized at the electrophysiological and gene levels by injecting the expression system with Xenopus oocytes. MATERIAL AND METHODS Cloning of plcln . Poly (A)/ mRNA was prepared using the MicroFast mRNA kit (Invitrogen, San Diego, CA) from rabbit ciliary bilayer isolated by perfusion and microdissection technique (18, 19). One hundred ng of Poly (A)/ mRNA was reverse transcribed to firststranded cDNA using random primer and Moloney murine lrukemia virus reverse transcriptase (Stratagene, La Jolla, CA). The cDNA pool (1 ml) and 100 pmol of each of a pair of specific primers 5*ATGAGCTTCCTCAAAAGTTTCCCG-3* and 5*-GTGATCAACATCTGCATCCTC-3*, which were designed based on the sequence of MDCK plcln (6), were added in 50 ml of PCR mixture. Thirty cycles of PCR were carried out: denaturation at 94 7C, 1 min; annealing at 55 7C, 1 min; extension at 72 7C, 1 min; final extension, 5 min. The amplified DNA fragment (708 bp containing entire coding region of plcln) was subcloned into PCR II (TA cloning kit, Invitrogen) and sequenced in both directions. Nucleotide sequence analyses were performed by the fluorescent chain-terminating dideoxynucleotide method (20). Northern analysis. RNA blot analysis was carried out by using 15 mg of total RNA, extracted from rabbit ciliary bilayer by the guanidinium isothiocyanate method (21), size fractionated by electropho-

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resis on denatured 1% agarose-formaldehyde gels, and transferred to nylon membrane. The full-length plcln probe was labeled with [a32P] dCTP (3000 Ci/mmol, Amersham, Arlington Heights, IL) by random primer DNA labeling system (Gibcobrl, Gaithersburg, MD) and used for hybridization. In situ hybridization. The labeled antisense and sense RNA probes were prepared by in vitro transcription using SP 6 and T7 RNA polymerase in the presence of [a32S] UTP (ú1000 Ci/mmol, Amersham) using full-length plcln cDNA as a template. Hybridization was done using antisense RNA as a probe and sense RNA as control. Tissue sections (5 mm) after removing paraffin were rehydrated by passing them through a descending ethanol series, and incubated with proteinase K (1mg / ml) for 30 min at 37 7C, then treated with freshly prepared 0.1 M triethanolamine-HCI (pH 8.0) at room temperature for 10 min and dehydrated in an ascending ethanol series. The probe was dissolved in a buffer containing 50% formamide, 2 1 SSC, 250 ng/ml tRNA, 1 1 Denhardt’s solution, 500 mg/ml singlestranded salmon sperm DNA, 10% dextra sulfate, 10 mM dithiothreitol; 50 ml of probe solution was applied to each slide. After incubation at 52 7C overnight in a humidified chamber, sections were washed several times in 4 1 SSC containing 10 mM dithiothreitol followed by dehydration through an ascending ethanol series containing 0.3 M ammonium acetate, and incubated them in 50 % formamide / 2 1 SSC at 52 7C for 15 min. Sections were then treated with RNAase A (30 mg/ml) at 37 7C for 30 min, and rinsed several times in 2 1 SSC containing 10 mM b-mercaptoethanol, followed for 15 min in 0.1 1 SSC at 52 7C before being dehydrated again . The sections were dipped in NTB-2 emulsion (1: 1 with water) (Eastman Kodak, Rochester, NY) at 42 7C, and exposed for 3 weeks, and developed with Kodak D-19 and Kodak fixer (Eastman Kodak), and then stained with hematoxylin and eosin for microscopic evaluation. Morphometry. To determine the distribution of plcln mRNA between NPE and PE, grain densities were evaluated for five different of sections (magnification, 1 1000 each) from both NPE and PE. Statistical difference was evaluated by Student’s t-test . Electrophysiology. The full-length plcln cDNA was subcloned into pSP 64 poly (A) vector (Promega, Piscataway, NJ) at Xba 1 and BamH 1 sites. Capped cRNA was synthesized from this construct using SP 6 polymerase after linearization of this construct by restriction with Pvu II. Oocytes were isolated from Xenopus laevis (Nasco, Atkinson, WI), defolliculated by collagenase treatment (Sigma, St. Louis, MO), and injected with either Ç50 ng cRNA or 50 nl nucleasefree water per oocyte. Whole oocyte currents were recorded using a two-electrode voltage clamp (OC-725A, Warner Instrument) in ND96 solution (96 mM NaCI, 2 mM KCI, 1.8 mM CaCI, 1 mM MgCI, 5 mM HEPES [pH 7.4]) at room temperature, 3 to 6 days after injection. Data were filtered at 1 kHz and sample at 4 kHz. Electrodes contained 3 M KCI and had resistances of 0.3-1 MV. Deta are expressed as mean { S. E. M. (n).

