Weltzin MM , George AA , Lukas RJ , Whiteaker P .

P20 GM103395 NIGMS NIH HHS , R01 DA043567 NIDA NIH HHS

                epilepsy

	Fig 1. Unitary amplitudes associated with human HS- and LS-isoform α4(R336H)β2-nAChR expressed in Xenopus laevis oocytes.Example single-channel ACh evoked response traces are shown below panels (A), (B), and (C), exhibiting a typical mixture of individual openings and short bursts of activity, interspersed with longer periods of inactivity. (A) Amplitudes of HS-isoform α4(R336H)β2-nAChR open events evoked by 1.3 μM ACh are characterized as a single population. (B and C) LS-isoform α4(R336H)β2-nAChR single-channel openings appeared to exhibit two distinct amplitudes, whether they were evoked in the presence of either a low ACh concentration (0.7 μM; Panel B) or a high ACh concentration (30 μM; Panel C). This finding was confirmed by two-way ANOVA, using ACh concentration and apparent amplitude class (small opening (OS) or large opening (OL)) as factors. A main effect of amplitude class was observed, confirming that amplitudes of OS and OL are distinctly different from each other (F1,22 = 144.7, ɫɫɫɫP < 0.0001). In contrast, no main effect of ACh concentration on open amplitude was observed (F1,22 = 0.04, P = 0.53), nor was there a significant interaction between the two factors (interaction ACh concentration x amplitude size F1,22 = 0.25, P = 0.25). Accordingly, the amplitudes of OS and large opening OL were not significantly altered by changes in the concentration of ACh applied. Amplitude histograms represent events collected across multiple individual single-channel patch recordings, for each illustrated combination of receptor construct and ACh concentration. Values are given as mean ± S.E.M., and were collected from 5–8 patches across a minimum of three separate experiments.
	Fig 2. Unitary amplitudes associated with human HS- and LS-isoform α4β2(V337G)-nAChR expressed in Xenopus laevis oocytes.As in Fig 1, example single-channel ACh evoked response traces are shown below panels (A), (B), and (C), which display a mixture of individual openings and short bursts of activity, which are interspersed with longer periods of inactivity. (A) HS-isoform α4β2(V337G)-nAChR stimulated with 1.3 μM ACh produced single-channel openings with a single characteristic amplitude. (A) Amplitudes of HS-isoform α4(R336H)β2-nAChR open events evoked by 1.3 μM ACh are characterized as a single population. (B and C) LS-isoform α4β2(V337G)-nAChR single-channel openings appeared to exhibit two distinct amplitudes, whether they were evoked in the presence of either a low ACh concentration (0.7 μM; Panel B) or a high ACh concentration (30 μM; Panel C). Similar to the findings for LS-isoform α4(R336H)β2-nAChR, this was confirmed by two-way ANOVA. A main effect of amplitude class was again observed, confirming that amplitudes of small openings (OS) and large openings (OL) are distinctly different from each other (F1,20 = 124.2, ɫɫɫɫP < 0.0001). In a further point of similarity with LS-isoform α4(R336H)β2-nAChR, the amplitudes of OS and OL of LS-isoform α4β2(V337G)-nAChR were not significantly altered by changes of applied ACh concentration (F1,22 = 0.04, P = 0.53), nor was there a significant interaction between the two factors (interaction ACh concentration x amplitude size F1,22 = 0.25, P = 0.25). Amplitude histograms represent events collected across multiple individual single-channel patch recordings, for each illustrated combination of receptor construct and ACh concentration. Values are given as mean ± S.E.M, and were collected from 5–7 patches across a minimum of three separate experiments.
