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Comparative Study
. 2011 Sep 28;31(39):13870-9.
doi: 10.1523/JNEUROSCI.2652-11.2011.

Single-channel and Structural Foundations of Neuronal α7 Acetylcholine Receptor Potentiation

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Free PMC article
Comparative Study

Single-channel and Structural Foundations of Neuronal α7 Acetylcholine Receptor Potentiation

Corrie J B daCosta et al. J Neurosci. .
Free PMC article

Abstract

Potentiation of neuronal nicotinic acetylcholine receptors by exogenous ligands is a promising strategy for treatment of neurological disorders including Alzheimer's disease and schizophrenia. To gain insight into molecular mechanisms underlying potentiation, we examined ACh-induced single-channel currents through the human neuronal α7 acetylcholine receptor in the presence of the α7-specific potentiator PNU-120596 (PNU). Compared to the unusually brief single-channel opening episodes elicited by agonist alone, channel opening episodes in the presence of agonist and PNU are dramatically prolonged. Dwell time analysis reveals that PNU introduces two novel components into open time histograms, indicating at least two degrees of PNU-induced potentiation. Openings of the longest potentiated class coalesce into clusters whose frequency and duration change over a narrow range of PNU concentration. At PNU concentrations approaching saturation, these clusters last up to several minutes, prolonging the submillisecond α7 opening episodes by several orders of magnitude. Mutations known to reduce PNU potentiation at the whole-cell level still give rise to multisecond-long single-channel clusters. However mutation of five residues lining a cavity within each subunit's transmembrane domain abolishes PNU potentiation, defining minimal structural determinants of PNU potentiation.

Figures

Figure 1.
Figure 1.
Macroscopic versus microscopic potentiation of wild-type (wt) α7 by PNU. a, Whole-cell currents elicited by separate applications of 100 μm ACh to a single HEK 293 cell expressing wild-type human α7 before (left) and after (right) a 60 s incubation with 3 μm PNU. b, Addition of 3 μm PNU to the extracellular solution surrounding a cell-attached patch of wild-type human α7 results in a marked increase in current in the continued presence of 100 μm ACh in the patch pipette. c, Expanded view of the boxed region in b showing that the rise in current results from a dramatic PNU-induced prolongation of channel openings. Note that in b the trace was filtered at 0.1 kHz, while in c the same trace was filtered at 5 kHz to better resolve brief openings.
Figure 2.
Figure 2.
Single-channel traces and dwell time histograms for wild-type α7 in the presence of increasing concentrations of PNU in the pipette solution. Recordings were made in the cell-attached patch configuration at an applied potential of −70 mV. Currents activated by 100 μm (a), 10 μm (b), and 1 μm (c) ACh are all shown at a bandwidth of 5 kHz, with channel openings as upward deflections. Corresponding histograms of dwell times are shown on logarithmic time axes with overlaid fits to the sum of exponentials (solid line, fits; dotted lines, individual components). Cluster duration histograms (far right) are open time histograms generated by summing open times with flanking closings briefer than a critical time (τcrit, indicated by arrows) determined from corresponding closed time histograms (see Materials and Methods).
Figure 3.
Figure 3.
Mutations that markedly reduce PNU potentiation at the whole-cell level exhibit potentiation at the single-channel level. Current traces with associated open time and cluster duration histograms from mutant α7 receptors (A226S; M254L; A226S+M254L double mutant), both in the absence (left) and presence (right) of 3 μm PNU in the pipette solution, are shown. Openings were elicited by 100 μm ACh; all recordings were made in the cell-attached patch configuration with an applied voltage of −70 mV. Single-channel traces are shown at a bandwidth of 5 kHz, and openings are upward deflections.
Figure 4.
Figure 4.
Combining individual mutations progressively reduces PNU potentiation. Single-channel traces and dwell time histograms from triple (SL+I281M; SL+V288F), single (I281M; V288F) and quadruple (SL+I281M+V288F) mutant α7 receptors in the absence (left) and presence (right) of 3 μm PNU in the pipette solution. All recordings were made in the cell-attached configuration at an applied voltage of −70 mV, with 100 μm ACh in the patch pipette. Single-channel openings are upward deflections, and all traces are shown at a bandwidth of 5 kHz.
Figure 5.
Figure 5.
Quintuple mutant eliminates potentiation by 3 μm PNU. Single-channel current traces and dwell time histograms from quintuple (SLMF+S223T) and single (S223T; S223V; S223A) mutant α7 receptors in the absence (left) and presence (right) of 3 μm PNU in the pipette solution. Openings were elicited by 100 μm ACh, and all recordings were made in the cell-attached patch configuration with an applied voltage of −70 mV. Openings are upward deflections, and all traces are shown at a bandwidth of 5 kHz.
Figure 6.
Figure 6.
Characterization of the quintuple TSLMF mutant. a, Addition of 3 μm PNU to the extracellular solution surrounding a cell-attached patch containing the TSLMF mutant, activated by 100 μm ACh, shows no PNU-induced change in single-channel activity. b, Whole-cell currents elicited by separate applications of 100 μm ACh for a single HEK 293 cell expressing the TSLMF mutant before (left) and after (right) a 60 s incubation with 3 μm PNU. c, PNU dose–response relationships for wild-type (black circles) and TSLMF mutant α7 (white triangles) determined from single-channel recordings (n = 2 or 3 recordings for each data point; see Materials and Methods). d, Ability of PNU to allosterically modulate agonist affinity. ACh binding was measured by its competition against the initial rate of α-bungarotoxin binding for both wild-type (circles) and TSLMF mutant (triangles) receptors in the presence (black circles, white upward triangles) and absence (gray circles and downward triangles) of 3 μm PNU (n = 3 for each data point). Error bars in c and d represent ± standard deviation of the means.
Figure 7.
Figure 7.
a, A possible mechanism for PNU potentiation. The scheme does not include agonist binding steps and assumes closed (C), open (O), and desensitized (D) states are bound with an optimal number of n agonists. The scheme also includes a minimum of two classes of PNU potentiation (p, pp) thought to relate to different levels of PNU occupancy. b, c, Structural determinants of PNU potentiation. Homology model (Cheng et al., 2006) based on Protein Data Bank identifier 2BG9 (Unwin, 2005) of the isolated transmembrane region of a single α7 subunit as viewed from the plane of the membrane (b) and the synaptic space (c). The side chains of the five amino acids mutated in the quintuple (TSLMF) mutant are shown as space-filling spheres. The four transmembrane helices (TM1-4) are also labeled. The dashed lines in b represent the approximate location of the plasma membrane.

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