RESULTS Cloning of rabbit plcln . A full length 708-bp plcln cDNA from rabbit ciliary epithelium mRNA was amplified with the PCR. A comparison of amino acid sequence of the rabbit plcln with the human plcln (17) is shown in Fig. 1, in which the rabbit plcln is composed of 236 amino acid residues, whereas the human plcln consists of 237 amino acid residues. The deduced amino acid of rabbit plcln is 93% identical to that of human plcln . The half including the C-terminus end of the rabbit plcln protein exhibits 100% homology with the human plcln protein. The first (amino acids 30-40) and second (amino acids 49-59) regions of the four putative

FIG. 1. Deduced amino acid sequence of plcln in ciliary epithelium of rabbit and human. Identical amino acids are indicated by periods. The four putative transmembrane b-sheets, positions I (30-40), II (49-59), III (60-68), and IV (77-87), are underlined.

transmembrane b-sheets are also conserved 100% whereas third (amino acids 60-68) and fourth (amino acids 77-87) regions differ in a single amino acid. The region of largest dissimilarity resides in a stretch of six amino acids (70-75). Tissue distribution. To determine whether plcln transcripts are expressed in the rabbit ciliary epithelium, a blot containing total RNA from rabbit ciliary body tissue was hybridized to the full-length rabbit plcln probe (708 bp). The major mRNA species of approximate 1.8 and 4.2 kb were identified. The 1.8 kb species was more abundant (Fig. 2). The transcript of 01.8 kb is consistent with the result observed in human ciliary body (17). Although the transcript of 04.2 kb was not detected in human ciliary body, This difference may reflect different products of mRNA processing in different species. Based on these results, we further localized the site of expression of plcln in the bilayered rabbit ciliary epithelium by in situ hybridization. Fig. 3 demonstrates that the marked expression of plcln mRNA in ciliary epithelium in both NPE and PE, but the expression is 50% more abundant in NPE than PE. Average silver grain counts/view x1000 of the NPE were 331{ 22 (15) and PE were 220 { 18 (15) a statistically significant difference (p õ 0.002). No signal was detected in a parallel control experiment using a sense RNA probe (data not shown). Functional expression. When oocytes were injected with plcln cRNA transcribed in vitro, outwardly rectifying currents [0700 { 220 nA (5) at 080 mV and 3840 { 160 nA (5) at / 80 mA] were observed, compared with water-injected oocytes [080 { 5 nA (5) at 080 mV and 210 { 8 nA (5) at / 80 mV]. The currents reversed at 020 mV. The outwardly rectifying current was detected in 67%, 20 of 30 oocytes injected with plcln cRNA, whereas only 2 of 50 (4%) oocytes injected with water had the similar outward current.

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FIG. 2. Expression of plcln mRNA. (A) Northern analysis of plcln in rabbit ciliary epithelium. Total RNA (15 mg) isolated from rabbit ciliary epithelium was hybridized in a Northern blot with the rabbit full-length plcln cDNA probe. The ribosomal RNA 28 S/18 S are indicated at right. (B and C) Localization of plcln in rabbit ciliary epithelium by in situ hybridization. Sections were hybridized with 35S-labeled antisense RNA probe specific for plcln . Sections were stained with hematoxylin/eosin after hybridization. (B) Darked-field photomicrograph of emulsion-dipped section of ciliary epithelium (1400). (C) Bright-field photomicrograph (11000).

