Critical Involvement of the E373–D434 Region in the Acid Sensitivity of a NhaB-Type Na⁺/H⁺ Antiporter from Vibrio alginolyticus

Abstract

It has been well established that VaNhaB, a NhaB-type Na(+)/H(+) antiporter found in Vibrio alginolyticus, exhibits a striking acid sensitivity. However, the molecular basis of the pH-dependent regulatory mechanism of the antiport activity is yet to be investigated. In this study, we generated various chimeric proteins composed of VaNhaB and a pH insensitive ortholog found in Escherichia coli (EcNhaB) and analyzed the pH responses of their Na(+)/H(+) antiport activities to search for the key residues or domains that are involved in the pH sensitivity of VaNhaB. Our results revealed the significant importance of a stretch of amino acid residues within the loop 8-loop 9 regions (E373-D434) responsible for the acid sensitivity of VaNhaB, along with the possible involvement of other unidentified residues that are widely spread in the primary structure of VaNhaB. Moreover, we demonstrated that the E373-D434 region of VaNhaB was able to confer some degree of acid sensitivity on our pH insensitive chimeric antiporter that is mainly composed of EcNhaB except for seven amino acid substitutions at the N-terminal end. This result strongly suggested the possibility that the E373-D434 region is able to act, at least partially, as machinery that diminishes the activity of the NhaB-type antiporter at an acidic pH.

Diversities and similarities in pH dependency among bacterial NhaB-like Na+/H+ antiporters

“…Earlier reports demonstrated that while EcNhaB from E. coli, the first characterized NhaB-like antiporter, maintains a certain amount of activity at wide ranges of pH, the activity of VaNhaB was almost undetectable at pH 6.5 when expressed in E. coli (Pinner et al, 1994;Nakamura et al, 2001). Likewise, we have also shown that the activity of VaNhaB was significantly decreased at a pH below 7.5, while the activity of EcNhaB from E. coli was only modestly affected by the change of pH at low concentration (2.5 mM) of NaCl when ectopically expressed in E. coli cells by using an identical expression system (Kiriyama et al, 2012). Similar pH sensitivity to VaNhaB was also reported for the NhaB-like antiporter from Pseudomonas aeruginosa (PaNhaB); the significant decrease of the activity was observed at a pH below 7.0 when measured in the presence of 0.5 mM NaCl (Kuroda et al, 2004).…”

“…After the DNA sequences had been confirmed (Macrogen), each nhaB-like gene was inserted into SalI and XmaI sites of the pHG165 expression vector (Messing & Vieira, 1982) and introduced into the TO114 strain. For 26haemagglutinin (26HA) epitope tagging of NhaB-like antiporters, each nhaB gene, except SbnhaB, was cloned into the XmaI and XhoI sites of pHG165-2HA, which contains an oligonucleotide linker that encodes a 26HA epitope tag between the BamHI and PstI sites of pHG165 (Kiriyama et al, 2012). For 26HA tagging of SbNhaB, the SbnhaB gene, which contains an internal XhoI site, was amplified by PCR using the SbnhaB forward and the SbnhaB reverse2 primers (see Table S1), subcloned into pGEM-T Easy, and then introduced into the XmaI and SalI sites of pHG165-2HA.…”

“…Everted membrane vesicles were prepared using a French press as previously described (Kiriyama et al, 2012). In brief, 2 ml overnight cultures of TO114 strains were inoculated into 200 ml fresh LBK medium and grown to OD 600 0.5-0.7.…”

“…Measurement of Na + /H + antiport activity with the acridine orange quenching assay. Na + /H + antiport activities were measured with the acridine orange quenching assay as previously described (Kiriyama et al, 2012). Membrane vesicles (corresponding to 100 mg protein) were suspended in 2 ml assay buffer containing 10 mM Tris/HCl (pH 6.5-9.0), 5 mM MgCl 2 , 140 mM choline chloride and 1.5 mM acridine orange, then 2 mM Tris-D,L-lactate (pH 9.0) was added to initiate respiration.…”

“…In addition, we have previously shown that the antiport activity of VaNhaB could still be observed even at pH 6.5 when a higher concentration (100 mM) of NaCl was used, which indicated that the antiport activity of VaNhaB did not completely cease at this pH (Kiriyama et al, 2012). This observation suggested the possibility that the mechanism of the pH-dependent regulation of VaNhaB might involve change in the affinity for Na + (Kiriyama et al, 2012); however, changes in the kinetic properties of VaNhaB at different pH values had not been investigated and still needed to be determined in order to come to this conclusion. Furthermore, it is currently uncertain whether NhaB-like antiporters share a common mechanism for their pHdependent regulation of activities.…”