Oligomeric structure of the neutral amino acid transporters KAAT1 and CAATCH1

Abstract

The highly homologous neutral amino acid transporters KAAT1 and CAATCH1, cloned from the midgut epithelium of the Manduca sexta larva, are members of the Na(+)/Cl(-)-dependent transporter family. Recent evidence indicates that transporters of this family form constitutive oligomers. CAATCH1 and KAAT1 give rise to specific kinds of current depending on the transported amino acid, cotransported ion, pH, and membrane voltage. Different substrates induce notably distinct transport-associated currents in the two proteins that represent useful tools in structural-functional studies. To determine whether KAAT1 and CAATCH1 form functional oligomers, we have constructed four concatameric proteins for electrophysiological analysis, consisting of one KAAT1 protein covalently linked to another KAAT1 (K-K concatamer) or to CAATCH1 (K-C concatamer) and vice versa (C-C concatamer and C-K concatamer), and eight constructs where the two transporters were linked to yellow or cyan fluorescent protein in the NH(2) or COOH terminus, to determine the oligomer formation and the relative distance between the different subunits by fluorescence resonance energy transfer (FRET) analysis. Heterologous expression of the concatenated constructs and coinjection of the original proteins in different proportions allowed us to compare the characteristics of the currents to those of the oocytes expressing only the wild-type proteins. All the constructs were fully active, and their electrophysiological behavior was consistent with the activity as monomeric proteins. However, the FRET studies indicate that these transporters form oligomers in agreement with the LeuT(Aa) atomic structure and confirm that the COOH termini of the adjacent subunits are closer than NH(2) termini.

Synergy and specificity of two Na+–aromatic amino acid symporters in the model alimentary canal of mosquito larvae

“…The upper bands are predominant in the gut tissues but not in the heterologous expression system (Fig.·1) suggesting that the putative homodimers are the prevalent structure for both aromatic transporters in the gut tissues. The facts that symmetrical dimers were identified in other member of SLC6 by similar analysis (Hastrup et al, 2001), by FRET (Bossi et al, 2007) and upon crystallization of bacterial NAT AaLeuT (Yamashita et al, 2005) supports our hypothesis and suggests that dimer formation is conserved and essential for the physiological role of SLC6 members. A detailed analysis would be required to determine the exact nature and functional roles of quaternary modifications of NATs.…”

Oligomeric Structure and Minimal Functional Unit of the Electrogenic Sodium Bicarbonate Cotransporter NBCe1-A

“…Other transport proteins known to be oligomers whose transport behavior is consistent with activity as monomers include lactose, permease (54), AE1 (79), aquaporin-1 (51), NKCC2 (80), KAAT1 (56), and CAATCH1 (56). An obvious question then arises from these studies and our data.…”

“…However, given the aforementioned assumptions and uncertainties involved, we elected to utilize a concatameric approach. This method was first utilized with voltage-gated K ϩ channels (50) and has since been applied to analyze other membrane proteins, including aquaporins (51), the Na ϩ ,K ϩ -ATPase (52), ligand-gated ion channels (53), lactose permease (54), NaP i 2 (55), KAAT1 (56), and CAATCH1 (56). The following concatameric constructs of wt-NBCe1-A and NBCe1-A T442C were analyzed by immunoblotting, immunocytochemistry, and functionally the following: 1) wt-NBCe1-A-linker-wt-NBCe1-A used as a negative con- trol; 2) wt-NBCe1-A-linker-NBCe1-A T442C ; 3) NBCe1-A T442C -linker-wt-NBCe1-A; and 4) NBCe1-A T442C -linkerNBCe1-A T442C used as a positive control.…”

Molecular basis of essential amino acid transport from studies of insect nutrient amino acid transporters of the SLC6 family (NAT-SLC6)

“…A pair of putative NAT from the B. mori genome also resides within the miNAT cluster (data not shown). Studies of chimerically bound Ms KAAT1 and Ms CAATCH1 showed functional expression of such constructs with possible formation of oligomeric structures (Bossi et al, 2007). Binary homo-dimerization is a documented phenomenon for several NTT-SLC6 members (Horschitz et al, 2003; Schmid et al, 2001) and is evident from the crystallization of the first bacterial SLC6 representative (Yamashita et al, 2005).…”