Increased Functional Diversity of Plant K+ Channels by Preferential Heteromerization of the Shaker-like Subunits AKT2 and KAT2

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.

Calcium-dependent modulation and plasma membrane targeting of the AKT2 potassium channel by the CBL4/CIPK6 calcium sensor/protein kinase complex

“…The YFP fluorescence formed by the assembly of AKT2-YC and YN-CIPK6 accumulated predominantly at ERrich regions of the cell (Supplementary information, Figure S1A). These results are in line with the observation that many characterized channel subunits are retained at the ER and require association with escort proteins, protein modification or oligomerization for trafficking beyond the ER [19][20][21] and confirm previous AKT2 localization studies [22].…”

FRET imaging in living maize cells reveals that plasma membrane aquaporins interact to regulate their subcellular localization

“…2). Similar protein relocalization from the ER or Golgi apparatus to the plasma membrane as a consequence of a heteromerization process has been reported in mammalian and plant cells for different channels or receptors (17,32,33), but not for AQPs.…”

Plant adaptation to fluctuating environment and biomass production are strongly dependent on guard cell potassium channels

“…Two-hybrid experiments in yeast and functional analyses after coexpression in Xenopus oocytes have provided evidence that KAT2 can interact and form heteromeric channels with other Shaker subunits, at least KAT1 and AKT2 (15,22). Expression of a domneg kat2 gene in a kat2 knockout background is shown to result in total suppression of GCK in activity.…”

“…Alternatively, we developed a dominant negative strategy based on the fact that Shaker channels are homotetrameric or heterotetrameric proteins, formed from subunits encoded by one or several Shaker genes (13)(14)(15)(16). WT plants were transformed with a dominant negative (domneg) kat2 construct (15) under control of the KAT2 promoter. The resulting transgenic plants, named domneg-1 (T 3 generation, homozygous for the domneg transgene), did not display any defect in transpiration (Fig.…”