A Single-Pore Residue Renders the Arabidopsis Root Anion Channel SLAH2 Highly Nitrate Selective

Abstract

In contrast to animal cells, plants use nitrate as a major source of nitrogen. Following the uptake of nitrate, this major macronutrient is fed into the vasculature for long-distance transport. The Arabidopsis thaliana shoot expresses the anion channel SLOW ANION CHANNEL1 (SLAC1) and its homolog SLAC1 HOMOLOGOUS3 (SLAH3), which prefer nitrate as substrate but cannot exclude chloride ions. By contrast, we identified SLAH2 as a nitrate-specific channel that is impermeable for chloride. To understand the molecular basis for nitrate selection in the SLAH2 channel, SLAC1 and SLAH2 were modeled to the structure of HiTehA, a distantly related bacterial member. Structure-guided site-directed mutations converted SLAC1 into a SLAH2-like nitrate-specific anion channel and vice versa. Our findings indicate that two pore-occluding phenylalanines constrict the pore. The selectivity filter of SLAC/SLAH anion channels is determined by the polarity of pore-lining residues located on alpha helix 3. Changing the polar character of a single amino acid side chain (Ser-228) to a nonpolar residue turned the nitrate-selective SLAH2 into a chloride/nitrate-permeable anion channel. Thus, the molecular basis of the anion specificity of SLAC/SLAH anion channels seems to be determined by the presence and constellation of polar side chains that act in concert with the two pore-occluding phenylalanines.

Investigation of the regulation mechanism of Arabidopsis thaliana anion channel SLAH2

“…A recent study showed that SLAH2 was able to specifically interact with and be activated by CPK21 and CIPK23 (Maierhofer et al, 2014). Therefore, SLAH2 could be activated and regulated by multiple CPKs in roots, similar to the activation of SLAC1 and SLAH3 by multiple CPKs in guard cells.…”

“…We then constructed constitutively active versions of these kinases. According to previous reports, the deletion of the EF hands of CPKs renders the kinases Ca 2+ -insensitive and thus leads to a constitutively active form of the CPKs (Geiger et al, 2010(Geiger et al, , 2011Scherzer et al, 2012;Maierhofer et al, 2014). We then generated the truncated versions of the CPK2, CPK4, and CPK11 by deleting their EF hand domains and named these truncated CPKs CPK2ΔEF, CPK4ΔEF, and CPK11ΔEF, respectively.…”

“…Xylem-slowly activating anion conductance (X-SLAC) has been reported in the plasma membranes of stele cells from barley roots (Kohler and Raschke, 2000). SLAH2 is expressed in the stele cells of Arabidopsis roots (Maierhofer et al, 2014). Owing to its strict nitrate selectivity, a role of SLAH2 in nitrate transport in roots has been hypothesized (Maierhofer et Typical whole-oocyte recordings of S-type anion currents in oocytes injected with water as a control or SLAH2 cRNA plus the cRNA of a kinase as indicated.…”

“…SLAH2, a close homolog of SLAH3, is expressed in the root stele cells, which determine the anion composition of the xylem sap. SLAH2 is strictly permeable to nitrate (Maierhofer et al, 2014), and this ion selectivity of SLAH2 is obviously different from that of SLAC1 (Geiger et al, 2009;Lee et al, 2009).…”

“…In recent years, a slow-type (S type) anion channel family including SLAC1 and four SLAC1 homologs (SLAHs), namely SLAH1-SLAH4, was reported in Arabidopsis thaliana (Negi et al, 2008;Vahisalu et al, 2008). All the members of the SLAC1/SLAHs family are localized in the plasma membrane of plant cells and SLAC1 is specifically expressed in guard cells (Negi et al, 2008;Vahisalu et al, 2008;Geiger et al, 2009Geiger et al, , 2011Lee et al, 2009;Maierhofer et al, 2014). The activation of SLAC1 causes anion efflux from guard cells and further leads to stomatal closure (Vahisalu et al, 2008).…”