	Fig 3. Closed-dwell time durations between single-channel openings of human HS- or LS-isoform α4(R336H)β2- or α4β2(V337G)-nAChR isoforms expressed in Xenopus laevis oocytes.(A) HS-isoform α4(R336H)β2-nAChR were stimulated with ACh (1.3 μM) and the resulting closed-dwell time durations between openings were best described with a pair of time constants. (B) When LS-isoform α4(R336H)β2-nAChR were stimulated at a low ACh concentration (0.7 μM), closed durations between openings were best described with three time constants. (C) The number of time constants required to best fit closed time distributions did not change as LS-isoform α4(R336H)β2-nAChR were stimulated at a higher ACh concentration (30 μM). (D) HS-isoform α4β2(V337G)-nAChR were stimulated with ACh (1.3 μM) and the resulting closed durations between openings were best described with a pair of time constants. (E) When LS-isoform α4β2(V337G)-nAChR were stimulated with a low ACh concentration (0.7 μM), closed durations between openings were best described with three time constants. (F) Increasing the ACh concentration to 30 μM did not change the number of time constants required to best fit closed duration distributions of LS-isoform α4β2(V337G)-nAChR. Closed-dwell duration histograms are representative examples collected from individual single-channel patch recordings. Individual τ values and percentage of total events corresponding to each closed duration (in parentheses) from these example patch recordings have been inserted into each panel to facilitate interpretation. Data were collected from 5–8 individual patches, across at least three separate experiments. Mean values of each property calculated from group data are summarized in Table 3, as mean ± SEM, together with any statistical analyses applied.
	Fig 4. Open-dwell time durations observed for HS- or LS-isoform human α4(R336H)β2- and α4β2(V337G)-nAChR expressed in Xenopus laevis oocytes.(A) Stimulation of HS-isoform α4(R336H)β2-nAChR with ACh (1.3 μM) resulted in open- dwell-time durations best described with a pair of time constants. (B) Simulation of LS-isoform α4(R336H)β2-nAChR at a low ACh concentration (0.7 μM) also resulted in open durations that were best fit using two time constants. (C) The number of time constants required to best fit the open time distributions did not change as LS-isoform α4(R336H)β2-nAChR were stimulated at a higher ACh concentration (30 μM). (D) Simulation of HS-isoform α4β2(V337G)-nAChR with ACh (1.3 μM) resulted in open durations again best described with a pair of time constants. (E) When LS-isoform α4β2(V337G)-nAChR were stimulated with a low ACh concentration (0.7 μM), open-dwell times were also best fit using two time constants. (F) Increasing the ACh concentration to 30 μM did not change the number of time constants required to best fit the open duration distribution of LS-isoform α4β2(V337G)-nAChR. Open-dwell time histograms are representative examples resulting from analysis of individual single-channel patch recordings. Individual τ values and percentage of total events (in parentheses) corresponding to each open duration from these example patch recordings have been inserted into each panel to facilitate interpretation. Data were collected from 5–8 individual patches, across at least three separate experiments. Mean values of each property calculated from group data are summarized in Table 4, as mean ± SEM, together with any statistical analyses applied.
	Fig 5. Closed-dwell time durations between burst activity of human LS-isoform α4(R336H)β2-nAChR.Closed-dwell time durations between bursts of activity (interburst durations) were measured for LS-isoform α4(R336H)β2-nAChR expressed in Xenopus laevis oocytes. Closed durations between bursts of either small (A) or large amplitude (A’) events evoked from LS-isoform α4(R336H)β2-nAChR at a low ACh concentration (0.7 μM) were best fit with four or three τ values, respectively. Increasing the ACh concentration to 30 μM resulted in LS-isoform α4(R336H)β2-nAChR closed state intervals between bursts of small (B) or large amplitude (B’) events that were best fit with five and four time constants, respectively. Interburst duration histograms are shown for pooled data from all recordings, which result from collection of data from 5 or 8 individual patches, across at least three separate experiments. The calculated τ values are summarized as mean ± SEM in Table 5, together with the statistical analyses applied.
