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

R01 DA043567 NIDA NIH HHS , R21 DA026627 NIDA NIH HHS

	Fig 1. Schematic representations of high-sensitivity (α4β2)2β2- and low-sensitivity (α4β2)2α4-nAChR isoforms.A) Configuration of nAChR α4 and β2 protein subunits in each of the two isoforms. In this case, fully-concatenated pentameric isoforms are illustrated (HSP and LSP, respectively, with the short A-G-S peptide linkers that are used to enforce subunit associations shown). Note that both isoforms contain two canonical agonist binding sites (magenta circles) at the interfaces between the (+)-face of an α4 subunit and the (-)-face of a β2 subunit. The LS-isoform (α4β2)2α4-nAChR contains an additional agonist binding site (blue diamond) at the interface between the (+)- and (-)-faces of two α4 subunits. In receptors assembled from unlinked α4 and β2 nAChR subunits, mixed populations of the same two isoforms are typically formed. Each isoform exhibits the same ratios and associations of subunits as shown for the corresponding concatenated assembly, but A-G-S linkers are absent. It is also possible to produce essentially-pure populations of HS- or LS-isoform α4β2-nAChR by expressing strongly-biased ratios of unlinked subunits (see methods section for details). B) Illustration (from top to bottom) of cDNA constructs used to express concatenated HSP (α4β2)2β2- and LSP (α4β2)2α4-nAChR isoforms. Each construct is flanked with AscI and EcoRV restriction sites (5’ and 3’, respectively; indicated by gray triangles) for subcloning into high expression oocyte vectors. A Kozac sequence and the β2 signal peptide (SP) were retained only for the 1st position. Flanking each subunit position are unique restriction sites (indicated by gray triangles) used in concatemer design (for example, NotI and EcoRV were used in exchanging nAChR subunits at position 5). The two concatemers differ in composition only at position 5, containing either the β2 or α4 subunits in the HSP or LSP constructs, respectively. C) Illustrations of the whole-cell concentration response relationships of each α4β2-nAChR isoform. On the left, high-sensitivity isoform (α4β2)2β2-nAChR produce a single-phase response (shown in magenta, and resulting from ACh binding to the pair of high-affinity α4(+)/(-)β2 agonist binding sites. On the right, low-sensitivity isoform (α4β2)2α4-nAChR produce two response phases. One, again shown in magenta, is also produced by ACh engagement of the pair of α4(+)/(-)β2 agonist binding sites that is conserved across the two isoforms. This exhibits a similar EC50 value, and results in a similar amount of function per receptor, as is seen for the high-sensitivity isoform. Accordingly, it is named “HS-phase.” However, at higher ACh concentrations, a second and significantly-larger phase of function is observed. This emerges as a result of additional ACh binding to the low-sensitivity agonist binding pocket formed at the unique α4(+)/(-)α4 subunit interface. The combined response of (α4β2)2α4-nAChR has a characteristic biphasic appearance, and is dominated by the larger low-sensitivity phase. This has led to (α4β2)2α4-nAChR being named the low-sensitivity isoform.
	Fig 2. Unitary amplitudes and conductances associated with human HS (α4β2)2β2-nAChR and LS (α4β2)2α4-nAChR expressed in X. laevis oocytes from unlinked subunits.(A) HS (α4β2)2β2-nAChR (grey bars) open amplitudes were found to fall into a single population. (B) LS (α4β2)2α4-nAChR (green bars) single channel openings were recorded in the presence of a low ACh concentration (0.7 μM). Under these conditions, openings exhibited two characteristic amplitudes (small and large). Both the small and large amplitudes were significantly different than that associated with opening of HS (α4β2)2β2-nAChR (F2,17 = 12.38, One-way ANOVA; Tukey’s multiple comparison test indicates ** P < 0.01, and *** P < 0.001). (C) LS (α4β2)2α4-nAChR stimulated with a high ACh concentration (30 μM) also displayed two distinct amplitudes, which were statistically indistinguishable from those recorded in panel (B), using a low ACh concentration (two-tailed unpaired Student’s t-test were applied to each amplitude class; df = 11–13 and P > 0.05 in each case). Amplitude histograms are all of the events for each construct and ACh concentration collected from individual single-channel patch recordings. Example 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. (D) Single channel slope conductance was calculated for openings of HS (α4β2)2β2-nAChR by calculating the mean single-channel amplitude across several transmembrane voltages. (E) The single-channel slope conductances associated with small and large openings of LS (α4β2)2α4-nAChR (gsmall and glarge, respectively) were also significantly different from each other (**** P < 0.0001; two-tailed unpaired Student’s t-test, df = 6). The conductance value gsmall was similar to that associated with openings of the HS isoform. Values are given as mean ± S.E.M, and were collected from 5–9 patches across a minimum of three separate experiments. All recordings were performed 3—6d following cRNA injection.
