Dissecting the proton transport pathway in electrogenic Na+/H+antiporters.
Sodium/proton exchangers of theSLC9family mediate the transport of protons in exchange for sodium to help regulate intracellular pH, sodium levels, and cell volume. In electrogenic Na+/H+antiporters, it has been assumed that two ion-binding aspartate residues transport the two protons that are later exchanged for one sodium ion. However, here we show that we can switch the antiport activity of the bacterial Na+/H+antiporter NapA from being electrogenic to electroneutral by the mutation of a single lysine residue (K305). Electroneutral lysine mutants show similar ion affinities when driven by [Formula: see text]pH, but no longer respond to either an electrochemical potential ([Formula: see text]) or could generate one when driven by ion gradients. We further show that the exchange activity of the human Na+/H+exchanger NHA2 (SLC9B2) is electroneutral, despite harboring the two conserved aspartic acid residues found in NapA and other bacterial homologues. Consistently, the equivalent residue to K305 in human NHA2 has been replaced with arginine, which is a mutation that makes NapA electroneutral. We conclude that a transmembrane embedded lysine residue is essential for electrogenic transport in Na+/H+antiporters.
Centre for Biomembrane Research, Department of Biochemistry and Biophysics, Stockholm University, SE-106 91 Stockholm, Sweden.
Povilas
P
Uzdavinys
Centre for Biomembrane Research, Department of Biochemistry and Biophysics, Stockholm University, SE-106 91 Stockholm, Sweden.
Mathieu
M
Coinçon
Centre for Biomembrane Research, Department of Biochemistry and Biophysics, Stockholm University, SE-106 91 Stockholm, Sweden.
Emmanuel
E
Nji
Centre for Biomembrane Research, Department of Biochemistry and Biophysics, Stockholm University, SE-106 91 Stockholm, Sweden.
Mama
M
Ndi
Centre for Biomembrane Research, Department of Biochemistry and Biophysics, Stockholm University, SE-106 91 Stockholm, Sweden.
Iven
I
Winkelmann
Department of Chemistry and Biochemistry, University of Bern, CH-3012 Bern, Switzerland david.drew@dbb.su.se christoph.vonballmoos@dcb.unibe.ch.
Christoph
0000-0002-4642-6088
C
von Ballmoos
Centre for Biomembrane Research, Department of Biochemistry and Biophysics, Stockholm University, SE-106 91 Stockholm, Sweden; david.drew@dbb.su.se christoph.vonballmoos@dcb.unibe.ch.
David
D
Drew
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D017920:Antiporters / Q000737:chemistry / Q000378:metabolism*; D001224:Aspartic Acid / Q000737:chemistry; D001419:Bacteria / Q000378:metabolism; D001665:Binding Sites; D003545:Cysteine / Q000737:chemistry; D004563:Electrochemistry; D006801:Humans; D006863:Hydrogen-Ion Concentration; D017136:Ion Transport; D008239:Lysine / Q000737:chemistry; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011522:Protons; D011994:Recombinant Proteins / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D012964:Sodium / Q000378:metabolism; D017923:Sodium-Hydrogen Exchangers / Q000378:metabolism*; D013045:Species Specificity; D016963:Thermus thermophilus / Q000378:metabolism*
D017920:Antiporters; D011522:Protons; D011994:Recombinant Proteins; C527589:SLC9B2 protein, human; D017923:Sodium-Hydrogen Exchangers; D001224:Aspartic Acid; D012964:Sodium; D008239:Lysine; D003545:Cysteine
Na+/H+exchangers; energetics; membrane protein; proton transport; secondary active transporters