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07. - 10.06.2022 | 2nd International Transmembrane Transporter Society: catching transport in motion

220610 event image ITTS 2022

Conference Venue:
The Maersk Tower
Blegdamsvej 3B
2200 Copenhagen N
Denmark

Go to the Conference website here.


Meet Andre Bazzone and Rocco Zerlotti 

Nanion Senior Scientist, Andre Bazzone and Ph.D. Student Rocco Zerlotti will each present a poster.

Presenter: Rocco Zerlotti
Date: Wednesday, June 8
Time slot: 12:30 PM – 14:15 PM CEST
Poster Number: 74
Title: Individual charge translocations upon GABA, Na+ and Cl- binding to human GAT-1 indicate conformational flexibility

Presenter: Andre Bazzone
Date: Thursday, June 9 
Time slot: 12:00 PM – 13:45 PM CEST
Poster Number: 07
Title: Sugar binding and translocation in SGLT1: SSM-based electrophysiology reveals sugar occluded intermediates 

Abstracts:

Presenter: Rocco Zerlotti
Title: Individual charge translocations upon GABA, Na+ and Cl- binding to human GAT-1 indicate conformational flexibility

Abstract:

Charge translocation across the cell membrane generate currents that can be studied for the functional characterization of a transporter. Solid-supported membrane electrophysiology (SSME) utilizes membrane vesicles to measure electrogenic steps within the reaction cycle, triggered by substrates, at 0 mV. The solution exchange with a time resolution of up to 3 ms allows detection of pre-steady state (PSS) currents.Charge translocation across the cell membrane generate currents that can be studied for the functional characterization of a transporter. Solid-supported membrane electrophysiology (SSME) utilizes membrane vesicles to measure electrogenic steps within the reaction cycle, triggered by substrates, at 0 mV. The solution exchange with a time resolution of up to 3 ms allows detection of pre-steady state (PSS) currents.Here we present a SSME-based study on the human Na+/Cl−/γ-aminobutyric acid (GABA) co-transporter GAT-1, that reveals individual electrogenic events triggered by substrate binding. The well-known Na+-induced PSS current shows a time constant of 13 ms, and an EC50 of 3284mM, in good agreement with the estimated Na+ Kd of 20 mM, concluding that this electrogenic event may be directly associated with Na+ binding. We also detected PSS currents that have not been observed before with conventional electrophysiology. One is induced by GABA, but only in presence of Na+, consistent with current models in literature that assume Na+ binding generates a state with high affinity for GABA. This PSS current EC50 is 65 μM, in good agreement with the estimated Kd for GABA of 40-100 μM, indicating that this PSS current is directly associated with GABA binding. Finally, we detected another PSS current induced by Cl-. The Cl- dependence of this current is well described by a double Michaelis-Menten plot, with a first EC50 of 9 mM and a second EC50 of 215 mM. The PSS currents induced by Na+ and Cl- are independent from the presence of other substrates, thus suggesting a random binding order for these two substrates in the transport cycle. The presence of three distinct substrate-induced electrogenic events can be the result of substrate binding or conformational rearrangements, such as alternating access or local conformational transitions, thus potentially indicating a high conformational flexibility of GAT-1.

Presenter: Andre Bazzone
Title: Sugar binding and translocation in SGLT1: SSM-based electrophysiology reveals sugar occluded intermediates 

Abstract:

Recently we described an electrophysiological assay revealing pre steady-state charge translocations in the human Na+/sugar cotransporter SGLT1 upon sugar binding. We postulated this correlates with local conformational transitions that transfer charged protein residues across the membrane. In general, this may be attributed to mechanisms such as induced fit, substrate occlusion, alternating access or the closure of the extracellular gate.

Here we applied SSM-based electrophysiology for a detailed functional characterization of SGLT1 revealing kinetic properties of sugar binding and translocation, including rate and 41 equilibrium constants, apparent KM values and relative Imax values for different sugar species. Based on the data we conclude a kinetic model describing the sugar translocation pathway in SGLT1, revealing new insights into the transport mechanism: (1) Sugar is able to bind to the outward facing, empty carrier. Hence, substrate binding may happen in random order, under high sugar and low sodium conditions. (2) Not sodium binding, but sugar binding leads to an electrogenic conformational transition within SGLT1 under 0 mV conditions, likely representing sugar occlusion with k+=60 s-1 and k-=200 s-1. (3) Following sugar occlusion, the rate limiting step in sugar translocation is the transition into the inward open conformation. It occurs at the slowest rate for D-glucose, but at higher rates for sugars with lower apparent affinities. (4) At 0 mV and saturating sugar concentrations, all reactions within the sugar translocation pathway are fast compared to the empty carrier translocation. After suger release, sodium release limits the slow transition of the empty carrier from the inward to the outward open state. A 10-state kinetic model has been established to describe the sugar translocation pathway involving three substrate induced electrogenic transitions. Further work is required to merge this model with existing models based on the knowledge about voltage driven transitions observed with conventional electrophysiology.


Additional Posters of Interest

Poster No.9
Presenter: Bjørn Pedersen Lab
Date: Thursday, June 9 
Time slot: 12:00 PM – 13:45 PM CEST
Title: The WAT1 protein in the transport and homeostasis of the plant hormone auxin

Poster Number 23
Presenter: Prof. Christoph Fahlke - Forschungszentrum Jülich
Date: Thursday, June 9 
Time slot: 12:00 PM – 13:45 PM CEST
Title: Exploring the transport cycle of a bacterial homolog of vesicular glutamate transporters

About the SURFE2R N1

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Research Ins]i[ghts: Nanion’s Transporter Webinar Series 2022

Join the next webinar on 28.06.2022


About the International Transmembrane Transporter Society (ITTS)

The International Transmembrane Transporter Society (ITTS) is a scientific society dedicated to promoting transporter biologists and transporter-related research.

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