AKT2/3 Subunits Render Guard Cell K(+) Channels Ca(2+) Sensitive

Inward-rectifying K(+) channels serve as a major pathway for Ca(2+)-sensitive K(+) influx into guard cells. Arabidopsis thaliana guard cell inward-rectifying K(+) channels are assembled from multiple K(+) channel subunits. Following the recent isolation and characterization of an akt2/3-1 knockout mutant, we examined whether the AKT2/3 subunit carries the Ca(2+) sensitivity of the guard cell inward rectifier. Quantification of RT-PCR products showed that despite the absence of AKT2 transcripts in guard cells of the knockout plant, expression levels of the other K(+) channel subunits (KAT1, KAT2, AKT1, and AtKC1) remained largely unaffected. Patch-clamp experiments with guard cell protoplasts from wild type and akt2/3-1 mutant, however, revealed pronounced differences in Ca(2+) sensitivity of the K(+) inward rectifier. Wild-type channels were blocked by extracellular Ca(2+) in a concentration- and voltage-dependent manner. Akt2/3-1 mutants lacked the voltage-dependent Ca(2+) block, characteristic for the K(+) inward rectifier. To confirm the akt2/3-1 phenotype, two independent knockout mutants, akt2-1 and akt2::En-1 were tested, demonstrating that the loss of AKT2/3 indeed affects the Ca(2+) dependence of guard cell inward rectifier. In contrast to AKT2 knockout plants, AKT1, AtKC1, and KAT1 loss-of-function mutants retained Ca(2+) block of the guard cell inward rectifier. When expressed in HEK293 cells, AKT2 channel displayed a pronounced susceptibility toward extracellular Ca(2+), while the dominant guard cell K(+) channel KAT2 was Ca(2+) insensitive. Thus, we conclude that the AKT2/3 subunit constitutes the Ca(2+) sensitivity of the guard cell K(+) uptake channel.