06.05.2021 - Secondary transport inhibition and regulation by SSM-electrophysiology
Secondary active transporters utilize existing concentration gradients in cells for the uphill transport of molecules. As the coupled substrates are typically ions, mostly sodium or protons, many of these transporters are electrogenic. Their characteristics can be investigated using electrophysiological techniques like SSM-based electrophysiology. In the second session of the Transporter Webinar Series we meet Christine Ziegler and Camilo Perez, who share their stories about using this technology to investigate inhibition and regulation of secondary active transporters.
Title: The K+ switch in BetP: from coupling to regulation
Professor Dr. Christine Ziegler
(University of Regensburg, Faculty of Biology and Pre-Clinics, Institute of Biophysics and physical Biochemistry, Structural Biology-Biophysics II)
The bacterial betaine transporter BetP is a prime example for an efficient osmotic stress sensor and regulated secondary transporter, respectively. BetP’s full activation depends on the presence of 300mM internal K+, however, K+ is not transported. Several K+ binding sites were identified at the osmo-sensor, but also close to a Na+ site. We introduced a point mutation based in one of the two sodium binding sites in order to switch BetP from Na+ to K+ coupling. Using cryoEM/X-ray crystallography combined with SSM/Stopped-Flow Trp-fluorescence we discovered an intriguing competition between Na+ and K+ binding in BetP, which hints towards a change in the functional role of K+ in LeuT-fold transporter during evolution. BetP shares its overall fold with SLC6 neurotransmitter transporters, which also show differences in their ability to facilitate K+-coupled antiport. Therefore, our structure-function study provides new insights into an evolutionary switching of K+ between coupling to regulatory ion.
Title: Selection of transporter-targeted inhibitory nanobodies by SSM-based electrophysiology
Professor Dr. Camilo Perez
(University of Basel, Biozentrum, Center for Molecular Life Sciences)
Single domain antibodies (nanobodies) have been extensively used in machanistic and structural studies of protein and pose an enormous potential as tools for developing clinical therapies, many of which depend on inhibition of membrane proteins such as transporters.However, most of the methods used to determine inhibition of transport activity are difficult to perform in high-throughput routines and depend on labeled substrates availability. This complicates the screening of large nanobody libraries. Solid-supported membrane (SSM) electrophysiology to select inhibitory and non-inhibitory nanobodies targeting an electrogenic secondary transporter and to calculate nanobodies inhibitory constants. This technique may be especially useful for selecting inhibitory nanobodies targeting transporters for which labeled substrates are not available.