	Fig 6. Closed-dwell time durations between burst activity of human LS-isoform α4β2(V337G)-nAChR.Closed-dwell time durations between bursts of activity (interburst closed durations) were measured for LS-isoform α4β2(V337G)-nAChR expressed in Xenopus laevis oocytes. Closed durations between bursts of either small (A) or large amplitude (A’) events evoked from LS-isoform α4β2(V337G)-nAChR at a low ACh concentration (0.7 μM) were best fit with either three or four τ values, respectively. Increasing the ACh concentration to 30 μM resulted in closed state intervals between bursts of LS-isoform α4β2(V337G)-nAChR small (B) or large amplitude (B’) events that were best fit with four and three time constants, respectively. Interburst closed duration histograms are shown for pooled data from all recordings, which result from collection of data from 5 or 7 individual patches, across at least three separate experiments. The calculated τ values are summarized as mean ± SEM in Table 5, together with the statistical analyses applied.
	Fig 7. Durations of individual openings within bursts of small or large amplitude openings of human LS-isoform α4(R336H)β2-nAChR.LS-isoform α4(R336H)β2-nAChR were expressed in Xenopus laevis oocytes and durations of individual openings within bursts of activity (intraburst openings) were measured. For LS-isoform α4(R336H)β2-nAChR stimulated with a low ACh concentration (0.7 μM), bursts of either small (A) or large amplitude (A’) events were seen. Openings within bursts of small amplitude events were best characterized using a single time constant, as were openings within bursts of large amplitude events. When the ACh concentration was increased to 30 μM, durations of individual openings of LS-isoform α4(R336H)β2-nAChR within small (B) or large amplitude (B’) bursts each remained associated with single time constants. Histogram panels each show pooled data, which were collected from 5 or 8 individual patches, across at least three separate experiments. The calculated τ values are summarized as mean ± SEM in Table 6, together with the statistical analyses applied.
	Fig 8. Durations of individual openings within bursts of small or large amplitude openings of human LS-isoform α4β2(V337G)-nAChR.LS-isoform α4β2(V337G)-nAChR were expressed in Xenopus laevis oocytes and durations of individual openings within bursts of activity (intraburst openings) were measured. When LS-isoform α4β2(V337G)-nAChR were stimulated with a low ACh concentration (0.7 μM), bursts of either small (A) or large (A’) amplitude events were seen. The open durations for individual events within bursts of small amplitude openings were best fit with two time constants (τ1 and τ2), while individual openings within bursts of large amplitude events were best fit with a single time constant. When the ACh concentration was increased to 30 μM, intraburst open durations of individual small amplitude openings of LS-isoform α4β2(V337G) nAChR within bursts (B) were best characterized using a single time constant. This contrasts with the two distinct time constants measured for small amplitude openings at the low ACh concentration. Individual open durations of large amplitude events within bursts remained best described with a single time constant, in the presence of the high ACh concentration (B’). Histogram panels each show pooled data, which were collected from 5 or 7 individual patches, across at least three separate experiments. The calculated τ values are summarized as mean ± SEM in Table 6, together with the statistical analyses applied.
	Fig 9. Human LS-isoform α4(R336H)β2-nAChR small and large amplitude burst properties.LS-isoform α4(R336H)β2-nAChR were expressed in Xenopus laevis oocytes. Function was evoked using 0.7 or 30 μM ACh and properties of the resulting bursts of open events were measured. (A) The proportions of small or large amplitude openings falling within bursts (as a fraction of the total numbers of each type of open event) were determined. (B) The mean number of openings per burst of LS-isoform α4(R336H)β2-nAChR was determined. (C) The durations of LS-isoform α4(R336H)β2-nAChR small or large amplitude bursts were measured at the low (0.7 μM) and high (30 μM) ACh concentrations. Noted statistical differences refer to the difference between the receptor property measured for LS-isoform α4(R336H)β2-nAChR versus its wildtype counterpart (i.e. fold change). (D) The Popen within bursts was also measured for bursts of small or large amplitude openings evoked from LS-isoform α4(R336H)β2-nAChR in the presence of the low or high ACh concentrations. Histograms within each panel show pooled data which were collected from 5 or 8 individual patches, respectively, across at least three separate experiments. Values and error bars represent the mean ± SEM of values across patches, and are summarized in Table 7, along with the statistical analyses applied.
