Figure 4.
Disulfide trapping of the S729C mutant. A, Test responses to 10 mm glutamate are inhibited following a 3.2 s exposure to 50 μm glutamate and 10 μm CuPhen (*); the response to the first application of 10 mm glutamate after trapping is too small to measure, but in the continuous presence of DTT full recovery occurs at a rate of 0.3 s−1, as shown by a single exponential fit to the envelope of the peak amplitude of subsequent responses to glutamate (filled red circles); extrapolation to the end of the application of 50 μm glutamate and 10 μm CuPhen (open red circle) was used to estimate the extent of trapping; the upper trace shows the piezo command voltage. B, The 1st and 22nd responses to 10 mm glutamate in DTT from panel A are essentially indistinguishable, as shown in the inset for which the traces are overlaid. These traces were chosen because of their similar amplitude, and in both, desensitization was well described by a single exponential function (red curves, kdes = 332 s–1). The upper trace is the open tip response recorded at the end of the experiment. C, Trapping in the absence of ligand is much slower than in the presence of glutamate. Exposure to 10 μm CuPhen for 12.8 s (*) traps 54% of receptors. The peak currents during recovery in DTT were fitted as in panel A (red curve, 0.3 s–1) and the trapping estimated from the fitted curve (open circle). D, The active fraction of receptors in the presence of 50 μm glutamate (filled circles), or at rest (open circles), was plotted against the period of trapping in 10 μm CuPhen; with 50 μm glutamate there was complete trapping; in contrast, for receptors at rest trapping remained incomplete at 360 s. The arrows represent the time intervals for the representative traces shown in B and C. For trapping in resting conditions, the solid line is a biexponential fit (τ1 = 3.5 ± 0.6 s (amplitude 76 ± 5%), τ2 = 50 ± 20 s; active fraction at t∞ = 32 ± 2%); the dashed line shows a monoexponential fit (τ = 7 ± 1 s).