Skip to main content
SpringerLink
Log in

Functional and Molecular Characterization of a Volume-activated Chloride Channel in Rabbit Corneal Epithelial Cells

  • Articles
  • Published:
The Journal of Membrane Biology Aims and scope Submit manuscript

Abstract

We characterized the functional and molecular properties of a volume-regulated anion channel (VRAC) in SV40-immortalized rabbit corneal epithelial cells (tRCE), since they mediate a robust regulatory volume decrease (RVD) response during exposure to a hypotonic challenge. Whole-cell patch clamp-monitored chloride currents and light- scattering measurements evaluated temporal cell- volume responsiveness to hypoosmotic challenges. Exposure to 200 mOsm medium elicited an outwardly-rectifying current (VACC), which was reversible upon reperfusion with isotonic (300 mOsm) medium. VACC and RVD were chloride-dependent because either chloride removal or application of NPPB (100 μM) suppressed these responses. VACC behavior exhibited voltage-dependent inhibition in the presence of DIDS (500 μM), whereas inhibition by both NPPB (100 μM) and niflumic acid (500 μM) was voltage-independent. VACC was insensitive to glibenclamide (250 μM), verapamil (500 μM) or removal of extracellular calcium. Phorbol dibutyrate, PDBu, (100 nM) had no effect on activated VACC. However, preincubation with PDBu prior to hypotonic challenge prevented VACC and RVD responses as well as prolonged characteristic time. An inactive phorbol ester analogue had no effect on RVD behavior. Moreover, Northern blot analysis verified expression of ClC-3 gene transcripts. The presence of ClC-3 transcripts along with the correspondence between the effects of known ClC-3 inhibitors on VACC and RVD suggest that ClC-3 activation underlies these responses to hypotonic-induced cell swelling.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8

Similar content being viewed by others

References

  • M.J. Ackerman K.D. Wickman D.E. Clapham (1994) ArticleTitleHypotonicity activates a native chloride current in Xenopus oocytes J. Gen. Physiol. 103 153–179 Occurrence Handle10.1085/jgp.103.2.153 Occurrence Handle1:CAS:528:DyaK2cXkslyru7w%3D Occurrence Handle8189203

    Article  CAS  PubMed  Google Scholar 

  • L. Al-Nakkash P.S. Reinach (2001) ArticleTitleActivation of a CFTR-mediated chloride current in a rabbit corneal epithelial cell line Invest. Ophthalmol. Vis. Sci. 42 2364–2370 Occurrence Handle1:STN:280:DC%2BD3MvosFyisA%3D%3D Occurrence Handle11527951

    CAS  PubMed  Google Scholar 

  • K. Araki-Sasaki Y. Ohashi T. Sasabe K. Hayashi H. Watanabe Y. Tano H. Handa (1995) ArticleTitleAn SV40-immortalized human corneal epithelial cell line and its charcteristics Invest. Ophthalmol. Vis. Sci. 36 614–621 Occurrence Handle1:STN:280:ByqC1Mjjs1E%3D Occurrence Handle7534282

    CAS  PubMed  Google Scholar 

  • V.N. Bildin Z. Wang P. Iserovich P.S. Reinach (2003) ArticleTitleHypertonicity-induced p38MAPK activation elicits recovery of corneal epithelial cell volume and layer integrity J. Membrane Biol. 193 1–13 Occurrence Handle10.1007/s00232-002-2002-8 Occurrence Handle1:CAS:528:DC%2BD3sXkvVequrk%3D

    Article  CAS  Google Scholar 

  • V.N. Bildin H. Yang R.B. Crook J. Fischbarg P.S. Reinach (2000) ArticleTitleAdaptation by corneal epithelial cells to chronic hypertonic stress depends on upregulation of Na:K:2Cl cotransporter gene and protein expression and ion transport activity J. Membrane Biol. 177 41–50 Occurrence Handle10.1007/s002320001098 Occurrence Handle1:CAS:528:DC%2BD3cXmsVOrt7g%3D

    Article  CAS  Google Scholar 

  • S.H. Boese M. Glanville M.A. Gray N.L. Simmons (2000) ArticleTitleThe swelling-activated anion conductance in the mouse renal inner medullary collecting duct cell line mIMCD-K2 J. Membrane Biol. 177 51–64 Occurrence Handle10.1007/s002320001099 Occurrence Handle1:CAS:528:DC%2BD3cXmsVOrt7k%3D

    Article  CAS  Google Scholar 

  • G. Borsani E.I. Rugarli M. Taglialatela C. Wong A. Ballabio (1995) ArticleTitleCharacterization of a human and murine gene (CLCN3) sharing similarities to voltage-gated chloride channels and to a yeast integral membrane protein Genomics 27 131–141 Occurrence Handle10.1006/geno.1995.1015 Occurrence Handle1:CAS:528:DyaK2MXlvVahs7s%3D Occurrence Handle7665160

