Article Figures & Data
Figures
- Fig. 1.
Comparison of embryonic (αβδγ) and adult (αβδε) type ACh receptor desensitization in excised outside-out patches from two different Xenopus oocytes.A, Macroscopic current activation for αβδγ and αβδε receptors in response to 400 msec pulses of ACh at four different concentrations. B, The semi-log representation of current decay is shown in response to 100 μm ACh. Current amplitudes were normalized to the same initial peak values for comparison. The indicated time constants are based on the least-squares linear fit to the current decay. C, The dose–response relationships between peak ACh-activated current and ACh concentration were fit to the Hill equation (R =I/(1 + (K/x)n), whereR is the peak response at any concentration,I is the peak response at 100 μm ACh,n is the Hill coefficient, and K is the half-maximal concentration. The fits yielded values for embryonic (EC50 = 4.2 μm; Hill coefficient = 1.4) and adult (EC50 = 9.4 μm; Hill coefficient = 1.3) receptor types.
- Fig. 2.
Time-dependent recovery from desensitization reveals two components. A, Two-pulse measurement of recovery from desensitization for ACh-activated current from α2βδγ (top traces) and α2βδε (bottom traces) receptor types. For both receptor types a 400 msec pulse of 100 μmACh was followed at varied intervals by a second 100 μmpulse of ACh. The final response shown was measured after a 10 sec interval (note the discontinuous time scale for this response).B, The fractional recovery of peak current at each recovery interval was determined on the basis of the ratio of pulse 2 to pulse 1 current. Recovery curves for single patches containing α2βδγ (top graph) and α2βδε (bottom graph) receptor types were constructed by fitting the data points to the sum of two exponential curves. The time constants and percentage of total amplitude for each component are indicated for the individual patches.
- Fig. 3.
8Br-cAMP treatment of oocytes speeds recovery from desensitization. A, Comparison of standard recovery traces from patches excised from a control (top traces) and 8Br-cAMP-treated (bottom traces) oocyte. Both pulses of ACh were 400 msec in duration and 100 μmconcentration. B, The amount of recovery, expressed as the ratio of pulse 2 to pulse 1 current measured at a fixed 1 sec interval, is plotted for all control patches.C, The fractional recovery plotted for patches from a single batch of oocytes exhibiting very slow recovery from desensitization. Shaded columns indicate the fractional recovery from control patches, and filled columns indicate patches from oocytes treated with 8Br-cAMP for 2–4 hr. D, Recovery from desensitization as a function of interpulse intervals. Short intervals are shown intop relationship, and the entire time course in shown in the bottom relationship. The top data set is fit to a single exponential, and the bottom set is fit to the sum of two exponentials with the indicated time constants.
- Fig. 4.
Inhibitor of PKA leads to a slowing of the recovery from desensitization. A, Comparison of ACh-activated currents for control αβδγ receptors (top; same data shown in Fig. 2 for comparison) with those co-injected with an RNA coding for a PKA inhibitor protein (bottom). The arrows indicate the time at which the pulse 2 ACh was applied. B, The fractional recovery for patches from control (squares) and PKA-inhibited (triangles) oocytes measured during the first 3 sec recovery interval. C, The full time course of recovery for patches from control and PKA-inhibited oocytes. The time constants and areas for PKA-inhibited patches corresponded to 0.5 sec (11%) for the fast component and 68.6 sec (89%) for the slow component.
- Fig. 5.
Time-dependent slowing of recovery from desensitization after exposure to phosphatase. A, Data sets obtained by using a two-pulse protocol to measure the time-dependent recovery from desensitization. Indicated are the times elapsed since the formation of the outside-out patch. The pipette solution contained 0.5 U/ml of potato acid phosphatase, pH-adjusted to 6.8. B, The fractional recovery of ACh-activated current measured with short interpulse intervals at various times after patch formation. Each data set corresponds to the indicated time after patch formation. C, Comparison of fractional recovery measured for untreated patches (same control data shown in Fig. 2 for comparison) and patches after a 20 min exposure to phosphatase. In phosphatase-treated patches 70% of the receptors recovered with a time constant of 103 sec.
- Fig. 6.