DISCUSSION In this study, plcln was cloned from ocular rabbit ciliary epithelium by PCR. The deduced amino acid sequence of plcln cloned from rabbit ciliary epithelium exhibits 93% identity to the plcln previously cloned from human NPE cell line. Moreover, the half including the C-terminus end of the rabbit plcln protein displays 100% homology with human plcln protein (17). The very high level of conservation indicates that any domain of plcln C-terminus probably has a functional role. Outwardly rectifying currents were recorded in oocytes overexpressing plcln , a find is consistent with a previous report (6). A consensus has not yet been reached concerning whether plcln is the chloride channel itself, with 2 plcln molecules combining to form a transmembrane chloride channel (6). Does plcln function as a swelling-induced chloride conductance regulatory protein rather than as the chloride channel (15)? Irrespective of this controversy, results from both the molecular biology and physiology indicate that plcln is critical for the activation of swelling-induced CI0 release in epithelial and cardiac cells, and Xenopus oocytes (6, 10, 15-17, 22). By using a polyclonal antiserum raised against human plcln , a widespread expression of plcln has been found in different cells and tissues (23). All these findings suggest that plcln can play an important role in regulating cell volume. The ciliary epithelium is composed of two cell layers (NPE and PE), but it is not clear what is the distribution of plcln there in. It was reported that freshly iso-

lated bovine NPE cells exhibited a regulatory volume decrease (RVD) when subjected to osmotic swelling, whereas PE cells did not display a regulatory volume response after hypnotic swelling (24). Miley et al. did report that PE cell displayed an RVD when using freshly isolated bovine cells (25). RVD has not been found in the rabbit ciliary epithelium, and, it should be pointed out that extracellular ciliary channels may exert volume regulation (26). Nonetheless, in this study of the rabbit, we localized the site of plcln expression in ciliary epithelium and directly observed distribution of plcln expression by in situ hybridization. A marked expression was observed both in NPE and PE cells, with a 50% more abundant expression of plcln in NPE than PE cells (Fig. 3). Perhaps that coincides with the larger surface area of the NPE compared with NE. Most importantly, outwardly rectifying chloride current were recorded both in NPE and PE cells of rabbit (data not shown). The chloride current is consistent with that expressed in Xenopus oocytes injected with plcln cRNA. The data presented above, widespread distribution and marked expression of plcln both in NPE and PE, accord with the findings of others who suggest that volume-sensitive plcln protein may be involved in the secretion of aqueous humor. This finding is in general agreement with syncytial model of aqueous humor secretion (24): ions are first taken up from the stroma of the ciliary process across the PE cell plasma membrane, a cell without tight junctions that uses its entire surface for inward transport. Then ions diffuse across

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FIG. 3. Electrophysiological characterization of plcln injected in oocytes. (A) Currents observed in plcln cRNA-injected oocytes (Ç50 ng per oocyte). (B) Currents in H2O-injected oocytes. Holding potential, 020 mV. Voltage steps (800 ms) were made from 080 mV to /80 mV in 20 mV steps. (C) Current-voltage relationship obtained from oocytes injected with plcln cRNA (closed circles) and water (closed circles) (mean { S.E.M., 5 oocytes).

gap junctions into the adjacent NPE cells. Finally, ions are then transported across the basolateral membrane of the NPE cell into the posterior chamber of the eye accompanied by water. A cAMP-induced chloride conductance channel was observed and reported for NPE (27, 28), integrating the known adrenergic components

for aqueous secretion (2). Its features are different from cystic fibrosis transmembrane conductance regulator (CFTR) CI0 channel (27). It is not clear whether there is a close link between volume-sensitive plcln protein and cAMP-induced CI0 channel, but we have recently observed that volume-sensitive CI0 currents can be

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blocked by cAMP (22), indicating strategic differences between cAMP sensitive rate limiting low conductance NPE CI0 channels (28) and the volume regulator plcln described here. In addition to plcln , a volume-activated CI0 current regulated by P-glycoprotein was reported in bovine NPE cells (13). Although there is no homology to be found between the amino acid sequence of plcln protein and P-glycoprotein, there is no necessary contradiction here. The plcln protein and P-glycoprotein may coexist in ocular epithelial cells and affect different volumeactivated CI0 channels. In summary, cDNA encoding a swelling-induced chloride conductance regulatory protein, plcln , was cloned and characterized from rabbit ciliary epithelium. The predicted protein has a sequence of 236 amino acids bearing a 93% homology to human plcln . More abundant expression of plcln mRNA was observed in NPE than PE by in situ hybridization. Outwardly rectifying currents were recorded in oocytes expressing plcln . These results are consistent with the idea that plcln plays an important role in regulating volume in these ocular secretory epithelial cells. ACKNOWLEDGMENTS We thank F. J. Sigworth and Steve A. N. Goldstein for kindly providing access to certain laboratory equipment and J. P. Zhang for assistance with in situ hybridization.