    Article  CAS  PubMed  Google Scholar 

  • O.A. Candia R. Montoreano S.M. Podos (1977) ArticleTitleEffect of the ionophore A23187 on chloride transport across isolated frog cornea Am. J. Physiol. 233 F94–101 Occurrence Handle1:CAS:528:DyaE2sXltlSqt7g%3D Occurrence Handle329688

    CAS  PubMed  Google Scholar 

  • O.A. Candia A.C. Zamudio (2002) ArticleTitleSecretagogues reduce basolateral K+ permeability in the rabbit corneal epithelium J. Membrane Biol. 190 197–205 Occurrence Handle10.1007/s00232-002-1037-1 Occurrence Handle1:CAS:528:DC%2BD3sXktFOgsg%3D%3D

    Article  CAS  Google Scholar 

  • P. Chomczynski N. Sacchi (1987) ArticleTitleSingle-step method of RNA isolation by acid guanidium thiocyanate-phenol-chloroform extraction Anal. Biolchem. 162 156–159 Occurrence Handle10.1016/0003-2697(87)90021-2 Occurrence Handle1:CAS:528:DyaL2sXitFSns7Y%3D

    Article  CAS  Google Scholar 

  • M. Coco-Prados J. Sanchez-Torres K. Peterson-Yantorno M.M. Civan (1996) ArticleTitleAssociation of ClC-3 channel with Cl− transport by human nonpigmented ciliary epithelial cells J. Membrane Biol. 150 197–208 Occurrence Handle10.1007/s002329900044 Occurrence Handle1:CAS:528:DyaK28XitVKlt7k%3D

    Article  CAS  Google Scholar 

  • D. Duan S. Cowley B. Horowitz J.R. Hume (1999) ArticleTitleA serine residue in ClC-3 links phosphorylation-dephosphorylation to chloride channel regulation by cell volume J. Gen. Physiol. 113 57–70 Occurrence Handle10.1085/jgp.113.1.57 Occurrence Handle1:CAS:528:DyaK1MXlvFShsw%3D%3D Occurrence Handle9874688

    Article  CAS  PubMed  Google Scholar 

  • D. Duan C. Winter S. Cowley J.R. Hume B. Horowitz (1997) ArticleTitleMolecular identification of a volume-regulated chloride channel Nature 390 417–421 Occurrence Handle10.1038/37151 Occurrence Handle1:CAS:528:DyaK2sXns12ru7g%3D Occurrence Handle9389484

    Article  CAS  PubMed  Google Scholar 

  • D. Filipovic H. Sackin (1991) ArticleTitleA calcium-permeable stretch-activated cation channel in renal proximal tubule Am. J. Physiol. 260 F119–129 Occurrence Handle1:CAS:528:DyaK3MXnvFWnsA%3D%3D Occurrence Handle1847010

    CAS  PubMed  Google Scholar 

  • M. Haas (1994) ArticleTitleThe Na-K-Cl cotransporters Am. J. Physiol. 267 C869–C885 Occurrence Handle1:CAS:528:DyaK2MXhvFWrtro%3D Occurrence Handle7943281

    CAS  PubMed  Google Scholar 

  • P. Iserovich P.S. Reinach H. Yang J. Fischbarg (1998) ArticleTitleA novel approach to resolve cellular volume responses to an anisotonic challenge Adv. Exp. Med. Biol. 438 687–692 Occurrence Handle1:STN:280:DyaK1c3pvVKqtw%3D%3D Occurrence Handle9634956

    CAS  PubMed  Google Scholar 

  • R. Itoh S. Kawamoto Y. Miyamoto S. Kinoshita K. Okubo (2000) ArticleTitleIsolation and characterization of a Ca2+ -activated chloride channel from human corneal epithelium Curr. Eye Res. 21 918–925 Occurrence Handle10.1076/ceyr.21.6.918.6983 Occurrence Handle1:STN:280:DC%2BD3M7msVSruw%3D%3D Occurrence Handle11262615

    Article  CAS  PubMed  Google Scholar 

  • T.J. Jentsch V. Stein F. Weinreich A.A. Zdebik (2002) ArticleTitleMolecular structure and physiologic function of chloride channels Phys. Reviews 82 503–568 Occurrence Handle1:CAS:528:DC%2BD38XjtFOnsL0%3D

