Identification of the key determinant of the transport promiscuity in Na+-translocating rhodopsins.

Bacterial Na+-transporting rhodopsins convert solar energy into transmembrane ion potential difference. Typically, they are strictly specific for Na+, but some can additionally transport H+. To determine the structural basis of cation promiscuity in Na+-rhodopsins, we compared their primary structures and found a single position that harbors a cysteine in strictly specific Na+-rhodopsins and a serine in the promiscuous Krokinobacter eikastus Na+-rhodopsin (Kr2). A Cys253Ser variant of the strictly specific Dokdonia sp. PRO95 Na+-rhodopsin (NaR) was indeed found to transport both Na+ and H+ in a light-dependent manner when expressed in retinal-producing Escherichia coli cells. The dual specificity of the NaR variant was confirmed by analysis of its photocycle, which revealed an acceleration of the cation-capture step by comparison with the wild-type NaR in a Na+-deficient medium. The structural basis for the dependence of the Na+/H+ specificity in Na+-rhodopsin on residue 253 remains to be determined.