	Fig 3. Unitary amplitudes and conductances associated with human HSP (α4β2)2β2-nAChR and LSP (α4β2)2α4-nAChR expressed in X. laevis oocytes from pentameric, fully-concatenated constructs (HSP and LSP, respectively).All statistical comparisons for this figure were performed using two-tailed unpaired Student’s t-tests, df = 8–13, significant differences were noted at P < 0.05. (A) HSP (α4β2)2β2-nAChR (magenta bars) stimulated with 1.3 μM ACh produced single-channel openings with a single characteristic amplitude which was indistinguishable from that measured in Fig 2A, for the same isoform expressed using unlinked subunits. (B) LSP (α4β2)2α4-nAChR (cyan bars) single-channel openings were recorded in the presence of a low ACh concentration (0.7 μM). These openings fell into two amplitude classes (small and large), associated with values indistinguishable from those determined in Fig 2B, for the same isoform expressed using unlinked subunits. (C) LSP (α4β2)2α4-nAChR were stimulated with a high ACh concentration (30 μM). Openings displayed two distinct amplitudes, which were statistically indistinguishable from those recorded in panel (B), using a low ACh concentration. Amplitude histograms are all of the events for each construct and ACh concentration from individual single-channel patch recordings. As in Fig 2, example traces are shown below panels (A), (B), and (C), which display a mixture of individual openings and short bursts of activity, interspersed with longer periods of inactivity. (D) Single-channel slope conductance was calculated for openings of HSP (α4β2)2β2-nAChR by calculating the mean single-channel amplitude across several transmembrane voltages. The value obtained was not statistically different to that recorded from (α4β2)2β2-nAChR expressed from unlinked subunits. (E) The single-channel slope conductances associated with small and large openings of LSP (α4β2)2α4-nAChR (gsmall and glarge, respectively) were also determined. The conductance value gsmall was significantly lower than that associated with openings of the HSP isoform (* P < 0.05) and significantly lower than that associated with the large openings of the LSP isoform (** P <0.01). Conductances of small and large amplitude events expressed using the concatemer approach did not differ from events produced by the loose subunit technique. Values are given as mean ± S.E.M, and were collected from 5–6 patches across a minimum of three separate experiments. All recordings were performed 3—6d following cRNA injection.
	Fig 4. Closed and open dwell durations associated with human HS or LS α4β2-nAChR isoforms expressed in X. laevis oocytes from unlinked subunits.HS (α4β2)2β2-nAChR stimulated with ACh (1.3 μM). Closed durations between openings were best described with a pair of time constants (A), as were open durations (D). When LS (α4β2)2α4-nAChR were stimulated at a low ACh concentration (0.7 μM), closed durations between openings were best described with three time constants (B), while open durations were best fit using two time constants (E). The numbers of time constants required to best fit closed (C) and open (F) time distributions did not change as LS isoform nAChR were stimulated at a higher ACh concentration (30 μM), but the closed dwell times durations did shorten significantly (see Table 1). The open duration constants did show a trend towards shortening as the ACh concentration was increased, but this did not reach significance in this preliminary analysis (which did not discriminate between open events within and outside of bursts). Closed and open 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 and open durations from an example patch recording have been inserted into each panel to facilitate interpretation. Data were collected from 6–9 individual patches, across at least three separate experiments. Calculated parameters are summarized in Table 1, together with the statistical analyses applied.
