Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1996 Dec 2;135(6):1619–1632. doi: 10.1083/jcb.135.6.1619

Identification of a cytoplasmic domain important in the polarized expression and clustering of the Kv2.1 K+ channel

PMCID: PMC2133974  PMID: 8978827

Abstract

The voltage-sensitive K+ channel Kv2.1 has a polarized and clustered distribution in neurons. To investigate the basis for this localization, we expressed wild-type Kv2.1 and two COOH-terminal truncation mutants, delta C318 and delta C187, in polarized epithelial MDCK cells. These functional channel proteins had differing subcellular localization, in that while both wild-type Kv2.1 and delta C187 localized to the lateral membrane in high density clusters, delta C318 was expressed uniformly on both apical and lateral membranes. A chimeric protein containing the hemagglutinin protein from influenza virus and the region of Kv2.1 that differentiates the two truncation mutants (amino acids 536-666) was also expressed in MDCK cells, where it was found in high density clusters similar to those observed for Kv2.1. Polarized expression and clustering of Kv2.1 correlates with detergent solubility, suggesting that interaction with the detergent insoluble cytoskeleton may be necessary for proper localization of this channel.

Full Text

The Full Text of this article is available as a PDF (6.1 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Almers W., Stanfield P. R., Stühmer W. Lateral distribution of sodium and potassium channels in frog skeletal muscle: measurements with a patch-clamp technique. J Physiol. 1983 Mar;336:261–284. doi: 10.1113/jphysiol.1983.sp014580. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Balcarova-Ständer J., Pfeiffer S. E., Fuller S. D., Simons K. Development of cell surface polarity in the epithelial Madin-Darby canine kidney (MDCK) cell line. EMBO J. 1984 Nov;3(11):2687–2694. doi: 10.1002/j.1460-2075.1984.tb02194.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Brewer C. B., Roth M. G. A single amino acid change in the cytoplasmic domain alters the polarized delivery of influenza virus hemagglutinin. J Cell Biol. 1991 Aug;114(3):413–421. doi: 10.1083/jcb.114.3.413. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chandy K. G., Gutman G. A. Nomenclature for mammalian potassium channel genes. Trends Pharmacol Sci. 1993 Dec;14(12):434–434. doi: 10.1016/0165-6147(93)90181-i. [DOI] [PubMed] [Google Scholar]
  5. Compton T., Ivanov I. E., Gottlieb T., Rindler M., Adesnik M., Sabatini D. D. A sorting signal for the basolateral delivery of the vesicular stomatitis virus (VSV) G protein lies in its luminal domain: analysis of the targeting of VSV G-influenza hemagglutinin chimeras. Proc Natl Acad Sci U S A. 1989 Jun;86(11):4112–4116. doi: 10.1073/pnas.86.11.4112. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Dotti C. G., Parton R. G., Simons K. Polarized sorting of glypiated proteins in hippocampal neurons. Nature. 1991 Jan 10;349(6305):158–161. doi: 10.1038/349158a0. [DOI] [PubMed] [Google Scholar]
  7. Dotti C. G., Simons K. Polarized sorting of viral glycoproteins to the axon and dendrites of hippocampal neurons in culture. Cell. 1990 Jul 13;62(1):63–72. doi: 10.1016/0092-8674(90)90240-f. [DOI] [PubMed] [Google Scholar]
  8. Doyle C., Sambrook J., Gething M. J. Analysis of progressive deletions of the transmembrane and cytoplasmic domains of influenza hemagglutinin. J Cell Biol. 1986 Oct;103(4):1193–1204. doi: 10.1083/jcb.103.4.1193. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Frech G. C., VanDongen A. M., Schuster G., Brown A. M., Joho R. H. A novel potassium channel with delayed rectifier properties isolated from rat brain by expression cloning. Nature. 1989 Aug 24;340(6235):642–645. doi: 10.1038/340642a0. [DOI] [PubMed] [Google Scholar]
  10. Froehner S. C. Regulation of ion channel distribution at synapses. Annu Rev Neurosci. 1993;16:347–368. doi: 10.1146/annurev.ne.16.030193.002023. [DOI] [PubMed] [Google Scholar]
