Biophysical Society Conference | Tahoe 2022

Molecular Biophysics of Membranes

Tuesday Speaker Abstracts

FLUORESCENT VISUALIZATION OF POTASSIUM FLUXES William R Kobertz 1 ;

1 UMASS Medical School, Biochemistry & Molecular Pharm, Worcester, MA, USA The fluorescent visualization of intracellular ions and metabolites has reimaged our basic understanding of the inner workings of cells, tissues, and living organisms. In contrast, there is a dearth of tools to fluorescently visualize extracellular fluxes. Part of the challenge stems from the fact that cellular egress is contrary to the pervasive intracellular-centric experimental paradigm. Recently, we have been using chemistry to target the cell’s glycocalyx, which ideally positions fluorescent sensors within nanometers of the extracellular vestibules of ion channels and membrane transporters. My laboratory’s efforts to fluorescently visualize potassium and other cations entering and exiting cells using these technologies will be presented. PHOSPHATE POSITION IS KEY IN MEDIATING THE TMEM16A-PI(4,5)P2 INTERACTION Maiwase Tembo 1 ; Rachel E Bainbridge 1 ; Grant J Daskivich 1 ; Jacob D Durrant 1 ; Joel C Rosenbaum 1 ; Anne E Carlson 1 ; 1 University of Pittsburgh, Biological Sciences, Pittsburgh, PA, USA TMEM16A is a Ca2+-activated Cl- channel that plays a critical role in regulating diverse physiologic processes. In addition to Ca2+, TMEM16A activation requires the membrane lipid phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). Here we interrogated the properties of the lipid that mediate its interaction with the channel using patch- and two-electrode voltage- clamp recordings on cells that endogenously express TMEM16A channels: oocytes from the African clawed frog Xenopus laevis. During continuous application of Ca2+ to excised inside- out patches, we found TMEM16A-conducted currents decayed shortly after patch excision. Following this rundown, the application of synthetic PI(4,5)P2 recovered current. Only lipids that include a phosphate at the 4’ position effectively recovered TMEM16A currents; lipids lacking the 4’ phosphate had minimal effects on channel gating. Docking PI(4,5)P2 into a homology model of the TMEM16A channel explained why the 4’ phosphate is required for this interaction. These findings improve our understanding of how PI(4,5)P2 binds to and potentiates TMEM16A channels.

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