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Plant Physiol, February 2002, Vol. 128, pp. 400-410
Electrophysiological Analysis of Cloned Cyclic Nucleotide-Gated
Ion Channels1
Qiang
Leng,2
Richard W.
Mercier,
Bao-Guang
Hua,
Hillel
Fromm, and
Gerald A.
Berkowitz*
Department of Plant Science, University of Connecticut, Storrs,
Connecticut 06269-4067 (Q.L., R.W.M., B.-G.H., G.A.B.); and Centre
for Plant Sciences, Leeds Institute for Biotechnology and Agriculture,
School of Biology, University of Leeds, Leeds LS2 9JT, United Kingdom
(H.F.)
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.
1
This work was supported by the National Science
Foundation (grant no. MCB-0090675) and by the U.S. Department of
Agriculture National Research Initiative (grant no. 2001-01945). This
is paper no. 2,052 of the Storrs Agricultural Experiment Station.
2
Present address: Yale School of Medicine, Department of
Cellular and Molecular Physiology, 333 Cedar Street, B121-SHM, New Haven, CT 06520.
*
Corresponding author; e-mail gerald.berkowitz{at}uconn.edu; fax
860-486-0682.
© 2002 American Society of Plant Physiologists
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