Skip to main content

Quantitative RT-PCR Methods for Investigation of Low Copy Potassium Channel Gene Expression in Native Murine Arteries

  • Protocol

Part of the Methods in Molecular Biology book series (MIMB,volume 491)

Summary

Voltage-gated K+ channels (KV channels) are encoded by the KCNx gene family and have such a wide range of properties that it is necessary to identify the precise expression profile that is instrumental in governing the electrical phenotype of a cell and its response to extrinsic factors. Real-time quantitative RT-PCR methodology has been developed and validated for specific RNA species in vascular smooth muscle cells. We have shown that most of the KCNA gene family, encoding the major KVα1 subunits, was markedly up-regulated in the resistance artery compared to the thoracic aorta, in line with reported patch-clamp recordings. Thus quantitative real-time RT-PCR data can be translated into physiological response.

Keywords

  • Potassium channel
  • Real-time RT-PCR
  • Smooth muscle

This is a preview of subscription content, access via your institution.

Buying options

Protocol
USD   49.95
Price excludes VAT (USA)
  • DOI: 10.1007/978-1-59745-526-8_2
  • Chapter length: 15 pages
  • Instant PDF download
  • Readable on all devices
  • Own it forever
  • Exclusive offer for individuals only
  • Tax calculation will be finalised during checkout
eBook
USD   139.00
Price excludes VAT (USA)
  • ISBN: 978-1-59745-526-8
  • Instant PDF download
  • Readable on all devices
  • Own it forever
  • Exclusive offer for individuals only
  • Tax calculation will be finalised during checkout
Softcover Book
USD   179.00
Price excludes VAT (USA)
Hardcover Book
USD   179.00
Price excludes VAT (USA)
Fig. 2.1.
Fig. 2.2.
Fig. 2.3.
Fig. 2.4.
Fig. 2.5.

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more

References

  1. Coetzee, W.A., Amarillo, Y., Chiu, J., Chow, A., Lau, D., McCormack, T., Moreno, H., Nadal, M.S., Ozaita, A., Pountney, D., Saganich, M., Vega-Saenz de Miera, E., and Rudy, B. (1999) Molecular diversity of K + channels. Ann. NY Acad. Sci. 868, 233–285.

    CAS  CrossRef  PubMed  Google Scholar 

  2. Cheong, A., Dedman, A.M., and Beech, D.J. (2001) Expression and function of native potassium channel K(V)alpha1 subunits in terminal arterioles of rabbit. J. Physiol. 534, 691–700.

    CAS  CrossRef  PubMed  Google Scholar 

  3. Albarwani, S., Nemetz, L.T., Madden, J.A., Tobin, A.A., England, S.K., Pratt, P.F., and Rusch, N.J. (2003) Voltage-gated K + channels in rat small cerebral arteries: molecular identity of the functional channels. J. Phys-iol. 551, 751–763.

    CAS  CrossRef  Google Scholar 

  4. Chen, T.T., Luykenaar, K.D., Walsh, E.J., Walsh, M.P., and Cole, W.C. (2006) Key role of Kv1 channels in vasoregulation. Circ. Res. 99, 53–60.

    CAS  CrossRef  PubMed  Google Scholar 

  5. Cox, R.H., Fromme, S.J., Folander, K.L., and Swanson, R.J. (2008) Voltage gated K( +) channel expression in arteries of Wis-tar—Kyoto and spontaneously hypertensive rats. Am. J. Hypertens. 21, 213–218.

    CAS  CrossRef  PubMed  Google Scholar 

  6. Fountain, S.J., Cheong, A., Flemming, R., Mair, L., Sivaprasadarao, A., and Beech, D.J. (2004) Functional up-regulation of KCNA gene family expression in murine mesenteric resistance artery smooth muscle. J. Physiol. 556, 29–42.

    CAS  CrossRef  PubMed  Google Scholar 

  7. Yuan, X.J., Wang, J., Juhaszova, M., Golovina, V.A., and Rubin, L.J. (1998) Molecular basis and function of voltage‐gated K + channels in pulmonary arterial smooth muscle cells. Am. J. Physiol. 274, L621–L635.

    CAS  PubMed  Google Scholar 

  8. Moudgil, R., Michelakis, E.D., and Archer, S.L. (2006) The role of k + channels in determining pulmonary vascular tone, oxygen sensing, cell proliferation, and apoptosis:implications in hypoxic pulmonary vaso-constriction and pulmonary arterial hypertension. Microcirculation 13, 615–632. 9. Chai, Q., Xu, X., Jia, Q., Dong, Q., Liu, Z., Zhang, W., and Chen, L. (2007) Molecular basis of dysfunctional Kv channels in small coronary artery smooth muscle cells of streptozotocin-induced diabetic rats. Chin. J. Physiol. 50, 171–177. 10. Fergus, D.J., Martens, J.R., and England, S.K. (2003) Kv channel subunits that contribute to voltage-gated K + current in renal vascular smooth muscle. Pflugers Arch. 445, 697–704.

    CAS  CrossRef  PubMed  Google Scholar 

  9. Chai, Q., Xu, X., Jia, Q., Dong, Q., Liu, Z., Zhang, W., and Chen, L. (2007) Molecular basis of dysfunctional Kv channels in small coronary artery smooth muscle cells of streptozotocin-induced diabetic rats. Chin. J. Physiol. 50, 171–177.

    CAS  PubMed  Google Scholar 

  10. Fergus, D.J., Martens, J.R., and England, S.K. (2003) Kv channel subunits that contribute to voltage-gated K + current in renal vascular smooth muscle. Pflugers Arch. 445, 697–704.

    CAS  PubMed  Google Scholar 

  11. Thorneloe, K.S., Chen, T.T., Kerr, P.M., Grier, E.F., Horowitz, B., Cole, W.C., and Walsh, M.P. (2001) Molecular composition of 4-aminopyridine-sensitive voltage-gated K( +) channels of vascular smooth muscle. Circ. Res. 89, 1030–1037.

    CAS  CrossRef  PubMed  Google Scholar 

  12. Fountain, S.J., Cheong, A., Li, J., Dondas, N.Y., Zeng, F., Wood, I.C., and Beech, D.J. (2007) K(v)1.5 potassium channel gene regulation by Sp1 transcription factor and oxidative stress. Am. J. Physiol. 293, H2719–H2725.

    CAS  Google Scholar 

  13. Oste, C. (1988) Polymerase chain reaction. Biotechniques 6, 162–167.

    CAS  PubMed  Google Scholar 

Download references

Acknowledgments

This work was supported by the British Hearth Foundation and the Wellcome Trust.

Author information

Authors and Affiliations

Authors

Editor information

Editors and Affiliations

Rights and permissions

Reprints and Permissions

Copyright information

© 2008 Springer Science+Business Media, LLC

About this protocol

Cite this protocol

Cheong, A., Fountain, S.J., Beech, D.J. (2008). Quantitative RT-PCR Methods for Investigation of Low Copy Potassium Channel Gene Expression in Native Murine Arteries. In: Lippiat, J.D. (eds) Potassium Channels. Methods in Molecular Biology, vol 491. Humana Press. https://doi.org/10.1007/978-1-59745-526-8_2

Download citation

  • DOI: https://doi.org/10.1007/978-1-59745-526-8_2

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-934115-65-7

  • Online ISBN: 978-1-59745-526-8

  • eBook Packages: Springer Protocols