	Fig 5. Closed and open durations associated with human HSP or LSP α4β2-nAChR isoforms expressed in X. laevis oocytes from pentameric, fully-concatenated constructs.HSP (α4β2)2β2-nAChR were stimulated with ACh (1.3 μM). Closed dwell durations between openings were best described with three time constants (A), while open durations were best described with a pair of time constants (D). When LSP (α4β2)2α4-nAChR were stimulated with a low ACh concentration (0.7 μM), closed durations between openings were best described with four time constants (B), while individual-event open times were best fit using three time constants (E). As was seen for unlinked subunits, increasing the ACh concentration to 30 μM did not change the number of time constants required to best fit closed (C) and open (F) duration distributions, but shortening of closed dwell durations was observed (see Table 2). Open dwell duration values did significantly shorten at the higher ACh concentration (Table 2). Closed and open dwell duration histograms are representative examples resulting from analysis of individual single-channel patch recordings. Individual τ values and percentage of total events corresponding to each closed and open duration from an example patch recording have been inserted into each panel to facilitate interpretation. Data were collected from 5–8 individual patches, across at least three separate experiments. Calculated parameters are summarized in Table 2, together with the statistical analyses applied.
	Fig 6. Closed durations between burst activity of human HS or LS α4β2-nAChR isoforms.Closed dwell durations between bursts of activity (interburst durations) were measured for HS or LS α4β2-nAChR isoforms expressed in X. laevis oocytes using unlinked subunits. (A) For HS (α4β2)2β2-nAChR, interburst closed durations evoked by 1.3 μM ACh were best described using four time constants. The same was true for closed durations between bursts of either small (B) or large amplitude (B’) events evoked from LS (α4β2)2α4-nAChR at a low ACh concentration (0.7 μM). Increasing the ACh concentration to 30 μM did not change the number of time constants associated with LS (α4β2)2α4-nAChR closed state intervals between bursts of small (C) or large (C’) events, but did result in significant shortening of the time constants in each case. Closed and open dwell duration pooled histograms are shown, which result from collection of data from 6–7 individual patches, across at least three separate experiments. The calculated τ values are summarized in Table 3, together with the statistical analyses applied.
	Fig 7. Durations of individual openings within bursts of small and large amplitude openings of human HS or LS α4β2-nAChR isoforms.HS or LS isoforms were expressed in X. laevis oocytes using unlinked subunits, and durations of individual openings within bursts (intraburst) of activity were measured. (A) HS (α4β2)2β2-nAChR stimulated with 1.3 μM ACh exhibited intraburst openings with a single, small, characteristic amplitude, as described previously. The open dwell durations associated with these openings were best described using a pair of time constants. For LS (α4β2)2α4-nAChR stimulated with a low ACh concentration (0.7 μM), bursts of either small (B) or large amplitude (B’) events were seen. Further, each type of burst was associated with a single, characteristic, intraburst duration of individual openings. The open durations for individual events within small amplitude bursts were significantly shorter than those associated with individual events within large amplitude bursts. When the ACh concentration was increased to 30 μM, the same general outcome was found. Intraburst open durations of individual openings of LS (α4β2)2α4-nAChR within small (C) and large amplitude (C’) bursts remained associated with single time constants, and the durations of small amplitude individual openings continued to be shorter than those of large amplitude individual openings. However, the durations of both event classes were significantly shortened in the presence of the higher ACh concentration. Histogram panels each show pooled data, which were collected from 6–7 individual patches, across at least three separate experiments. Representative traces of each category of bursts are shown below the corresponding histogram panels. Scale bars are 1 pA in height, and 10 ms in width. The calculated τ values are summarized in Table 3, together with the statistical analyses applied.
