gramicidin-a and 2-aminoethoxydiphenyl-borate

Many applications utilizing artificial lipid bilayers require the ability to exchange the bilayer's solution environment. However, because of the instability of the bilayer, the rate of solution exchange is limited, which significantly hinders the measurement rate and throughput. We have developed an artificial bilayer system that can withstand high flow speeds, up to 2.1 m/s, by supporting the bilayer with a hydrogel. We demonstrated the ability to measure during flow by measuring the conductance of gramicidin-A channels while switching between solutions of two different compositions, recording a time to measure 90% change in current of approximately 2.7 seconds at a flow rate of 0.1 m/s. We also demonstrated a potential application of this system by measuring the conductance modulation of the rat TRPM8 ion channel by an agonist and antagonist at varying concentrations, obtaining 7-point IC50 and EC50 values in approximately 7 minutes and 4-point values within 4 minutes.

Topics: Animals; Boron Compounds; Electric Conductivity; Gramicidin; Hydrogel, Polyethylene Glycol Dimethacrylate; Lipid Bilayers; Liposomes; Membrane Potentials; Menthol; Rats; Solutions; Time Factors; TRPM Cation Channels

Pulmonary surfactant with surfactant-associated proteins (PS+SAP) decreases pulmonary inflammation by suppressing neutrophil activation. We have observed that PS+SAP inserts channels into artificial membranes, depolarizes neutrophils, and depresses calcium influx and function in stimulated neutrophils. We hypothesize that PS+SAP suppresses neutrophil activation by depletion of internal Ca(++) stores and that PS+SAP induces depletion through release of Ca(++) stores and through inhibition of Ca(++) influx. Our model predicts that PS+SAP releases Ca(++) stores through insertion of channels, depolarization of neutrophils, and activation of a G protein-dependent pathway. If the model of channel insertion and membrane depolarization is accurate, then gramicidin-a channel protein with properties similar to those of PS+SAP-is expected to mimic these effects. Human neutrophils were monitored for [Ca(++)] responses after exposure to one of two different PS+SAP preparations, a PS-SAP preparation, gramicidin alone, and gramicidin reconstituted with phospholipid (PLG). [Ca(++)] responses were reexamined following preexposure to inhibitors of internal Ca(++) release or the G protein pathway. We observed that (i) 1% PS+SAP-but not PS-SAP-causes transient increase of neutrophil [Ca(++)] within seconds of exposure; (ii) 1% PLG-but not gramicidin alone-closely mimics the effect of PS+SAP on Ca(++) response; (iii) PS+SAP and PLG equally depolarize neutrophils; (iv) direct inhibition of internal Ca(++) stores releases or of G protein activation suppresses Ca(++) responses to PS+SAP and PLG; and (v) preexposure to either PS+SAP or PLG inhibits Ca(++) influx following fMLP stimulation. We conclude that PS+SAP independently depolarizes neutrophils, releases Ca(++) from internal stores by a G protein-mediated pathway, and alters subsequent neutrophil response to physiologic stimulants by depleting internal Ca(++) stores and by inhibiting Ca(++) influx during subsequent fMLP activation. The mimicking of these results by PLG supports the hypothesis that PS+SAP initiates depolarization via channel insertion into neutrophil plasma membrane.

Topics: Boron Compounds; Calcium; Cell Membrane; Cytosol; Gramicidin; GTP-Binding Proteins; Humans; Imidazoles; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Pertussis Toxin; Phospholipids; Potentiometry; Pulmonary Surfactant-Associated Proteins; Pulmonary Surfactants