A novel ABCC9 variant in a Greek family with Cantu syndrome affecting multiple generations highlights the functional role of the SUR2B NBD1.

Cantu syndrome (CS) (OMIM #239850) is an autosomal dominant multiorgan system condition, associated with a characteristic facial phenotype, hypertrichosis, and multiple cardiovascular complications. CS is caused by gain-of-function (GOF) variants in KCNJ8 or ABCC9 that encode pore-forming Kir6.1 and regulatory SUR2 subunits of ATP-sensitive potassium (KATP) channels. A novel heterozygous ABCC9 variant, c.2440G>T; p.Gly814Trp, was identified in three individuals from a four generation Greek family. The membrane potential in cells stably expressing hKir6.1 and hSUR2B with p.Gly814Trp was hyperpolarized compared to cells expressing WT channels, and inside-out patch-clamp assays of KATP channels formed with hSUR2B p.Gly814Trp demonstrated a decreased sensitivity to ATP inhibition, confirming a relatively mild GOF effect of this variant. The specific location of the variant reveals an unrecognized functional role of the first glycine in the signature motif of the nucleotide binding domains in ATP-binding cassette (ABC) protein ion channels.

Molecular and functional characterization of the first nucleobase transporter gene from African trypanosomes.

African trypanosomes are unable to synthesize purines and depend upon purine nucleoside and nucleobase transporters to salvage these compounds from their hosts. To understand the crucial role of purine salvage in the survival of these parasites, a central objective is to identify and characterize all of the purine permeases that mediate uptake of these essential nutrients. We have cloned and functionally expressed in a purine nucleobase transport deficient strain of Saccharomyces cerevisiae a novel nucleobase transporter gene, TbNT8.1, from Trypanosoma brucei. The permease encoded by this gene mediates the uptake of hypoxanthine, adenine, guanine, and xanthine with Kms in the low micromolar range. The TbNT8.1 protein is a member of the equilibrative nucleoside transporter (ENT) family of permeases that occur in organisms as diverse as protozoa and mammals. TbNT8.1 is distinct from other ENT permeases that have been identified in trypanosomes in utilizing multiple purine nucleobases, rather than purine nucleosides, as substrates and is hence the first bona fide nucleobase permease identified in these parasites. Furthermore, unlike the mRNAs for other purine transporters, TbNT8.1 mRNA is significantly more abundant in insect stage procyclic forms than in mammalian stage bloodstream forms, and the TbNT8.1 permease thus may represent a major route for purine nucleobase uptake in procyclic trypanosomes.

Functional expression and characterization of a purine nucleobase transporter gene from Leishmania major.

Leishmania major, like all the other kinetoplastid protozoa, are unable to synthesize purines and rely on purine nucleobase and nucleoside acquisition across the parasite plasma membrane by specific permeases. Although, several genes have been cloned that encode nucleoside transporters in Leishmania and Trypanosoma brucei, much less progress has been made on nucleobase transporters, especially at the molecular level. The studies reported here have cloned and expressed the first gene for a L. major nucleobase transporter, designated LmaNT3. The LmaNT3 permease shows 33% identity to L. donovani nucleoside transporter 1.1 (LdNT1.1) and is, thus, a member of the equilibrative nucleoside transporter (ENT) family. ENT family members identified to date are nucleoside transporters, some of which also transport one or several nucleobases. Functional expression studies in Xenopus laevis oocytes revealed that LmaNT3 mediates high levels of uptake of hypoxanthine, xanthine, adenine and guanine. Moreover, LmaNT3 is an high affinity transporter with K(m) values for hypoxanthine, xanthine, adenine and guanine of 16.5 +/- 1.5, 8.5 +/- 0.6, 8.5 +/- 1.1, and 8.8 +/- 4.0 microM, respectively. LmaNT3 is, thus, the first member of the ENT family identified in any organism that functions as a nucleobase rather than nucleoside or nucleoside/nucleobase transporter.

Six related nucleoside/nucleobase transporters from Trypanosoma brucei exhibit distinct biochemical functions.

Purine nucleoside and nucleobase transporters are of fundamental importance for Trypanosoma brucei and related kinetoplastid parasites because these protozoa are not able to synthesize purines de novo and must salvage the compounds from their hosts. In the studies reported here, we have identified a family of six clustered genes in T. brucei that encode nucleoside/nucleobase transporters. These genes, TbNT2/927, TbNT3, TbNT4, TbNT5, TbNT6, and TbNT7, have predicted amino acid sequences that show high identity to each other and to TbNT2, a P1 type nucleoside transporter recently identified in our laboratory. Expression in Xenopus laevis oocytes revealed that TbNT2/927, TbNT5, TbNT6, and TbNT7 are high affinity adenosine/inosine transporters with K(m) values of <5 microm. In addition, TbNT5, and to a limited degree TbNT6 and TbNT7, also mediate the uptake of the nucleobase hypoxanthine. Ribonuclease protection assays showed that mRNA from all of the six members of this gene family are expressed in the bloodstream stage of the T. brucei life cycle but that TbNT2/927 and TbNT5 mRNAs are also expressed in the insect stage of the life cycle. These results demonstrate that T. brucei expresses multiple purine transporters with distinct substrate specificities and different patterns of expression during the parasite life cycle.