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6. Paulmichl, M., Li, Y., Wickman, K., Ackerman, M., Peralta, E., and Clapham, D. (1992) Nature 356, 238–241. 7. Valverde, M. A., Diaz, M., Sepulveda, F. V., Gill, D. R., Hyde, S. C., and Higgins, C. F. (1992) Nature 355, 830–833. 8. Gru¨nder, S., Thiemann, A., Pusch, M., and Jentsch, T. J. (1992) Nature 360, 759–762. 9. Kawasaki, M., Suzuki, M., Uchida, S., and Marumo, F. (1995) Neuron 14, 1285–1291. 10. Coca-Prados, M., Anguita, J,. Chalfant, M. L., and Civan, M. M. (1995) Am. J. Physiol. 268, C572–C579. 11. Yantorno. R. E., Carre, D. A., Coca-Prados, M., Krupin, T., and Civan, M. M. (1992) Am. J. Physiol. 262, C501–C509. 12. Coca-Prados, M., Sanchez-Torres, J., Peterson-Yantorno, and Civan, M. M. (1996) J. Membra. Biol. 150, 197–208. 13. Wu, J., Zhang, J. J., Koppel, H., and Jacob, T. J. C. (1996) J. Physiol. 491, 743–755. 14. Zhang, J. J., and Jacob, T. J. C. (1996) J. Physiol. 499, 379–389. 15. Krapivinsky, G. B., Ackerman, M. J., Gordon, E. A., Krapivinsky, L. D., and Clapham, D. E. (1994) Cell 76, 439–448. 16. Abe, T., Takeuchi, K., Ishii, K., and Abe, K. (1993) Biochim. Biophys. Acta. 1173, 353–356. 17. Anguita, J., Chalfant, M. L., Civan, M. M., and Coca-prados, M. (1995) Biochem. Biophys. Res. Commun. 208, 89–95. 18. Sears, M. L., Yamada, E., Cummins, D., Mori, N., Mead, A., and Murakami, M. (1991) Tr. Am. Ophth. Soc. 89, 132–154. 19. Wan, X. L., Sears, J., Chen, S., and Sears, M. L. (1997) Exp. Eye Res. 64, 1005–1011. 20. Prober, J. M., Trainor, G. L., Dam, R. J., Hobbs, F. W., Robertson, C. W., Zagursky, R. J., Cocuzza, A. J., Jensen, M. A., and Baumeister, K. (1987) Science 238, 336–341. 21. Chomczynski, P., and Sacchi, N. (1987) Anal. Biochem. 162, 156– 159. 22. Ackerman, M. J., Wickman, K. D., and Clapham, D. E. (1994) J. Gen. Physiol. 103, 153–179. 23. Buyes, G., Greef, C. D., Raeymaekers, L., Droogmans, G., Nilius, B., and Eggermont, J. (1996) Biochem. Biophys. Res. Commun. 218, 822–827. 24. Edelman, J. L., Sachs, G., and Adorante, J. S. (1994) Am. J. Physiol. 266, C1210–C1221. 25. Miley, H. E., Walker, V. E., Pollard, C. E., and Jacob, T. J. C. (1995) Invest. Ophthalmol. Visual. Sci. 36, S586. 26. Fujita, H., Kondo, K., and Sears, M. (1984) Klin. Monatsbl. F. Augenheilk. 185, 28–34. 27. Chen, S., Inoue, R., Inomata, H., and Ito, Y. (1994) Br. J. Pharmacol. 112, 1137–1145. 28. Chen, S., and Sears, M. L. (1997) Curr. Eye Res. 16, 710–718.

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