Volume 20, Issue 3, Pages (March 1998)

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Volume 20, Issue 3, Pages 575-588 (March 1998) Mode Switching Kinetics Produced by a Naturally Occurring Mutation in the Cytoplasmic Loop of the Human Acetylcholine Receptor ε Subunit  Margherita Milone, Hai-Long Wang, Kinji Ohno, Richard Prince, Takayasu Fukudome, Xin-Ming Shen, Joan M. Brengman, Robert C. Griggs, Steven M. Sine, Andrew G. Engel  Neuron  Volume 20, Issue 3, Pages 575-588 (March 1998) DOI: 10.1016/S0896-6273(00)80996-4

Figure 1 Light Microscopy Localization of AChR and AChE at the EP Paired localization of AChR with rhodamine-labeled α-bgt (red signal) and of acetylcholinesterase with a monoclonal antibody (green signal) at patient ([A] and [B]) and control ([C] and [D]) EPs. Note attenuated reaction for AChR at the patient EP. Scale bar, 50 μm. Neuron 1998 20, 575-588DOI: (10.1016/S0896-6273(00)80996-4)

Figure 2 EP Fine Structure and Localization of AChR Patient ([A] and [B]) and control (C) EP regions. In (B) and (C), AChR is localized with peroxidase-labeled α-bgt. Note small and simplified postsynaptic region and paucity of secondary synaptic clefts in (A) and patchy and markedly attenuated reaction for AChR (arrows) in (B). Sections imaged in (B) and (C) are unstained. Magnification = 25,900×. Scale bar, 1 μm. Neuron 1998 20, 575-588DOI: (10.1016/S0896-6273(00)80996-4)

Figure 3 Channel Events at Patient and Control EPs Single channel currents from control and patient EPs are compared to currents from HEK cells expressing either AChR harboring wild-type ε or ε1254ins18. Left column shows single channel currents elicited by low concentrations of ACh with openings as upward deflections. Note presence of both 60 pS and 46 pS (asterisks) channels at the patient EP. ACh concentration was 1 μM for the EPs and 100 nM for the HEK cells. Right column shows the corresponding logarithmically binned burst-duration histograms with arrows indicating mean durations of major components of bursts. Time constant, τn, and fractional areas, an, for each component of bursts: control EP: τ1 = 0.05 ms, a1 = 0.14, τ2 = 3.24 ms, a2 = 0.86, total events, 1444. Patient EP, 60 pS channels: τ1 = 0.15 ms, a1 = 0.26, τ2 = 1.59 ms, a2 = 0.74, total events, 614. Patient EP, 46 pS channels: τ1 = 0.64 ms, a1 = 0.49, τ2 = 7.8 ms, a2 = 0.51, total events, 930. Wild-type HEK cells: τ1=0.03 ms, a1 = 0.17, τ2 = 0.20 ms, a2 = 0.17, τ3 = 2.8 ms, a3 = 0.66, total events, 1149. ε1245ins18 HEK cells: τ1 = 0.12, a1 = 0.15, τ2 = 0.95 ms, a2 = 0.85, total events, 510. Neuron 1998 20, 575-588DOI: (10.1016/S0896-6273(00)80996-4)

Figure 4 Genetic Analysis of the Mutations (A) Forward automated sequencing of ε exon 5. Heterozygous T-to-A transversion at nucleotide 382 converts codon 128 from a disulfide-loop-forming cysteine to a serine (εC128S, arrow). (B) Reverse automated sequencing of ε exon 11. A train of double peaks appears with the heterozygous inframe 18 bp duplication in the long cytoplasmic loop of ε (ε1254ins18, underlined). (C) Restriction analysis (εC128S) and gel size fractionation (ε1254ins18) of PCR products using genomic DNA from patient's muscle and relatives' blood. For εC128S, the wild-type allele gives rise to 444 bp (open arrowhead) and 79 bp fragments, whereas the mutant allele remains at 523 bp (closed arrowhead). The 79 bp fragment is not shown. For ε1254ins18, the sizes of the respective wild-type and mutant fragments are 83 bp (open arrowhead) and 101 bp (closed arrowhead). The patient and her two affected siblings harbor both mutations. Her unaffected mother is heterozygous for εC128S, and her two children are heterozygous for ε1254ins18. Arrow indicates patient, closed symbols show affected individuals. Neuron 1998 20, 575-588DOI: (10.1016/S0896-6273(00)80996-4)

