Beyond the patch‐clamp resolution: functional activity of nonelectrogenic vacuolar NHX proton/potassium antiporters and inhibition by phosphoinositides

Abstract

Summary We combined the patch‐clamp technique with ratiometric fluorescence imaging using the proton‐responsive dye BCECF as a luminal probe. Upon application of a steep cytosol‐directed potassium ion (K+) gradient in Arabidopsis mesophyll vacuoles, a strong and reversible acidification of the vacuolar lumen was detected, whereas no associated electrical currents were observed, in agreement with electroneutral cation/H+ exchange. Our data show that this acidification was generated by NHX antiport activity, because: it did not distinguish between K+ and sodium (Na+) ions; it was sensitive to the NHX inhibitor benzamil; and it was completely absent in vacuoles from nhx1 nhx2 double knockout plants. Our data further show that NHX activity could be reversed, was voltage‐independent and specifically impaired by the low‐abundance signaling lipid PI(3,5)P2, which may regulate salt accumulation in plants by acting as a common messenger to coordinately shut down secondary active carriers responsible for cation and anion uptake inside the vacuole. Finally, we developed a theory based on thermodynamics, which supports the data obtained by our novel experimental approach. This work, therefore, represents a proof‐of‐principle that can be applied to the study of proton‐dependent exchangers from plants and animals, which are barely detectable using conventional techniques.

Structure-Guided Identification of Critical Residues in the Vacuolar Na+,K+/H+ Antiporter NHX1 from Arabidopsis thaliana

“…EcNhaA and TtNapA, this ND-motif is exchanged for a DD-motif (D163-D164 and D200-D201, respectively) (Hunte et al 2005; Lee et al 2013b; Padan et al 2009) (Figure 1B). These results are consistent with the placement of AtNHX1 in the CPA1 clade and electroneutral exchange activity (Gradogna et al 2021; Venema et al 2002).…”

“…Recently Uzdavinyz et al ( 2017) have demonstrated that the electrogenic properties of CPA2 family members is not due to this DD-motif as previously thought, but to the presence of a conserved Lys in TM10, which in CPA1 members is exchanged for an Arg that promotes electroneutral activity (Figure 1B). This position in AtNHX1 is occupied by R353, which is coherent with the electroneutral exchange of AtNHX1 (Gradogna et al 2021;Venema et al 2002). Mutagenic studies of this conserved Arg in TM10 in different CPA1 family members have been performed but results were inconclusive (Hellmer et al 2003;Schushan et al 2010;Wang et al 2014).…”

The Discovery of Naringenin as Endolysosomal Two-Pore Channel Inhibitor and Its Emerging Role in SARS-CoV-2 Infection

“…In Figure 3 , it can be observed that human TPC2 fused to EGFP was targeted to the membrane of the large central vacuole and that hTPC2-mediated currents were activated by nanomolar concentrations of the phosphoinositide PI(3,5)P 2 [ 61 ] and did not have a strong voltage dependence. The plant homolog AtTPC1 is not modulated by PI(3,5)P 2 [ 61 ], which acts as a high-affinity inhibitor (nanomolar range) of tonoplast anion (AtCLCa [ 62 ]) and cation (AtNHXs [ 63 ]) transporters. Human TPC1 is also functional when expressed in vacuoles ( Figure 4 ); similarly to hTPC2, nanomolar concentrations of PI(3,5)P 2 are required to activate hTPC1.…”

Electrophysiology and fluorescence to investigate cation channels and transporters in isolated plant vacuoles

“…However, BCECF detects a very significant increase in proton concentration. This increase is completely absent in vacuoles isolated from Arabidopsis knockout plants for NHX1/2 transporters (Gradogna et al 2021). Therefore, this approach is able to detect the activity of non-electrogenic transporters and to investigate their possible modulators.…”