C-Terminus-Mediated Voltage Gating of Arabidopsis Guard Cell Anion Channel QUAC1

Abstract

Anion transporters in plants play a fundamental role in volume regulation and signaling. Currently, two plasma membrane-located anion channel families—SLAC/SLAH and ALMT—are known. Among the ALMT family, the root-expressed ALuminium-activated Malate Transporter 1 was identified by comparison of aluminum-tolerant and Al(3+)-sensitive wheat cultivars and was subsequently shown to mediate voltage-independent malate currents. In contrast, ALMT12/QUAC1 (QUickly activating Anion Channel1) is expressed in guard cells transporting malate in an Al(3+)-insensitive and highly voltage-dependent manner. So far, no information is available about the structure and mechanism of voltage-dependent gating with the QUAC1 channel protein. Here, we analyzed gating of QUAC1-type currents in the plasma membrane of guard cells and QUAC1-expressing oocytes revealing similar voltage dependencies and activation–deactivation kinetics. In the heterologous expression system, QUAC1 was electrophysiologically characterized at increasing extra- and intracellular malate concentrations. Thereby, malate additively stimulated the voltage-dependent QUAC1 activity. In search of structural determinants of the gating process, we could not identify transmembrane domains common for voltage-sensitive channels. However, site-directed mutations and deletions at the C-terminus of QUAC1 resulted in altered voltage-dependent channel activity. Interestingly, the replacement of a single glutamate residue, which is conserved in ALMT channels from different clades, by an alanine disrupted QUAC1 activity. Together with C- and N-terminal tagging, these results indicate that the cytosolic C-terminus is involved in the voltage-dependent gating mechanism of QUAC1.

Cytosolic Nucleotides Block and Regulate the Arabidopsis Vacuolar Anion Channel AtALMT9

“…Moreover, the only attempt to remove ATP from the cytosolic solution revealed a change of the bell-shaped I-V characteristic to monotonic one (13). As a corollary our data also provide a simple and straightforward interpretation of a phenomenon that R-type currents are activated by extracellular malate (23)(24)(25). In this work we found that vacuolar malate (which correspond in our configuration to extracellular malate) is able to release the ATP block (Figs.…”

The ALMT Gene Family Performs Multiple Functions in Plants

“…Using a similar mutational approach, Furuichi et al [78] concluded that three acidic residues, E274Q, D275N and E284Q, located on the hydrophilic C-terminal region of TaALMT1 are important for the Al 3+ -activated transport activity because mutations at these sites decreased the Al-dependent responses without affecting basal transport activity [78]. A comparable mutation in AtALMT1, E284Q, has the same effect [78] and the mutations E256Q in AtALMT1 and E276Q in AtALMT12 are comparable to E274Q in TaALMT1 and show the same decreases to Al-dependent transport function [72]. However subsequent studies demonstrated that those three residues in TaALMT1 (E274, D275 and E284) are highly conserved throughout the entire ALMT family, even in ALMTs that are not activated by Al.…”

“…It was concluded that both the C-terminal and N-terminal tails were orientated extracellularly [70]. More recent studies have questioned this structure and instead have suggested that the C-terminal end is oriented toward the intracellular space [20,[71][72][73][74]. Secondary structures of ALMT proteins with the ALMT domain (PF11744) vary.…”

“…In fact, the topology of ALMT transporters remains unclear and doubt still remains whether the N and the C-terminal ends are intracellular or extracellular [6,8,20,70,72,79]. Recently Ramesh et al [80] showed that the transport activity of several ALMTs expressed in Xenopus oocytes is very sensitive to inhibition by gamma-aminobutyric acid (GABA) and muscimol, an agonist of GABA A receptor.…”

Molecular mechanisms of Al tolerance in gramineous plants

“…Furthermore, it was found that both the N-and C-domains are involved in Al-mediated enhancement of transport activity. It is argued that the C-terminal half is, at least in part, oriented towards the intracellular space, and is thus incapable of direct interaction with extracellular Al (Dreyer et al 2012;Mumm et al 2013). In addition, a recent comprehensive phytogenetic analysis showed that these three residues are highly conserved throughout the entire ALMT family, in which some members do not show Al-activation.…”