Expression of a cloned plant K⁺ channel in Xenopus oocytes: analysis of macroscopic currents

Abstract

SummaryThe open reading frame from the Arabidopsis thaliana KAT1 cDNA was cloned in a transcription plasmid between the 3' and 5' untranslated regions of a J3-globin cDNA from Xanopus oocyte. The polyedenylated transcripts resulting from in vitro transcription gave rise to high levels of expression of KAT1 channel when injected in Xanopus oocytes. Upon hyperpolarization, a slow activating current could be recorded, inwardly-or outwardly-directed, depending on K + external concentration. Predictions of the voltage-gated channel theory were shown to fit the data well. The equivalent gating charge and the half-activation potential ranged around 2 and -145 mV, respectively. KAT1 gating characteristics did not depend on K + external concentration nor on external pH in the 5.0-7.5 range. KAT1 conductance was, however, increased (40%) when external pH was decreased from 6.5 to 5.0. The apparent affinity constant of KAT1 for K + lay in the range 15-30 raM, at external pH 7.4. As for many K + channels of animal ceils, external caesium caused a voltage-dependent blockage of inward (but not outward) KAT1 current, whereas tetraethylammonium caused a voltage-independent block of both inward and outward KAT1 currents. In conclusion, high levels of expression made it possible to carry out the first quantitative analysis of KAT1 macroscopic currents. KAT1 channel was shown to display features similar to those of as yet uncloned inward-rectifying voltage-gated channels described in both plant cells (namely guard cells) and animal cells.

Functional expression of the plant K+ channel KAT1 in insect cells

“…Furthermore, the shift of the activation potential in insect cells is accompanied by a shift of the deactivation kinetics towards more positive potentials. This results in a slower deactivation at more hyperpolarized potentials in Sf9 cells than in oocytes [12,25,26]. Similar observations were also made for animal ion channels, e.g.…”

“…As in other expression systems in Sf9 cells, KAT1 channels are characterized by high K + selectivity, strong rectification as well as slow hyperpolarizationdependent activation. The voltage-dependent properties of KAT1 expressed in oocytes [12,14,21,25,26] are similar to inward-rectifying K+ channels in vivo [10]. The major difference of the in vitro studies of Schachtman et al [21] and Hedrich et al [12] to those reported here for Sf9 cells concerns the relative permeability of KAT1 for NH~ over K +.…”

“…With respect to the expression system the threshold potential of KAT1 activation shifted along the voltage axis. Compared to the KAT1 properties in oocytes, the activation potential of KAT1 is less negative in Sf9 cells but more negative in yeast [4,12,25,26]. Furthermore, the shift of the activation potential in insect cells is accompanied by a shift of the deactivation kinetics towards more positive potentials.…”

“…In order to examine their electrical properties, expression systems were selected which tolerate transient membrane polarization in the range of-80 mV to -250 mV, characteristic for plant cells rather than for animal cells (-40 mV to -80 mV). So far, KAT1 could be functionally expressed in Xenopus oocytes and yeast [4,12,21,25,26], systems which allow the voltage-dependent stimulation of KAT1.…”

Roles of Higher Plant K+ Channels

“…Whereas the Rb+ and NH,' conductances of KATl expressed in oocytes are approximately 20 to 30% of the K+ conductance (Schachtman et al, 1992), the mutations T256D and T256G evoked Rb+ and NH,' conductances that were 10 times larger than the K+ conductance (Uozumi et al, 1995). However, this large inversion in selectivity, as measured by conductance ratios, did not apply to the selectivity as measured by reversal voltage analysis (Uozumi et al, 1995), emphasizing the fact that the two different types of analysis are not comparable (Hille, 1992;Very et al, 1995).…”