Effects of extracellular Ca2+ concentration on hair-bundle stiffness and gating-spring integrity in hair cells

When a hair cell is stimulated by positive deflection of its hair bundle, increased tension in gating springs opens transduction channels, permitting cations to enter stereocilia and depolarize the cell. Ca2+ is thought to be required in mechanoelectrical transduction, for exposure of hair bundles to Ca2+ chelators eliminates responsiveness by disrupting tip links, filamentous interstereociliary connections that probably are the gating springs. Ca2+ also participates in adaptation to stimuli by controlling the activity of a molecular motor that sets gating-spring tension. Using a flexible glass fiber to measure hair-bundle stiffness, we investigated the effect of Ca2+ concentration on stiffness before and after the disruption of gating springs. The stiffness of intact hair bundles depended nonmonotonically on the extracellular Ca2+ concentration; the maximal stiffness of ≈1200 μN⋅m−1 occurred when bundles were bathed in solutions containing 250 μM Ca2+, approximately the concentration found in frog endolymph. For cells exposed to solutions with sufficient chelator capacity to reduce the Ca2+ concentration below ≈100 nM, hair-bundle stiffness fell to ≈200 μN⋅m−1 and no longer exhibited Ca2+-dependent changes. Because cells so treated lost mechanoelectrical transduction, we attribute the reduction in bundle stiffness to tip-link disruption. The results indicate that gating springs are not linearly elastic; instead, they stiffen with increased strain, which rises with adaptation-motor activity at the physiological extracellular Ca2+ concentration.

Characterization of Spermidine Binding to Solubilized Plasma Membrane Proteins from Zucchini Hypocotyls1

In this work [14C]spermidine binding to total proteins solubilized from plasma membrane purified from zucchini (Cucurbita pepo L.) hypocotyls was investigated. Proteins were solubilized using octyl glucoside as a detergent. Specific polyamine binding was thermolabile, reversible, pH dependent with an optimum at pH 8.0, and had a Kd value of 5 μm, as determined by glass-fiber-filter assays. Sephadex G-25 M gel-filtration assays confirmed the presence of a spermidine-protein(s) complex with a specific binding activity. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis and native polyacrylamide gel electrophoresis of collected fractions having the highest specific spermidine-binding activity, several protein bands (113, 75, 66, and 44 kD) were identified. The specificity of spermidine binding was examined by gel-filtration competition experiments performed using other polyamines and compounds structurally related to spermidine. Partial purification on Sephadex G-200 led to the identification of 66- and 44-kD protein bands, which may represent the putative spermidine-binding protein(s) on the plasmalemma.