Fig. 3: PIP₂ binding site of BTR1 in the outward-facing state.

a BTR1_OF/APO dimer with one monomer shown as electrostatic surface representation. PIP₂ molecules are shown as gold sticks. b, c Close-up views of the PIP₂ binding site. The PIP₂ density is shown in mesh. The PIP₂ molecule and its surrounding residues are shown in stick representation. TMD, NTD and PIP₂ are colored in blue, pink and gold, respectively. d Structural comparison of the PIP₂ binding sites of BTR1_OF/APO (blue, with PIP₂ colored in gold) and AE1 (gray, with PIP₂ colored in gray) (PDB ID: 8CT3) aligned by the gate domains. Helices of BTR1 and AE1 are labeled in blue and gray, respectively. e Sequence alignment of the NTD residues forming the PIP₂ binding site from Homo sapiens BTR1, Homo sapiens AE1, Homo sapiens AE2, Homo sapiens NBCe1, Homo sapiens NDCBE and Mus musculus BTR1. Residues that are important for PIP₂ coordination and involved in forming the PIP₂ binding pocket (L site) are colored in red and orange, respectively. f Functional analysis of the residues in the PIP₂ binding site. The current density values of wild-type and mutant BTR1 proteins after the addition of 5 mM NH₄Cl at pHe 7.4 and 8.0 as measured by whole-cell patch-clamp. Data shown are mean values ± s.d. of n biologically independent experiments and p-values were calculated by two-sided unpaired t-tests. g The current density values of wild-type BTR1 and BTR1-R125H proteins at different intracellular pH (pHi 7.0 and 7.4), measured by whole-cell patch-clamp. Data shown are mean values ± s.d. of n biologically independent experiments and p-values were calculated by two-sided unpaired t-tests.