Biophysical Society Thematic Meeting - October 13-15, 2015

Biophysics of Proteins at Surfaces: Assembly, Activation, Signaling

Poster Abstracts

36-POS Board 36 Peptide Binding to Membranes and Tension-dependent Penetration Are Involved in the Inhibition of GsMTx4 on Mechanosensitive Ion Channels Radhakrishnan Gnanasambandam 1 , Chiranjib Ghatak 2 , Anthony Yasmann 3 , Alexey Ladokhin 2 , Frederick Sachs 1 , Sergei Sukharev 3 , Thomas Suchyna 1 . 3 University of Maryland, College Park, MD, USA. 1 University of New York at Buffalo, Buffalo, NY, USA, 2 The University of Kasnsas, Lawrence, KS, USA, GsMTx4, a 34 amino acid cysteine-knot peptide from spider venom, is highly specific at inhibiting cationic mechanosensitive ion channels (MSCs). GsMTx4 does not seem to interact stereospecifically with the channel protein, but acts from the boundary lipids. GsMTx4 has a hydrophobic face with tryptophans (Trp) surrounded by a ring of charged amino acids, including six lysines. To investigate the mechanism of MSC inhibition, we created lysine to glutamate mutants and tested them on Piezo1 channels in patches. Four mutants showed a 20-40% loss of activity. The free energy of binding to liposomes determined by isothermal titration calorimetry (ITC) varied with the mutation and was highest in the most functionally compromised K8E mutant. The binding affinity determined independently by aqueous Trp quenchers agreed with ITC. The penetration depth determined using brominated lipid quenchers indicated a shallow penetration, but the mutants generally penetrated deeper. ITC suggested there was more than one binding reaction, consistent with predicted shallow and deep modes. The effect of membrane tension on penetration depth was studied using lipid monolayers in a Langmuir trough. All peptides bound to expanded monolayers and occupied a large proportion of the area. Compression near the monolayer-bilayer equivalence pressure forced WT peptide from the monolayer, but the inactive mutants remained bound. These data place GsMTx4 near the polar interface of the outer monolayer at depth set by the lysines, and occupying a small fraction of membrane area at resting tensions. As the lateral pressure of lipids decreases during membrane stretching, the peptides penetrate deeper acting as “area reservoirs” effectively clamping the surface tension and preventing activation of MSCs.

84