The F_o-a subunit of the Na⁺-transporting F_oF₁ ATP synthase from Propionigenium modestum plays a key role in Na⁺ transport. It forms half channels that allow Na⁺ to enter and leave the buried carboxyl group on F_o-c subunits. The essential Arg residue R226, which faces the carboxyl group of F_o-c subunits in the middle of transmembrane helix 5 of the F_o-a subunit, separates the cytoplasmic side and periplasmic half-channels. To elucidate contributions of other amino acid residues of transmembrane helix 5 using hybrid F_oF₁ (F_o from P. modestum and F₁ from thermophilic Bacillus PS3), 25 residues were individually mutated to Cys, and effects of modification with the SH-modifying agent N-ethylmaleimide (NEM) on ATP synthesis and hydrolysis activity were analyzed. NEM significantly inhibited ATP synthesis and hydrolysis as well as proton pumping activities of A214C, G215C, A218C, I223C (cytoplasmic side from R226), and N230C (periplasmic side from R226) mutants and inhibited ATP synthesis activity of the K219C mutant (cytoplasmic side from R226). Thus, these residues contribute to the integrity of the Na⁺ half channel, and both half channels are present in the F_o-a subunit.