2.A.49 The Ammonium or Ammonia Transporter (Amt) Family

The proteins of the Amt family vary in size from 391 to 622 amino acyl residues and possess 11 (N-terminus out; most members) or 12 (N-terminus in) transmembrane α-helical spanners. The E. coli AmtB is a trimer (Blakey et al., 2002). It appears to have a dual function, transporting NH₃ and regulating nitrogen metabolism by directly interacting with regulatory proteins such as the PII protein and its homologue, GlnK (Blauwkamp and Ninfa, 2003). amtB and glnK form an operon, and GlnK regulates the activity of AmtB. Homologues occur in Gram-negative and Gram-positive bacteria, archaea, yeast, plants and animals and are probably ubiquitous. The eukaryotic proteins are, in general, larger than the prokaryotic proteins. Most functionally characterized members of the family are ammonia uptake transporters, but some may be CO₂ channels (Soupene et al., 2002b). Some, but not other Amt proteins also transport methylammonium.

The structure of the E. coli AmtB at 1.35 Å has been determined (Khademi et al., 2004). It is a gas channel with two structurally similar halves that span the membrane with opposite polarity. There is a vestibule that recruits NH₄⁺/NH₃, a binding site for NH₄⁺ or CH₃-NH₃⁺ that uses π-cation interactions, and a 20 Å long hydrophobic channel that lowers the NH₄⁺ pKa to below 6 using weak interactions with C-H hydrogen bond donors such as those provided by conserved histidines. Reconstitution of AmtB into vesicles showed that it conducts uncharged NH₃, releasing H⁺ on the outside.

The 11 TMSs (M1-M11) of AmtB form a right handed helical bundle around each channel. Residues from helices M1, M6, M7, M8 and M9 of one monomer interact with residues from helices M1, M2 and M3 of the neighboring subunit to form an interacting surface area of 2716 Ų. Polar aromatic residues (Y and W) comprise part of the membrane-aqueous phase interface.

As expected for a channel, NH₃ uniport occurs by energy-independent, non-concentrative, bidirectional diffusion (Soupene et al., 2002a). However, the tomato homologue, LeAMT1, is reported to be a potential-driven NH₄⁺ uptake uniporter (Ludewig et al., 2002), and in Corynebacterium glutamicum and Arabidopsis thaliana, uptake via the Amt1 homologues of AmtB has been reported to be driven by the pmf. In C. glutamicum, an NH₄⁺ uniport mechanism has been proposed. Members of the Amt family may thus be channels or secondary carriers.

Many organisms from all major kingdoms of living organisms possess multiple homologues. Rhodobacter capsulatus has two Amt family homologues, AmtB and AmtY. The former, but not the latter, has been reported to be an NH₄⁺ sensor as well as a transporter (Yakunin and Hallenbeck, 2002). Mep2 of Saccharomyces cerevisiae has been shown to function both as a transporter and as a sensor, generating a signal that regulates filamentous growth (pseudohyphal differentiation) in response to ammonium starvation (Lorenz and Heitman, 1998). This protein has an N-terminal, asparaginyl-linked glycosylated domain where only Asn-4 is glycosylated. Mep2, but not Mep1 or Mep3, has an extracytoplasmic N-terminus (Marini and André, 2000). This N-terminal domain is not required for either transport or sensing. Of the three S. cerevisiae Amt family paralogues, Mep2 exhibits higher affinity for NH₄⁺ (1 μM) than Mep1 (10 μM), and Mep1 exhibits higher affinity than Mep3 (1 mM).

The Amt family includes the Rhesus (Rh) family of proteins, both erythroid (RhaG, RhD and RhCE) and non-erythroid (RhCG, RhBG and RhGK). In the mammalian kidney collecting duct, RhBG is in the basolateral membrane while RhCG is in the apical membrane. Some of these proteins may be CO₂ channels (Soupene et al., 2002). Because of their channel-like properties, this family might more appropriately be classified in TC class 1.A. However, because of several reports suggest that some members exhibit carrier properties, the family is retained in class 2.A.

The generalized transport reactions catalyzed by members of the Amt family are suggested to be:

(1) NH₃ (out) NH₃ (in)

[In E. coli, NH₃ is the putative substrate, but NH₄⁺ is proposed for other systems. In the latter cases, K⁺ possibly serves as a counter ion in an antiport process with NH₄⁺.]

(2) CO₂ (in) CO₂ (out)

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