	Fig 10. Human LS-isoform α4β2(V337G)-nAChR burst properties.LS-isoform α4β2(V337G)-nAChR were expressed in Xenopus laevis oocytes. Function was evoked using 0.7 or 30 μM ACh, and properties of the resulting bursts of open events were measured. (A) The proportions of small or large amplitude openings falling within bursts (as a fraction of the total numbers of each type of open event) were determined. Noted statistical difference refers to the difference between the receptor property measured for the LS-isoform α4β2(V337G)-nAChR versus its wildtype counterpart. (B) The mean number of openings per burst of LS-isoform α4β2(V337G)-nAChR were measured at each of the two ACh concentrations, for bursts of small or large amplitude openings. (C) The durations of LS-isoform α4β2(V337G)-nAChR small or large amplitude bursts were measured at the low (0.7 μM) and high (30 μM) ACh concentrations. (D) The Popen within bursts was also measured for bursts of small or large amplitude openings evoked from LS-isoform α4β2(V337G)-nAChR in the presence of the low or high ACh concentrations. Histograms within each panel show pooled data, which were collected from 5 and 7 individual patches respectively, across at least three separate experiments. Values and error bars represent the mean ± SEM of values across patches, and are summarized in Table 8, along with the statistical analyses applied.
	Fig 11. Illustration of the α4(R336H)β2- and α4β2(V337G)-nAChR SHE-associated mutant locations substituted into the known cryo-EM structures of 5-HT3AR apo and conducting states.(A) Sequence alignments of 5-HT3AR and nAChR subunits are displayed with numbering based on a start at the translation initiation methionine. The locations of the intracellular loop SHE-associated mutations are highlighted. The α4(R336H) mutation (green) is located in the initial portion of the M3-M4 loop, whereas the β2(V337G) mutation (magenta) is located in the MX helix. (B) Schematic of the major structural domains of a single nAChR subunit. The large extracellular N-terminal agonist-binding domain is followed by four transmembrane helices (M1–M4). The major intracellular domain is located between the M3 and M4 helices and consists of a short initial portion of sequence that connects the M3 helix to the MX helix, the MX helix itself, an extended region of undefined structure, followed by the final M4 transmembrane helix. The locations of both SHE-associated mutations are shown in the initial portion of the M3-M4 domain and the MX helix subdomain of the major intracellular domain, along with that of a likely interacting residue in the MA helix (β2E449; orange, putative interaction denoted with a dotted blue line). Note that β2E449 is not shown in Panel A since it is located far in the linear sequence from the MX helix. (C) The apo (non-ligand-bound; PDB ID: 6BE1) state of the recent cryo-EM structure of the 5-HT3AR [45] was used as the basis to illustrate the locations of the α4(R336H)β2- and α4β2(V337G)-nAChR mutations, since equivalent structural information is not yet available for these intracellular regions of α4β2-nAChR. Position numbers refer to those of the human α4 or β2 nAChR subunit, as shown in the accompanying sequence alignment. Left Panel depicts the positioning of wildtype nAChR α4(R336) and β2(V337) residues, substituted into the known 5-HT3AR structure. The location of the initial portion of the M3-M4 domain, followed by the MX, MA, and M4 helices are labeled. Right Panel illustrates the locations of the SHE-associated α4(R336H) and β2(V337G) residues in the same context. Also shown in both panels is the location of the β2E449 residue thought to interact with α4(R336). (D) Same as (C), except that the residues of interest are substituted into the conducting (ligand-bound; PDB ID: 6DG8) 5-HT3AR cryo-EM structure [31]. Note the upward movement of the MX helix in the conducting, compared to apo, state.

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