  11. Gottardi C. J., Caplan M. J. Delivery of Na+,K(+)-ATPase in polarized epithelial cells. Science. 1993 Apr 23;260(5107):552–556. doi: 10.1126/science.8386395. [DOI] [PubMed] [Google Scholar]
  12. Gottardi C. J., Dunbar L. A., Caplan M. J. Biotinylation and assessment of membrane polarity: caveats and methodological concerns. Am J Physiol. 1995 Feb;268(2 Pt 2):F285–F295. doi: 10.1152/ajprenal.1995.268.2.F285. [DOI] [PubMed] [Google Scholar]
  13. Hamill O. P., Marty A., Neher E., Sakmann B., Sigworth F. J. Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflugers Arch. 1981 Aug;391(2):85–100. doi: 10.1007/BF00656997. [DOI] [PubMed] [Google Scholar]
  14. Hwang P. M., Fotuhi M., Bredt D. S., Cunningham A. M., Snyder S. H. Contrasting immunohistochemical localizations in rat brain of two novel K+ channels of the Shab subfamily. J Neurosci. 1993 Apr;13(4):1569–1576. doi: 10.1523/JNEUROSCI.13-04-01569.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Kim E., Niethammer M., Rothschild A., Jan Y. N., Sheng M. Clustering of Shaker-type K+ channels by interaction with a family of membrane-associated guanylate kinases. Nature. 1995 Nov 2;378(6552):85–88. doi: 10.1038/378085a0. [DOI] [PubMed] [Google Scholar]
  16. Li S., Polonis V., Isobe H., Zaghouani H., Guinea R., Moran T., Bona C., Palese P. Chimeric influenza virus induces neutralizing antibodies and cytotoxic T cells against human immunodeficiency virus type 1. J Virol. 1993 Nov;67(11):6659–6666. doi: 10.1128/jvi.67.11.6659-6666.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Maletic-Savatic M., Lenn N. J., Trimmer J. S. Differential spatiotemporal expression of K+ channel polypeptides in rat hippocampal neurons developing in situ and in vitro. J Neurosci. 1995 May;15(5 Pt 2):3840–3851. doi: 10.1523/JNEUROSCI.15-05-03840.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Morrow J. S., Cianci C. D., Ardito T., Mann A. S., Kashgarian M. Ankyrin links fodrin to the alpha subunit of Na,K-ATPase in Madin-Darby canine kidney cells and in intact renal tubule cells. J Cell Biol. 1989 Feb;108(2):455–465. doi: 10.1083/jcb.108.2.455. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Neame S. J., Isacke C. M. The cytoplasmic tail of CD44 is required for basolateral localization in epithelial MDCK cells but does not mediate association with the detergent-insoluble cytoskeleton of fibroblasts. J Cell Biol. 1993 Jun;121(6):1299–1310. doi: 10.1083/jcb.121.6.1299. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Nelson W. J., Hammerton R. W. A membrane-cytoskeletal complex containing Na+,K+-ATPase, ankyrin, and fodrin in Madin-Darby canine kidney (MDCK) cells: implications for the biogenesis of epithelial cell polarity. J Cell Biol. 1989 Mar;108(3):893–902. doi: 10.1083/jcb.108.3.893. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Nelson W. J., Shore E. M., Wang A. Z., Hammerton R. W. Identification of a membrane-cytoskeletal complex containing the cell adhesion molecule uvomorulin (E-cadherin), ankyrin, and fodrin in Madin-Darby canine kidney epithelial cells. J Cell Biol. 1990 Feb;110(2):349–357. doi: 10.1083/jcb.110.2.349. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Nelson W. J., Veshnock P. J. Dynamics of membrane-skeleton (fodrin) organization during development of polarity in Madin-Darby canine kidney epithelial cells. J Cell Biol. 1986 Nov;103(5):1751–1765. doi: 10.1083/jcb.103.5.1751. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Nelson W. J., Veshnock P. J. Modulation of fodrin (membrane skeleton) stability by cell-cell contact in Madin-Darby canine kidney epithelial cells. J Cell Biol. 1987 Jun;104(6):1527–1537. doi: 10.1083/jcb.104.6.1527. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Niethammer M., Kim E., Sheng M. Interaction between the C terminus of NMDA receptor subunits and multiple members of the PSD-95 family of membrane-associated guanylate kinases. J Neurosci. 1996 Apr 1;16(7):2157–2163. doi: 10.1523/JNEUROSCI.16-07-02157.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Pietrini G., Suh Y. J., Edelmann L., Rudnick G., Caplan M. J. The axonal gamma-aminobutyric acid transporter GAT-1 is sorted to the apical membranes of polarized epithelial cells. J Biol Chem. 1994 Feb 11;269(6):4668–4674. [PubMed] [Google Scholar]
