Abstract
Assembly of plant Shaker subunits as heterotetramers, increasing channel functional diversity, has been reported. Here we focus on a new interaction, between AKT2 and KAT2 subunits. The assembly as AKT2/KAT2 heterotetramers is demonstrated by (i) a strong signal in two-hybrid tests with intracytoplasmic C-terminal regions, (ii) the effect of KAT2 on AKT2 subunit targeting in tobacco cells, (iii) the complete inhibition of AKT2 currents by co-expression with a dominant-negative KAT2 subunit in Xenopus oocytes, and reciprocally, and (iv) the appearance, upon co-expression of wild-type AKT2 and KAT2 subunits, of new channel functional properties that cannot be explained by the co-existence of two kinds of homotetrameric channels. In particular, the instantaneous current, characteristic of AKT2, displayed new functional features when compared with those of AKT2 homotetramers: activation by external acidification (instead of inhibition) and weak inhibition by calcium. Single channel current measurements in oocytes co-expressing AKT2 and KAT2 revealed a strong preference for incorporation of subunits into heteromultimers and a diversity of individual channels. In planta, these new channels, which may undergo specific regulations, are likely to be formed in guard cells and in the phloem, where they could participate in the control of membrane potential and potassium fluxes.
Highlights
Animal voltage-gated Kϩ channels of the Shaker superfamily [1] and their plant homologs [2] result from the assembly of four so-called ␣-subunits
The interaction between AKT2 and KAT2 was further studied in a series of two-hybrid tests in which the C-terminal region of each of these two putative partners was fused either to the DNA-binding domain or to the activation domain of the GAL4 transcription factor (Fig. 1B)
When AKT2 is used as the bait, a much stronger signal is observed with KAT2 in the prey construct than with AKT2 (Fig. 1B)
Summary
Animal voltage-gated Kϩ channels of the Shaker superfamily [1] and their plant homologs [2] result from the assembly of four so-called ␣-subunits. Little is known on the actual interactions and assembly patterns of Shaker subunits underlying the voltagedependent currents recorded in plant cells and even less is known on the gating properties of the resulting heterotetrameric channels.
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