Functional roles of histidine and tyrosine residues in the H+-peptide transporter PepT1

Histidyl residues in peptide transporters PepT1 and PepT2 are believed to p articipate in proton and substrate binding and to be crucial to the transpo rters' functional activities. In the present study, we performed mutagenesi s of rabbit PepT1. We mutated three histidine residues (H57, H111, and H121 ) predicted to reside in transmembrane segments, as well as tyrosine residu es adjacent to H57. Functional analysis of wild-type and mutant PepT1 expre ssed in Xenopus oocytes, using both the radiotracer methods and two-microel ectrode voltage-clamping, revealed that not only the H57 but also the aroma tic residues near H57 were essential for the normal function of PepT1, in a greement with the concept that aromatic residues stabilize the charge on H when interacting with H57. While mutagenesis at H111 did not significantly affect the activity of PepT1, mutagenesis at H121 had profound implication s. The substrate affinities for H121 mutants were decreased depending both on the charge of the substrate and the charge on the substituted residues a t position 121. We propose that H57 and H121 are intimately involved in the binding of the coupling ion H+ and the recognition of transportable peptid e substrates, respectively. (C) 2000 Academic Press.