Patch-clamp recording has revolutionized the study of ion channels, transporters, and the electrical activity of small cells. Vital to this method is formation of a tight seal between glass recording pipette and cell membrane. To better understand seal formation and improve practical application of this technique, we examine the effects of divalent ions, protons, ionic strength, and membrane proteins on adhesion of membrane to glass and on seal resistance using both patch-clamp recording and atomic force microscopy. We find that H +, Ca2+, and Mg2+ increase adhesion force between glass and membrane (lipid and cellular), decrease the time required to form a tight seal, and increase seal resistance. In the absence of H+ (10-10 M) and divalent cations (<10-8 M), adhesion forces are greatly reduced and tight seals are not formed. H+ (10-7 M) promotes seal formation in the absence of divalent cations. A positive correlation between adhesion force and seal formation indicates that high resistance seals are associated with increased adhesion between membrane and glass. A similar ionic dependence of the adhesion of lipid membranes and cell membranes to glass indicates that lipid membranes without proteins are sufficient for the action of ions on adhesion.
Bibliographical noteFunding Information:
This work was supported by grants from The Israel Science Foundation founded by the Israel Academy of Sciences and Humanities and the Zlotowski Center for Neuroscience to S.D.S., from the National Institutes of Health (GM55611) to V.T.M., and from the National Institutes of Health (AR32805) and the Muscular Dystrophy Association to K.L.M. Z.G. was a recipient of the Kreitman Doctoral Fellowship and the Folks Foundation Graduate Fellowship.