Desensitization of receptors lacking PKA phosphorylation sites. A, Normalized responses of α2βδγ, α2βδ2, and α2βδγ mutant receptor responses to slow application of 100 μm ACh. The responses were recorded from the entire oocyte by using two-microelectrode voltage-clamp (Dagan TEV-200) and slow perfusion techniques. B, Current traces comparing the recovery of control (top) and mutant (bottom) receptors. The timing of pulse 2 application of 100 μm ACh is indicated byarrows. C, Dose–response relations for αβδγ mutant receptors. The fractional response was computed on the basis of the response to 100 μm ACh. The data were fit to the Hill equation (see Fig. 1), yielding an EC50 of 3.2 μm. The wild-type receptor dose–response from Figure1C is shown as a dashed line for comparison. D, The time constant of desensitization onset was determined by using fast perfusion for wild-type (squares) and mutant (triangles) receptors. The time constants, obtained by fitting the current decay to a single exponential curve, are indicated for four different ACh concentrations. E, The time course of recovery determined by a two-pulse recovery protocol. Top, The short interpulse recovery data, shown for wild-type (squares; same data shown in Fig. 2 for comparison) and mutant (triangle) receptors, were fit to a single exponential curve. Bottom, The full time course of recovery measured with the two-pulse protocol. These data sets were fit with the sum of two exponentials, yielding time constants of 1 sec (38%) and 40 sec (62%) for the mutant receptors.
- Fig. 7.
Slow recovery from desensitization increases with ACh pulse duration. A, Recovery from desensitization is compared for short (400 msec) and long (4 sec) P1 pulses of 100 μm ACh. The P2 pulse was set to the standard 400 msec. The recovery data were fit to the sum of two exponentials, and the time constants and area for each are indicated. B, Recovery from desensitization as a function of ACh pulse duration. Recovery for wild-type and mutant receptors was measured by using a variable-length P1, followed by a standard 400-msec-long pulse of 100 μmACh. The data were fit by a single exponential curve with the indicated time constants. The dashed line indicates the recovery expected with the standard 400 msec pulse, the duration that was used routinely to measure recovery.
- Fig. 8.
Desensitization measured with repetitive brief pulses of ACh. A, Responses of mutant (top) and wild-type (bottom) α2βδγ receptors to 3 msec pulses of 100 μm ACh at a rate of 10 Hz. The inset shows three sequential responses from wild-type receptors. The solid lines indicate the peak amplitude for each of the three responses, and the dashed lines indicate the amplitude at the termination of the 3 msec pulse. B, The peak response for mutant (triangles) and wild-type (circles) receptors measured for each repetitive ACh application. The dashed lines indicate the amount of desensitization that is predicted for wild-type and mutant receptors on the basis of the following relationship: Y = (e−t1/τ) + (a · (1 −e−t2/τ1) + (b · (1 −e−t2/τ2), whereY represents the predicted peak response ratio,t1 is the pulse duration (3 msec), t2 is the pulse interval (100 msec), τ is the time constant of desensitization (65 msec) determined by fitting the current decay in response to a 400 msec pulse of ACh, a is the proportion of fast-recovering receptors (76% for circles and 32% for triangles), b is the proportion of slow-recovering receptors, τ1 is the average time constant for fast recovery (1.7 sec), and τ2 is the average time constant for slow recovery (30 sec). The solid lines indicate the predicted fit after a correction for ACh pulse duration to 9.6 msec. This value was determined specifically for this experiment by using the equation D = (At/Ap) · D2, whereD represents the corrected duration, Atrepresents the total integrated current area measured during each individual response, Ap represents the integrated current area measured during the 3 msec exposure to ACh, andD2 is the pulse duration (3 msec).
Tables
Onset τ (sec) Recovery τf (sec) Amplitude fast Recovery τs (sec) Amplitude slow Untreated NA 0.35 ± 0.21 (3) 14 ± 6% 77.9 ± 10 86 ± 6% 8-Br-cAMP 0.068 ± 0.010 (10) 0.40 ± 0.10 (3) 66 ± 9%* 24.4 ± 11.0 (3)* 34 ± 9%* Control 0.064 ± 0.024 (54) 1.43 ± 0.45 (4) 78 ± 9% 40.5 ± 9.5 22 ± 9% PKA inhibitor 0.053 ± 0.017 (11) 0.33 ± 0.40 (3)* 10 ± 4%* 80.2 ± 13.5* 90 ± 4%* ser-ala mutant 0.062 ± 0.013 (23) 1.16 ± 0.92 (3) 38 ± 15%* 36.9 ± 22.4 62 ± 15%* plus phosphatase NA 1.1 (1) 30% 105 70% For statistical treatment 8-Br-cAMP patches were compared to the untreated patches, and all other experimental groups were compared to the control patches. Statistically different values are indicated by an asterisk (see Materials and Methods for details).