    CAS  Google Scholar 

  • N.G. Jin J.K. Kim D.K. Yang S.J. Cho J.M. Kim E.J. Koh H.C. Jung I. So K.W. Kim (2003) ArticleTitleFundamental role of ClC-3 in volume-sensitive Cl− channel function and cell volume regulation in AGS cells Am. J. Physiol. 285 G938–948 Occurrence Handle1:CAS:528:DC%2BD3sXpt1Kis7o%3D

    CAS  Google Scholar 

  • M. Kawasaki S. Uchida T. Monkawa A. Miyawaki K. Mikoshiba F. Marumo S. Sasaki (1994) ArticleTitleCloning and expression of a protein kinase C-regulated chloride channel abundantly expressed in rat brain neuronal cells Neuron 12 597–604 Occurrence Handle10.1016/0896-6273(94)90215-1 Occurrence Handle1:CAS:528:DyaK2cXmt1Cntrs%3D Occurrence Handle8155321

    Article  CAS  PubMed  Google Scholar 

  • S.D. Klyce (1972) ArticleTitleElectrical profiles in the corneal epithelium J. Physiol. 226 407–429 Occurrence Handle1:STN:280:CSyD3snlsFE%3D Occurrence Handle4538944

    CAS  PubMed  Google Scholar 

  • S.D. Klyce A.H. Neufeld J.A. Zadunaisky (1973) ArticleTitleThe activation of chloride transport by epinephrine and Db cyclic-AMP in the cornea of the rabbit Invest. Ophthalmol. Vis. Sci. 12 127–139 Occurrence Handle1:CAS:528:DyaE3sXhtF2msbY%3D

    CAS  Google Scholar 

  • S.D. Klyce R.K.S. Wong (1977) ArticleTitleSite and mode of adrenaline action on chloride transport across the rabbit corneal epithelium J. Physiol. 266 777–799 Occurrence Handle1:CAS:528:DyaE2sXktlers7s%3D Occurrence Handle864618

    CAS  PubMed  Google Scholar 

  • W.S. Marshall J.W. Hanrahan (1991) ArticleTitleAnion channels in the apical membrane of mammalian corneal epithelium primary cultures Invest. Ophthalmol. Vis. Sci. 32 1562–1568 Occurrence Handle1:STN:280:By6B3c7gt10%3D Occurrence Handle1707860

    CAS  PubMed  Google Scholar 

  • B. Nilius G. Droogmans (2003) ArticleTitleAmazing chloride channels: an overview Acta Physiol. Scand 177 119–147 Occurrence Handle10.1046/j.1365-201X.2003.01060.x Occurrence Handle1:CAS:528:DC%2BD3sXhtF2ltbg%3D Occurrence Handle12558550

    Article  CAS  PubMed  Google Scholar 

  • R. Patarca O.A. Candia P.S. Reinach (1983) ArticleTitleMode of inhibition of active chloride transport in the frog cornea by furosemide Am. J. Physiol. 245 F660–669 Occurrence Handle1:CAS:528:DyaL2cXksFSqtg%3D%3D Occurrence Handle6606983

    CAS  PubMed  Google Scholar 

  • L. Reuss P.S. Reinach S.A. Weinman T.P. Grady (1983) ArticleTitleIntracellular ion activities and Cl− transport mechanisms in bullfrog corneal epithelium Am. J. Physiol. 244 C336–C347 Occurrence Handle1:CAS:528:DyaL3sXktVemtrY%3D Occurrence Handle6601915

    CAS  PubMed  Google Scholar 

  • J.R. Riordan B. Forbush J.R. Hanrahan (1994) ArticleTitleThe molecular basis of chloride transport in shark rectal gland J. Exp. Biol. 196 405–418 Occurrence Handle1:CAS:528:DyaK2MXivFWrurc%3D Occurrence Handle7529818

    CAS  PubMed  Google Scholar 

  • A. Sardini J.S. Amey K.H. Weylandt M. nobles M.A. Valverde C.F. Higgins (2003) ArticleTitleCell volume regulation and swelling-activated chloride channels Biochim. Biophys. Acta. 1618 153–162 Occurrence Handle1:CAS:528:DC%2BD2cXjsV2qtg%3D%3D Occurrence Handle14729152

    CAS  PubMed  Google Scholar 

  • K. Strange F. Emma P.S. Jackson (1996) ArticleTitleCellular and molecular physiology of volume-sensitive anion channels Am. J. Physiol. 270 C711–730 Occurrence Handle1:CAS:528:DyaK28XitVajsLk%3D Occurrence Handle8638650

    CAS  PubMed  Google Scholar 

  • A. Thiemann S. Grunder M. Pusch T.J. Jentsch (1992) ArticleTitleA chloride channel widely expressed in epithelial and non-epithelial cells Nature 356 57–60 Occurrence Handle10.1038/356057a0 Occurrence Handle1:CAS:528:DyaK38Xks1Cqsbw%3D Occurrence Handle1311421

