Assembly of Plant Shaker-Like Kout Channels Requires Two Distinct Sites of the Channel α-Subunit

Abstract

SKOR and GORK are outward-rectifying plant potassium channels from Arabidopsis thaliana. They belong to the Shaker superfamily of voltage-dependent K(+) channels. Channels of this class are composed of four alpha-subunits and subunit assembly is a prerequisite for channel function. In this study the assembly mechanism of SKOR was investigated using the yeast two-hybrid system and functional assays in Xenopus oocytes and in yeast. We demonstrate that SKOR and GORK physically interact and assemble into heteromeric K(out) channels. Deletion mutants and chimeric proteins generated from SKOR and the K(in) channel alpha-subunit KAT1 revealed that the cytoplasmic C-terminus of SKOR determines channel assembly. Two domains that are crucial for channel assembly were identified: i), a proximal interacting region comprising a putative cyclic nucleotide-binding domain together with 33 amino acids just upstream of this domain, and ii), a distal interacting region showing some resemblance to the K(T) domain of KAT1. Both regions contributed differently to channel assembly. Whereas the proximal interacting region was found to be active on its own, the distal interacting region required an intact proximal interacting region to be active. K(out) alpha-subunits did not assemble with K(in) alpha-subunits because of the absence of interaction between their assembly sites.

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

“…The AKT2 and KAT2 cDNAs were cloned in pGEMHE (54). The AKT2 and KAT2 cDNA dominant-negative mutant constructs (pGEMHE-AKT2-DNM and pGEMHE-KAT2-DNM) were obtained as described previously (30).…”

“…Plant ␣-subunits have a long C-terminal region constituting more than half of the protein (28). This C-terminal region includes three remarkable domains believed to be involved in molecular interactions: (i) a putative cyclic nucleotide-binding domain, which participates in interactions between subunits within the channel tetramer (2,30); (ii) an ankyrin domain (found in six out of the nine Arabidopsis Shaker subunits), which may bind to other proteins (31); and (iii) a distal region, involved in tetramerization (2,30,32) and in clustering of channels via the conserved K HA domain (33). These ␣-subunits can generally assemble as homotetramers, but evidence has also been provided, by functional analyses in Xenopus oocytes or two-hybrid tests in yeast, for heterotetramerization.…”

“…Heterotetramerization has been reported only between subunits of the same functional type (inward or outward). It has been shown that the C-terminal region is involved in discrimination between outward and inward channel-forming subunits (30), preventing formation of heteromeric structures between the two subunit types. In the case of AKT1 and KAT1, heteromer formation has been reported in oocytes (34) and in yeast using the split ubiquitin system (38), but this phenomenon is not observed in two-hybrid tests and in vitro under conditions where homotetrameric associations are detected, suggesting that some discrimination can exist for the formation of stable associations (39).…”

Identification of regions responsible for the function of the plant K+ channels KAT1 and AKT2 in Saccharomyces cerevisiae and Xenopus laevis oocytes

“…It has been reported that the interaction between the cNBD and the K T /K HA domains is important for subunit assembly and the formation of functional channels. 7,8 In C-5, the fusion site of KAT1 and AKT2 is located directly downstream the cNBD, which confers an "extended" amino acid sequence between cNBD and K HA , in particular due to the addition of an ankyrin domain. The ankyrin domain, which is present in AKT2 but not in KAT1, is proposed to be important for the interaction of the channel with kinases.…”

“…5 The highest conservation among plant voltage-gated K C channels is found in the P loop, which contains the hallmark motif GYGD, the signature of highly selective K C channels. 6 The long C-terminal region harbors a putative cyclic nucleotide-binding domain (cNBD) and at the extreme C-terminus the K T region, which is involved in channel tetramerization, 7 and the so-called K HA region (rich in hydrophobic and acidic residues), which might be involved in channel clustering in the membrane. 8,9 An ankyrin domain, hypothesized to be a site of interaction with regulatory proteins, 10 is present in many plant K C channels between the cNBD and the K HA region.…”