Electrophysiological analysis of cloned cyclic nucleotide-gated ion channels

Plant Physiol. 2002 Feb;128(2):400-10. doi: 10.1104/pp.010832.

Abstract

Electrophysiological studies were conducted on the cloned plant cyclic nucleotide-gated ion channels AtCNGC2 and AtCNGC1 from Arabidopsis, and NtCBP4 from tobacco (Nicotiana tobacum). The nucleotide coding sequences for these proteins were expressed in Xenopus laevis oocytes or HEK 293 cells. Channel characteristics were evaluated using voltage clamp analysis of currents in the presence of cAMP. AtCNGC2 was demonstrated to conduct K(+) and other monovalent cations, but exclude Na(+); this conductivity profile is unique for any ion channel not possessing the amino acid sequence found in the selectivity filter of K(+)-selective ion channels. Application of cAMP evoked currents in membrane patches of oocytes injected with AtCNGC2 cRNA. Direct activation of the channel by cyclic nucleotide, demonstrated by application of cyclic nucleotide to patches of membranes expressing such channels, is a hallmark characteristic of this ion channel family. Voltage clamp studies (two-electrode configuration) demonstrated that AtCNGC1 and NtCBP4 are also cyclic nucleotide-gated channels. Addition of a lipophilic analog of cAMP to the perfusion bath of oocytes injected with NtCBP4 and AtCNGC1 cRNAs induced inward rectified, noninactivating K(+) currents.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Arabidopsis / genetics
  • Arabidopsis / physiology
  • Calmodulin-Binding Proteins / genetics
  • Calmodulin-Binding Proteins / metabolism
  • Cell Line
  • Cloning, Molecular
  • Cyclic AMP / pharmacology*
  • Cyclic Nucleotide-Gated Cation Channels
  • Gene Expression
  • Humans
  • Ion Channels / genetics
  • Ion Channels / physiology*
  • Ion Transport / drug effects
  • Ion Transport / physiology
  • Ligands
  • Membrane Potentials / drug effects
  • Membrane Potentials / physiology
  • Molecular Sequence Data
  • Oocytes / physiology
  • Patch-Clamp Techniques
  • Plant Proteins / genetics
  • Plant Proteins / physiology
  • Potassium / pharmacology
  • RNA, Complementary / genetics
  • Sequence Homology, Amino Acid
  • Sodium / pharmacology
  • Tobacco / genetics
  • Tobacco / physiology
  • Xenopus laevis

Substances

  • CBP4 protein, Nicotiana tabacum
  • Calmodulin-Binding Proteins
  • Cyclic Nucleotide-Gated Cation Channels
  • Ion Channels
  • Ligands
  • Plant Proteins
  • RNA, Complementary
  • Sodium
  • Cyclic AMP
  • Potassium