	Fig 8. ACh unitary amplitudes and conductances associated with human LSP α4P5W182A-nAChR expressed in X. laevis oocytes from pentameric, fully-concatenated constructs.All statistical comparisons for this figure were performed using two-tailed unpaired Student’s t-tests, df = 11–12, significant differences were noted at P < 0.05. (A) LSP α4P5W182A-nAChR were stimulated with a low ACh concentration (0.7 μM ACh). Evoked unitary responses produced openings that fell into two amplitude classes (small and large). These were statistically indistinguishable from those observed from LSP α4β2-nAChR constructs that did not harbor the α4P5W182A, when these were stimulated with the same concentration of ACh. (Fig 3B). (B) LSP α4P5W182A-nAChR were stimulated with a high (30 μM) ACh concentration. Single-channel openings displayed two amplitude classes that were indistinguishable from those in panel A using a low ACh concentration, and those of LSP-nAChR produced using the same high ACh concentration (Fig 3C). Example traces are shown below panels (A) and (B), demonstrating a mixture of individual openings at each amplitude level, and short bursts of activity followed by longer periods of inactivity. Values are given as mean ± S.E.M. Amplitude Data were collected across a minimum of three separate experiments from 7 patches, across a minimum of three separate experiments. All recordings were performed 3—6d following cRNA injection.
	Fig 9. Closed dwell durations between small and large amplitude bursts of human LSP-α4p5W182A-nAChR.LSP (α4β2)2α4-nAChR were expressed in X. laevis oocytes from a concatenated cDNA construct in which the critical tryptophan-182 amino-acid was mutated to alanine (W182A) in only the low-affinity α4/α4-interface agonist binding site (red bars). Closed durations between bursts of activity in the presence of a low ACh concentration (0.7 μM) were determined for both small (A) and large amplitude (A’) bursts. As for the parent LSP construct, interburst closed durations at the low ACh concentration were best described using four time constants, regardless of whether small or large amplitude bursts were considered. The effects of changing to a higher ACh concentration (30 μM) on interburst closed durations differed between the two amplitude classes of bursts. For bursts of small amplitude events (B), the number of time components was reduced to three, likely due to disappearance of the second-shortest time component (τS2). In this interpretation, the shortest time constant increased significantly while small, but significant, shortenings of the two longest time constants (τS3, τS4) were also seen. These shortenings were much less dramatic than those observed for the parent LSP construct (compare to Fig 6). For bursts of large amplitude events (B’), the increased ACh concentration did not alter the number of interburst closed dwell duration components detected. However, each time constant was significantly lengthened; this is the opposite of the behavior of the parent LSP construct. Data were collected from 7 individual patches, across at least three separate experiments, for each panel of the Fig. The calculated τ values are summarized in Table 4, together with the statistical analyses applied.
	Fig 10. Durations of individual openings within small or large amplitude event bursts of human LSP-α4p5W182A-nAChR.LSP (α4β2)2α4-nAChR harboring the α4p5W182A mutation, which significantly reduces macroscopic LSP function, were expressed in X. laevis oocytes using a concatenated cDNA construct. Similar to the parent LSP construct, when stimulated with a low ACh concentration (0.7 μM), bursts of either small (A) or large amplitude (A’) events were seen, and individuals openings within each type of burst were associated with a single, characteristic, open duration. Also as seen for the parent construct, the intraburst open durations for individual events within small amplitude bursts were significantly shorter than those associated with individual events within large amplitude bursts. In a further similarity to the parent construct, when the ACh concentration was increased to 30 μM, intraburst open durations of individual openings of LSP (α4β2)2α4-nAChR within small (B) and large amplitude (B’) bursts remained associated with single time constants, and the individual openings with small amplitude bursts continued to be shorter than those within large amplitude bursts. However, in direct opposition to the outcome for the unmutated LSP construct, durations of both small and large amplitude individual openings were significantly lengthened in the presence of the higher ACh concentration. Intraburst open duration τ values have been inserted into each panel to facilitate interpretation. Representative examples of bursts obtained from this mutant construct are shown below the corresponding histogram panels. Scale bars are 1 pA in height, and 10 ms in width. Data were collected from 7 individual patches, across at least three separate experiments. The calculated τ values are summarized in Table 4, together with the statistical analyses applied.
