PubMed · 16723996
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Ian J Jackson. 2006-05-24. Identifying the genes causing human diversity.. https://doi.org/10.1038/sj.ejhg.5201659
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K(+)-dependent Na(+)/Ca(2+) exchangers (NCKX) catalyze cytosolic Ca(2+) extrusion and are particularly important for neuronal Ca(2+) signaling. Of the five mammalian isoforms, the detailed functional characteristics have only been reported for NCKX1 and -2. In the current study, the functional characteristics of recombinant NCKX3 and -4 expressed in HEK293 cells were determined and compared with those of NCKX2. Although the apparent affinities of the three isoforms for Ca(2+) and Na(+) were similar, NCKX3 and -4 displayed approximately 40-fold higher affinities for K(+) ions than NCKX2. Functional analysis of various NCKX2 mutants revealed that mutation of Thr-551 to Ala, the corresponding residue in NCKX4, resulted in an apparent K(+) affinity shift to one similar to that of NCKX4 without a parallel shift in apparent Ca(2+) affinity. In the converse situation, when Gln-476 of NCKX4 was converted to Lys, the corresponding residue in NCKX2, both the K(+) and Ca(2+) affinities were reduced. These results indicate that the apparently low K(+) affinity of NCKX2 requires a Thr residue at position 551 that may reduce the conformational flexibility and/or K(+) liganding strength of side-chain moieties on critical neighboring residues. This interaction appears to be specific to the structural context of the NCKX2 K(+) binding pocket, because it was not possible to recreate the K(+)-specific low affinity phenotype with reciprocal mutations in NCKX4. The results of this study provide important information about the structure and function of NCKX proteins and will be critical to understanding their roles in neuronal Ca(2+) signaling.
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The staphylococcal TetA(K) tetracycline exporter is classified within the major facilitator superfamily of transport proteins and contains 14 alpha-helical transmembrane segments (TMS). Using cysteine-scanning mutagenesis, 27 amino acid residues across and flanking putative TMS 10 of the TetA(K) transporter were individually replaced with cysteine. The level of solvent accessibility to each of the targeted amino acid positions was determined as a measure of fluorescein maleimide reactivity and demonstrated that TMS 10 of TetA(K) has a cytoplasmic boundary at G313 and is likely to extend from at least V298 on the periplasmic side. TMS 10 was found to be amphiphilic containing at least partially solvent accessible amino acid residues along the length of one helical face, suggesting that this helix may line a solvent-exposed channel. Functional analyses of these cysteine mutants demonstrated a significant role for a number of amino acid residues, including a predominance of glycine residues which were further analyzed by alanine substitution. These residues are postulated to allow interhelical interactions between TMS 10 and distal parts of TetA(K) that are likely to be required for the tetracycline transport mechanism in TetA(K) and may be a general feature required by bacterial tetracycline transporters for activity.