  26. Rhodes K. J., Keilbaugh S. A., Barrezueta N. X., Lopez K. L., Trimmer J. S. Association and colocalization of K+ channel alpha- and beta-subunit polypeptides in rat brain. J Neurosci. 1995 Jul;15(7 Pt 2):5360–5371. doi: 10.1523/JNEUROSCI.15-07-05360.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Rodriguez Boulan E., Pendergast M. Polarized distribution of viral envelope proteins in the plasma membrane of infected epithelial cells. Cell. 1980 May;20(1):45–54. doi: 10.1016/0092-8674(80)90233-0. [DOI] [PubMed] [Google Scholar]
  28. Rodriguez-Boulan E., Powell S. K. Polarity of epithelial and neuronal cells. Annu Rev Cell Biol. 1992;8:395–427. doi: 10.1146/annurev.cb.08.110192.002143. [DOI] [PubMed] [Google Scholar]
  29. Sharma N., D'Arcangelo G., Kleinlaus A., Halegoua S., Trimmer J. S. Nerve growth factor regulates the abundance and distribution of K+ channels in PC12 cells. J Cell Biol. 1993 Dec;123(6 Pt 2):1835–1843. doi: 10.1083/jcb.123.6.1835. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Sheng M., Liao Y. J., Jan Y. N., Jan L. Y. Presynaptic A-current based on heteromultimeric K+ channels detected in vivo. Nature. 1993 Sep 2;365(6441):72–75. doi: 10.1038/365072a0. [DOI] [PubMed] [Google Scholar]
  31. Sheng M., Tsaur M. L., Jan Y. N., Jan L. Y. Contrasting subcellular localization of the Kv1.2 K+ channel subunit in different neurons of rat brain. J Neurosci. 1994 Apr;14(4):2408–2417. doi: 10.1523/JNEUROSCI.14-04-02408.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Sheng M., Tsaur M. L., Jan Y. N., Jan L. Y. Subcellular segregation of two A-type K+ channel proteins in rat central neurons. Neuron. 1992 Aug;9(2):271–284. doi: 10.1016/0896-6273(92)90166-b. [DOI] [PubMed] [Google Scholar]
  33. Shi G., Kleinklaus A. K., Marrion N. V., Trimmer J. S. Properties of Kv2.1 K+ channels expressed in transfected mammalian cells. J Biol Chem. 1994 Sep 16;269(37):23204–23211. [PubMed] [Google Scholar]
  34. Trimmer J. S. Immunological identification and characterization of a delayed rectifier K+ channel polypeptide in rat brain. Proc Natl Acad Sci U S A. 1991 Dec 1;88(23):10764–10768. doi: 10.1073/pnas.88.23.10764. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. VanDongen A. M., Frech G. C., Drewe J. A., Joho R. H., Brown A. M. Alteration and restoration of K+ channel function by deletions at the N- and C-termini. Neuron. 1990 Oct;5(4):433–443. doi: 10.1016/0896-6273(90)90082-q. [DOI] [PubMed] [Google Scholar]
  36. Vega-Salas D. E., Salas P. J., Gundersen D., Rodriguez-Boulan E. Formation of the apical pole of epithelial (Madin-Darby canine kidney) cells: polarity of an apical protein is independent of tight junctions while segregation of a basolateral marker requires cell-cell interactions. J Cell Biol. 1987 Apr;104(4):905–916. doi: 10.1083/jcb.104.4.905. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Wang H., Kunkel D. D., Martin T. M., Schwartzkroin P. A., Tempel B. L. Heteromultimeric K+ channels in terminal and juxtaparanodal regions of neurons. Nature. 1993 Sep 2;365(6441):75–79. doi: 10.1038/365075a0. [DOI] [PubMed] [Google Scholar]
  38. Weiss R. E., Roberts W. M., Stühmer W., Almers W. Mobility of voltage-dependent ion channels and lectin receptors in the sarcolemma of frog skeletal muscle. J Gen Physiol. 1986 Jun;87(6):955–983. doi: 10.1085/jgp.87.6.955. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Zurcher T., Luo G., Palese P. Mutations at palmitylation sites of the influenza virus hemagglutinin affect virus formation. J Virol. 1994 Sep;68(9):5748–5754. doi: 10.1128/jvi.68.9.5748-5754.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Zurzolo C., Rodriguez-Boulan E. Delivery of Na+,K(+)-ATPase in polarized epithelial cells. Science. 1993 Apr 23;260(5107):550–556. doi: 10.1126/science.8386394. [DOI] [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES