EXPRESSION OF A CLONED PLANT K- ANALYSIS OF MACROSCOPIC CURRENTS( CHANNEL IN XENOPUS OOCYTES )

The open reading frame from the Arabidopsis thaliana KAT1 cDNA was clo ned in a transcription plasmid between the 3' and 5' untranslated regi ons of a beta-globin cDNA from Xenopus oocyte. The polyadenylated tran scripts resulting from in vitro transcription gave rise to high levels of expression of KAT1 channel when injected in Xenopus oocytes. Upon hyperpolarization, a slow activating current could be recorded, inward ly- or outwardly-directed, depending on K+ external concentration. Pre dictions of the voltage-gated channel theory were shown to fit the dat a well. The equivalent gating charge and the half-activation potential ranged around 2 and -145 mV, respectively. KAT1 gating characteristic s did not depend on K+ external concentration nor on external pH in th e 5.0-7.5 range. KAT1 conductance was, however, increased (40%) when e xternal pH was decreased from 6.5 to 5.0. The apparent affinity consta nt of KAT1 for K+ lay in the range 15-30 mM, at external pH 7.4. As fo r many K+ channels of animal cells, external caesium caused a voltage- dependent blockage of inward (but not outward) KAT1 current, whereas t etraethylammonium caused a voltage-independent block of both inward an d outward KAT1 currents. In conclusion, high levels of expression made it possible to carry out the first quantitative analysis of KAT1 macr oscopic currents. KAT1 channel was shown to display features similar t o those of as yet uncloned inward-rectifying voltage-gated channels de scribed in both plant cells (namely guard cells) and animal cells.