	Fig 11. Human LS α4β2-nAChR small and large amplitude burst parameters.LS α4β2-nAChR isoforms were expressed in X. laevis oocytes using unlinked subunits and burst parameters were measured. Comparisons were made using Student’s two-tailed, unpaired, t-tests (df = 9–11, significant differences noted at p < 0.05). A) The mean proportion of small or large amplitude openings falling within bursts, compared to the total number of each type of open event, was determined at a low ACh (0.7 μM) and a high ACh (30 μM) concentration. For both small and large amplitude openings, the proportion of events within bursts of the corresponding amplitude appeared to increase with application of a high ACh concentration. A significant increase in the small amplitude burst proportion was observed with a high ACh concentration compared to the low ACh concentration (*). B) The mean number of openings per burst of LS isoform small or large amplitude openings was determined at both the low and high ACh concentrations. Altering the concentration of ACh had no significant effect on the numbers of openings within either class of bursts. C) The durations of LS isoform small or large amplitude bursts were measured at the low and high ACh concentrations. Increasing the ACh concentration significantly shortened the duration of both small and large amplitude bursts (*). D) The Popen within bursts was indistinguishable between bursts of small or large amplitude events, and was not significantly altered by an increase in applied ACh concentration from 0.7 μM to 30 μM.
	Fig 12. Comparison of mechanisms considered to explain the observed functional effects of ACh binding at the α4(+)/(-)α4 agonist binding site within LS-isoform α4β2-nAChR.In all panels, red shapes denote receptors in a closed state while bound by ACh. Intermediate “flip” states (also closed) are represented by orange shapes, while open states are indicated with green shapes (low- and high-conductance open states are shown as small and large black dots, respectively, within these green shapes). Where each model suggests emergence of a distinctly-different pathway, shapes change from circles to octagons. Receptor transitions between states are indicated with double-ended arrows, and bolder lines indicate increased probability of that transition occurring. ACh bound to the pair of canonical, high-affinity, α4(+)/(-)β2 agonist binding pockets is symbolized by blue stars, and a blue pentagon represents ACh bound to the low-affinity α4(+)/(-)α4 agonist binding site uniquely found within LS-isoform α4β2-nAChR. A) Since high-conductance openings are uniquely associated with LS-isoform α4β2-nAChR, the simplest potential explanation is that ACh binding to the α4(+)/(-)α4 agonist binding site directly produces entry into the high-conductance state. While this mechanism can explain the observation that almost all bursts contain either low or high-conductance openings, it is refuted since it predicts that the ratio of high- to low-conductance individual openings (and bursts thereof) should rise as the ACh concentration is increased. B) In contrast, a mechanism in which ACh binding to the α4(+)/(-)α4 agonist binding site produces increased entry into a common “flip” state would be expected to produce the observed phenomenon of increased bursting at a higher ACh concentration, while also maintaining similar ratios of high- to low-conductance individual openings across a range of ACh concentrations. However, this mechanism is refuted since it predicts that returning from either open state to the common “flip” state within a burst would be followed by a fixed probability of next moving to either of the two observed open-conductance states. In turn, this would be expected frequently to produce bursts typically containing mixtures of low- and high-conductance individual openings. C) A mechanism containing two distinct flip states, each of which is linked to only one of the observed low- or high-conductance open states is compatible with the highly-correlated conductance states of individual openings observed within bursts of LS-isoform α4β2-nAChR. In this mechanism, ACh binding to the α4(+)/(-)α4 agonist binding site similarly enhances entry into either “flip” state, which explains the remaining observed features of similar ratios of high- to low-conductance individual openings (and bursts thereof) at higher ACh concentrations.

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