    Article  CAS  PubMed  Google Scholar 

  • B. Verdon J.P. Winpenney K.J. Whitfield B.E. Argent M.A. Gray (1995) ArticleTitleVolume-activated chloride currents in pancreatic duct cells J. Membrane Biol. 147 173–183 Occurrence Handle10.1007/BF00233545 Occurrence Handle1:CAS:528:DyaK2MXosVWqsrc%3D

    Article  CAS  Google Scholar 

  • S.F. Weikersthal Particlevon M.A. Barrand S.B. Hladky (1999) ArticleTitleFunctional and molecular characterization of a volume-sensitive chloride current in rat brain endothelial cells J. Physiol. 516 75–84 Occurrence Handle10.1111/j.1469-7793.1999.075aa.x Occurrence Handle10066924

    Article  PubMed  Google Scholar 

  • L. Wang L. Chen T.J. Jacob (2000) ArticleTitleThe role of ClC-3 in volume-activated chloride currents and volume regulation in bovine epithelial cells demonstrated by antisense inhibition J. Physiol. 524 63–75 Occurrence Handle10.1111/j.1469-7793.2000.t01-1-00063.x Occurrence Handle1:CAS:528:DC%2BD3cXislKrtL4%3D Occurrence Handle10747184

    Article  CAS  PubMed  Google Scholar 

  • J.M. Wolosin O.A. Candia (1987) ArticleTitleSecretagogues increase basolateral K+ conductance of frog corneal epithelium Am. J. Physiol. 253 C550–560

    Google Scholar 

  • X. Wu H. Yang P. Iserovich J. Fischbarg P.S. Reinach (1997) ArticleTitleRegulatory volume decrease by SV40-transformed rabbit corneal epithelial cells require ryanodine-sensitive Ca2+-induced Ca2+ release J. Membrane Biol. 15 127–136 Occurrence Handle10.1007/s002329900250

    Article  Google Scholar 

  • H. Xiong C. Li E. Garami Y. Wang M. Ramjeesingh K. Galley C.E. Bear (1999) ArticleTitleClC-2 activation modulates regulatory volume decrease J. Membrane Biol. 167 215–221 Occurrence Handle10.1007/s002329900485 Occurrence Handle1:CAS:528:DyaK1MXhtFGntbk%3D

    Article  CAS  Google Scholar 

  • H. Yang Z. Wang Y. Miyamoto P.S. Reinach (2001) ArticleTitleCell signalling pathways mediating epidermal growth factor stimulation of Na:K:2Cl cotransport activity in rabbit corneal epithelial cells J. Membrane Biol. 183 93–101 Occurrence Handle10.1007/s00232-001-0057-6 Occurrence Handle1:CAS:528:DC%2BD3MXntFWiu7w%3D

    Article  CAS  Google Scholar 

Download references

Acknowledgments

The authors extend their gratitude to Dr. L. J. Rubin and Dr. T.-C. Hwang (University of Missouri-Columbia) for their generous use of patch-clamp setups. We are thankful to Dr. Araki Sasaki (Kinki University, Hyogo, Japan) for provision of the SV40-virus transformed rabbit corneal epithelial cell line. This work was supported by the Children’s Miracle Network Fund and NIH Training Grant (to L. A.) and National Institutes of Health, NEI, EY04795 (to P. S. R.).

Author information

Authors and Affiliations

  1. Dept. of Physiology, Midwestern University, Glendale Campus, 19555 N. 59th Avenue, Glendale, AZ, 85308, USA

    L. Al-Nakkash

  2. Dept. of Ophthalmology, Columbia University, New York, NY, 10032, USA

    P. Iserovich

  3. Dept. Ophthalmology and Visual Science, Yale University School of Medicine, New Haven, CT, 06510, USA

    M. Coca-Prados

  4. Dept. Biological Sciences, SUNY College of Optometry, New York, NY, 10036, USA

    H. Yang & P.S. Reinach

Authors
  1. L. Al-Nakkash
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. P. Iserovich
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. M. Coca-Prados
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. H. Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. P.S. Reinach
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to L. Al-Nakkash.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Al-Nakkash, L., Iserovich, P., Coca-Prados, M. et al. Functional and Molecular Characterization of a Volume-activated Chloride Channel in Rabbit Corneal Epithelial Cells. J Membrane Biol 201, 41–49 (2004). https://doi.org/10.1007/s00232-004-0706-5

Download citation

  • Received:

  • Revised:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00232-004-0706-5

Keywords

Search

Navigation