Figure 5 Acetylcholine Binding to Intact Cells Transfected with the Indicated AChR cDNAs Determined by Competition against the Initial Rate of 125I α-bgt Binding In (A), the data are fitted to the Hill equation which describes binding to two sites with different affinity (See 2 in Experimental Procedures). For α2βδ2, KA = 6.28 × 10–8, KB = 1.46 × 10–2, and fractA = 0.58. For βαδ + εC128S, KA = 5.04 × 10–8, Kb = 1.39 × 10–2, and fractA = 0.55. In (B), the smooth curves are fits to the Hill equation (1 in Experimental Procedures). For α2βδε, KOV = 7.51 × 10–7, n = 1.20. For βαδ + ε1254ins18, KOV = 5.31 × 10–7, n = 0.99. Neuron 1998 20, 575-588DOI: (10.1016/S0896-6273(00)80996-4)

Figure 6 Separation of Kinetic Modes for Receptors Containing ε1254ins18 (A) Example of a single cluster showing a mode switch from the ε1254ins18 receptor. Activity begins in a mode with high Popen, but after 116 detected events, abruptly switches to a mode with low Popen (264 detected events). (B) Corresponding plot of relative window mean (RWM), which peaks at the point of transition between the kinetic modes. RWM was calculated as described in Experimental Procedures. (C, D, and E) Plots of open probability (Popen), mean open time (τ open), and mean closed time (τ closed) are calculated as described in Experimental Procedures. (A) through (E) are plotted on the same time scale. (F) Distribution of Popen for all clusters in the recording obtained at 100 μM ACh. The entry for each cluster is weighted according to its total number of events (see Experimental Procedures). Neuron 1998 20, 575-588DOI: (10.1016/S0896-6273(00)80996-4)

Figure 7 Illustration of Kinetic Modes for Receptors Activated by 30 μM ACh Left column compares cluster Popen distributions for receptors containing wild-type ε and ε1254ins18. Clusters were defined using both intercluster closed time and mode switch criteria as described in Experimental Procedures. For the ε1254ins18 receptor, there were 166 total clusters and 11 mode switches within clusters, whereas for wild-type there were 53 total clusters. Wild-type receptors (A) show a single major peak in the Popen distribution with a minor contribution from clusters with high Popen (closed bars). By contrast, receptors containing ε1254ins18 (B) show a broad Popen distribution with three distinct peaks shown in red, green, and blue. Right columns show the corresponding closed and open duration histograms fitted by the sum of exponentials with the data separated on the basis of the Popen distribution. Neuron 1998 20, 575-588DOI: (10.1016/S0896-6273(00)80996-4)

Figure 8 Illustration of Kinetic Modes for Receptors Activated by the Saturating Concentration of 300 μM ACh Left columns plot relative cluster mean (RCM; see Experimental Procedures) and Popen distributions for receptors containing wild-type ε and ε1254ins18. Clusters were defined using both intercluster closed time and mode switch criteria as described in Experimental Procedures. For the ε1254ins18 receptor, there were 120 total clusters and 15 mode switches within clusters, while for wild-type there were 66 total clusters. Wild-type receptors (A) show a single major peak in both the RCM and Popen distributions. By contrast, receptors containing ε1254ins18 (B) show broad RCM and Popen distributions. Three apparent peaks in the RCM distribution are shown in red, green, and blue. Corresponding portions of the Popen distribution and open and closed histograms are coded according to color. Neuron 1998 20, 575-588DOI: (10.1016/S0896-6273(00)80996-4)

Figure 9 Kinetics of Activation of the Three Modes of the ε1254ins18 Mutant Receptor Upper panels show histograms of closed and open durations for each of the three modes recorded from one patch at 30 μM ACh. The smooth curves are results of the Scheme 1 fit with parameters given in Table 5. The lower panel shows the ACh concentration dependence of Popen for wild-type and the three kinetic modes of the ε1254ins18 receptor. The smooth curves are calculated dose-response relationships using parameters obtained in the global fitting analysis of all of the data according to Scheme 1 (see Table 5). Square root of events per bin is shown. Neuron 1998 20, 575-588DOI: (10.1016/S0896-6273(00)80996-4)