Electrophysiological Analysis of Cloned Cyclic Nucleotide-Gated Ion Channels

Abstract

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.