{"id":10829,"date":"2023-03-22T15:02:31","date_gmt":"2023-03-22T15:02:31","guid":{"rendered":"https:\/\/www.nanion.de\/?post_type=resource_library&#038;p=10829"},"modified":"2024-01-11T13:21:12","modified_gmt":"2024-01-11T13:21:12","slug":"2023-functional-characterization-of-sglt1-using-ssm-based-electrophysiology-kinetics-of-sugar-binding-and-translocation","status":"publish","type":"resource_library","link":"https:\/\/nanion.com\/zh-hans\/resource_library\/2023-functional-characterization-of-sglt1-using-ssm-based-electrophysiology-kinetics-of-sugar-binding-and-translocation\/","title":{"rendered":"2023- Functional characterization of SGLT1 using SSM-based electrophysiology: Kinetics of sugar binding and translocation"},"content":{"rendered":"<p>Beside the ongoing efforts to determine structural information, detailed functional studies on transporters are essential to entirely understand the underlying transport mechanisms. We recently found that solid supported membrane-based electrophysiology (SSME) enables the measurement of both sugar binding and transport in the Na<sup>+<\/sup>\/sugar cotransporter SGLT1 (<a href=\"https:\/\/www.frontiersin.org\/articles\/10.3389\/fphys.2023.1058583\/full#B7\" target=\"_blank\" rel=\"noopener\">Bazzone et al, 2022a<\/a>). Here, we continued with a detailed kinetic characterization of SGLT1 using SSME, determining K<sub>M<\/sub>\u00a0and K<sub>D<\/sub><sup>app<\/sup>\u00a0for different sugars, k<sub>obs<\/sub>\u00a0values for sugar-induced conformational transitions and the effects of Na<sup>+<\/sup>, Li<sup>+<\/sup>, H<sup>+<\/sup>\u00a0and Cl<sup>\u2212<\/sup>\u00a0on sugar binding and transport. We found that the sugar-induced pre-steady-state (PSS) charge translocation varies with the bound ion (Na<sup>+<\/sup>, Li<sup>+<\/sup>, H<sup>+<\/sup>\u00a0or Cl<sup>\u2212<\/sup>), but not with the sugar species, indicating that the conformational state upon sugar binding depends on the ion. Rate constants for the sugar-induced conformational transitions upon binding to the Na<sup>+<\/sup>-bound carrier range from 208\u00a0s<sup>\u22121<\/sup>\u00a0for D-glucose to 95\u00a0s<sup>\u22121<\/sup>\u00a0for 3-OMG. In the absence of Na<sup>+<\/sup>, rate constants are decreased, but all sugars bind to the empty carrier. From the steady-state transport current, we found a sequence for sugar specificity (V<sub>max<\/sub>\/K<sub>M<\/sub>): D-glucose &gt; MDG &gt; D-galactose &gt; 3-OMG &gt; D-xylose. While K<sub>M<\/sub>\u00a0differs 160-fold across tested substrates and plays a major role in substrate specificity, V<sub>max<\/sub>\u00a0only varies by a factor of 1.9. Interestingly, D-glucose has the lowest V<sub>max<\/sub>\u00a0across all tested substrates, indicating a rate limiting step in the sugar translocation pathway following the fast sugar-induced electrogenic conformational transition. SGLT1 specificity for D-glucose is achieved by optimizing two ratios: the sugar affinity of the empty carrier for D-glucose is similarly low as for all tested sugars (K<sub>D,K<\/sub><sup>app<\/sup>\u00a0= 210\u00a0mM). Affinity for D-glucose increases 14-fold (K<sub>D,Na<\/sub><sup>app<\/sup>\u00a0= 15\u00a0mM) in the presence of sodium as a result of cooperativity. Apparent affinity for D-glucose during transport increases 8-fold (K<sub>M<\/sub>\u00a0= 1.9\u00a0mM) compared to K<sub>D,Na<\/sub><sup>app<\/sup>\u00a0due to optimized kinetics. In contrast, K<sub>M<\/sub>\u00a0and K<sub>D<\/sub><sup>app<\/sup>\u00a0values for 3-OMG and D-xylose are of similar magnitude. Based on our findings we propose an 11-state kinetic model, introducing a random binding order and intermediate states corresponding to the electrogenic transitions detected\u00a0<em>via<\/em>\u00a0SSME upon substrate binding.<\/p>\n<p>&nbsp;<\/p>\n<p>Read more in the paper and find out more about other Solute Carriers in the article collection <a href=\"https:\/\/www.frontiersin.org\/articles\/10.3389\/fphys.2023.1058583\/full\" target=\"_blank\" rel=\"noopener\">here<\/a>.<\/p>\n<p>&nbsp;<\/p>\n","protected":false},"featured_media":0,"template":"","products_tags":[36],"resource_type":[75],"application_areas":[108],"therapeutic_areas":[],"target_categories":[60],"target_families":[],"targets":[303],"resource_keyword":[],"class_list":["post-10829","resource_library","type-resource_library","status-publish","hentry","products_tags-surfe2r-n1","resource_type-publication","application_areas-transporters","target_categories-transporters","targets-sglt1-slc5a1"],"acf":[],"_links":{"self":[{"href":"https:\/\/nanion.com\/zh-hans\/wp-json\/wp\/v2\/resource_library\/10829","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/nanion.com\/zh-hans\/wp-json\/wp\/v2\/resource_library"}],"about":[{"href":"https:\/\/nanion.com\/zh-hans\/wp-json\/wp\/v2\/types\/resource_library"}],"wp:attachment":[{"href":"https:\/\/nanion.com\/zh-hans\/wp-json\/wp\/v2\/media?parent=10829"}],"wp:term":[{"taxonomy":"products_tags","embeddable":true,"href":"https:\/\/nanion.com\/zh-hans\/wp-json\/wp\/v2\/products_tags?post=10829"},{"taxonomy":"resource_type","embeddable":true,"href":"https:\/\/nanion.com\/zh-hans\/wp-json\/wp\/v2\/resource_type?post=10829"},{"taxonomy":"application_areas","embeddable":true,"href":"https:\/\/nanion.com\/zh-hans\/wp-json\/wp\/v2\/application_areas?post=10829"},{"taxonomy":"therapeutic_areas","embeddable":true,"href":"https:\/\/nanion.com\/zh-hans\/wp-json\/wp\/v2\/therapeutic_areas?post=10829"},{"taxonomy":"target_categories","embeddable":true,"href":"https:\/\/nanion.com\/zh-hans\/wp-json\/wp\/v2\/target_categories?post=10829"},{"taxonomy":"target_families","embeddable":true,"href":"https:\/\/nanion.com\/zh-hans\/wp-json\/wp\/v2\/target_families?post=10829"},{"taxonomy":"targets","embeddable":true,"href":"https:\/\/nanion.com\/zh-hans\/wp-json\/wp\/v2\/targets?post=10829"},{"taxonomy":"resource_keyword","embeddable":true,"href":"https:\/\/nanion.com\/zh-hans\/wp-json\/wp\/v2\/resource_keyword?post=10829"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}