Voltage-gated sodium （Nav） channels are essential for the rapid upstroke of action potentials and the propa- gation of electrical signals in nerves and muscles. Defects of Nav channels are associated with a variety ...Voltage-gated sodium （Nav） channels are essential for the rapid upstroke of action potentials and the propa- gation of electrical signals in nerves and muscles. Defects of Nav channels are associated with a variety of channelopathies. More than 1000 disease-related muta- tions have been identified in Nay channels, with Nay1.1 and Nay1.5 each harboring more than 400 mutations. Nay channels represent major targets for a wide array of neurotoxins and drugs. Atomic structures of Nav chan- nels are required to understand their function and dis- ease mechanisms. The recently determined atomic structure of the rabbit voltage-gated calcium （Car） channel Carl.1 provides a template for homology-based structural modeling of the evolutionarily related Nay channels. In this Resource article, we summarized all the reported disease-related mutations in human Nav channels, generated a homologous model of human Nay1.7, and structurally mapped disease-associated mutations. Before the determination of structures of human Nay channels, the analysis presented here serves as the base framework for mechanistic investi- gation of Nav channelopathies and for potential struc- ture-based drug discovery.展开更多
Equilibrative nucleoside transporters （ENTs）, which facilitate cross-membrane transport of nucleosides and nucleoside-derived drugs, play an important role in the salvage pathways of nucleotide synthesis, cancer che...Equilibrative nucleoside transporters （ENTs）, which facilitate cross-membrane transport of nucleosides and nucleoside-derived drugs, play an important role in the salvage pathways of nucleotide synthesis, cancer chemotherapy, and treatment for virus infections. Functional characterization of ENTs at the molecular level remains technically challenging and hence scant. In this study, we report successful purification and bio- chemical characterization of human equilibrative nucle- oside transporter 1 （hENT1） in vitro. The HEK293F- derived, recombinant hENT1 is homogenous and func- tionally active in proteoliposome-based counter flow assays, hENT1 transports the substrate adenosine with a Km of 215 ＋ 34 pmol/L and a Vmax of 578 ＋ 23.4 nmol mg-1 min-1. Adenosine uptake by hENT1 is competi- tively inhibited by nitrobenzylmercaptopurine ribonu- cleoside （NBMPR）, nucleosides, deoxynucleosides, and nucleoside-derived anti-cancer and anti-viral drugs. Binding of hENT1 to adenosine, deoxyadenosine, and adenine by isothermal titration calorimetry is in general agreement with results of the competitive inhibition assays. These results validate hENT1 as a bona fide target for potential drug target and serve as a useful basis for future biophysical and structural studies.展开更多
文摘Voltage-gated sodium （Nav） channels are essential for the rapid upstroke of action potentials and the propa- gation of electrical signals in nerves and muscles. Defects of Nav channels are associated with a variety of channelopathies. More than 1000 disease-related muta- tions have been identified in Nay channels, with Nay1.1 and Nay1.5 each harboring more than 400 mutations. Nay channels represent major targets for a wide array of neurotoxins and drugs. Atomic structures of Nav chan- nels are required to understand their function and dis- ease mechanisms. The recently determined atomic structure of the rabbit voltage-gated calcium （Car） channel Carl.1 provides a template for homology-based structural modeling of the evolutionarily related Nay channels. In this Resource article, we summarized all the reported disease-related mutations in human Nav channels, generated a homologous model of human Nay1.7, and structurally mapped disease-associated mutations. Before the determination of structures of human Nay channels, the analysis presented here serves as the base framework for mechanistic investi- gation of Nav channelopathies and for potential struc- ture-based drug discovery.
文摘Equilibrative nucleoside transporters （ENTs）, which facilitate cross-membrane transport of nucleosides and nucleoside-derived drugs, play an important role in the salvage pathways of nucleotide synthesis, cancer chemotherapy, and treatment for virus infections. Functional characterization of ENTs at the molecular level remains technically challenging and hence scant. In this study, we report successful purification and bio- chemical characterization of human equilibrative nucle- oside transporter 1 （hENT1） in vitro. The HEK293F- derived, recombinant hENT1 is homogenous and func- tionally active in proteoliposome-based counter flow assays, hENT1 transports the substrate adenosine with a Km of 215 ＋ 34 pmol/L and a Vmax of 578 ＋ 23.4 nmol mg-1 min-1. Adenosine uptake by hENT1 is competi- tively inhibited by nitrobenzylmercaptopurine ribonu- cleoside （NBMPR）, nucleosides, deoxynucleosides, and nucleoside-derived anti-cancer and anti-viral drugs. Binding of hENT1 to adenosine, deoxyadenosine, and adenine by isothermal titration calorimetry is in general agreement with results of the competitive inhibition assays. These results validate hENT1 as a bona fide target for potential drug target and serve as a useful basis for future biophysical and structural studies.
