Nicotine up-regulates α4β2 nicotinic receptors and ER exit sites via stoichiometry-dependent chaperoning

May combine two claims.

The ER fluorescence in α4eGFP plus β2wt-transfected cells was two- to threefold brighter than PM and displayed little to no vesicular dynamics.

“The ER fluorescence in α4eGFP plus β2wt–transfected cells was two- to threefold brighter than PM and displayed little to no vesicular dynamics (Video 1).”

May combine two claims.

The vesicles in α4eGFP plus β4wt-transfected cells were pleomorphic, with diameters ranging from 0.5 to 1 μm, some elongated 2 to 5 μM, and either stationary or showing spatial displacements of 0.5-5 μm/s.

“Vesicles were pleomorphic, with diameters ranging from 0.5 to 1 µm. Some vesicles appeared as elongated structures ranging from 2 to 5 µM in length. Vesicles were either stationary or showed spatial displacements of 0.5–5 µm/s within the TIRF footprint.”

May combine two claims.

The footprint/ER ratio was 1.69 for α4eGFP plus β2wt and 5.01 for α4eGFP plus β4wt-transfected cells, indicating an approximately threefold increase in ER-localized nAChRs for α4β2.

“Measured ratios revealed an approximately threefold increase in ER-localized nAChRs for α4eGFP plus β2wt (footprint/ER ratio = 1.69) when compared with α4eGFP plus β4wt–transfected cells (footprint/ER ratio = 5.01; Fig. 1 D, 3).”

May combine two claims.

α4eGFP plus β4wt-transfected cells showed much less reticulate fluorescence and displayed vesicles within the TIRF footprint.

“In contrast, α4eGFP plus β4wt–transfected cells showed much less reticulate fluorescence, but these cells also displayed vesicles within the TIRF footprint (Video 2).”

Classified as probably not a substantial claim. May combine two claims.

The β2enhanced-ER-export mutant subunit mimics two regions of the β4 subunit sequence: the presence of an ER export motif and the absence of an ER retention/retrieval motif.

“To aid in the mechanistic analysis of these phenomena, we generated a β2enhanced-ER-export mutant subunit that mimics two regions of the β4 subunit sequence: the presence of an ER export motif and the absence of an ER retention/retrieval motif.”

May combine two claims.

In control solutions, the footprint/ER ratio for wild-type nAChRs was 3.42; nicotine treatment reduced the footprint/ER ratio to 2.19.

“In control solutions, the footprint/ER ratio was 3.42; nicotine treatment reduced the footprint/ER ratio to 2.19.”

May combine two claims.

Neuro-2a cells expressing α4-eGFPβ2-365AAQA368 nAChRs show an increase in the number of PM-localized pixels, an effect enhanced after treatment with 0.1 μM nicotine for 48 h.

“Neuro-2a cells expressing α4-eGFPβ2-365AAQA368 nAChRs showed an increase in the number of PM-localized pixels, an effect that was enhanced after treatment with 0.1 µM nicotine for 48 h.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Two ER fluorescence peaks are observed in Fig. 1 D (1).

“Two ER fluorescence peaks are observed in Fig. 1 D (1).”

May combine two claims.

The footprint/ER ratio for α4-eGFPβ2-L349M is 2.09, a 1.25-fold increase over α4-eGFPβ2wt (1.69), whereas α4L358A-eGFPβ2wt-transfected cells show a small reduction (1.43).

“Results showed a 1.25-fold increase in the ratio of α4-eGFPβ2-L349M (footprint/ER ratio = 2.09) as compared with α4-eGFPβ2wt nAChRs (footprint/ER ratio = 1.69), whereas α4L358A-eGFPβ2wt–transfected cells showed a small reduction in the integrated density ratio (footprint/ER ratio = 1.43).”

Classified as probably not a substantial claim. May combine two claims.

PM-localized pCS2-mcherry fluorescence was detected as a cellular footprint with uniform fluorescence, whereas ER-Tracker Green exhibited a reticulate pattern.

“PM-localized pCS2-mcherry fluorescence was detected as a cellular footprint with uniform fluorescence, whereas ER-Tracker Green exhibited a reticulate pattern (Fig. 1 A, first and third columns).”

May combine two claims.

Receptors in ∼90% of α4L358A-eGFPβ2wt-transfected cells primarily localize to the ER, and the remaining 10% of cells do not yield clear TIRF footprints.

“In contrast, receptors in ∼90% of α4L358A-eGFPβ2wt–transfected cells primarily localized to the ER (Fig. 4 A, fourth column), and the remaining 10% of cells did not yield clear TIRF footprints.”

Classified as probably not a substantial claim.

Merged images showed that the ER fluorescence was readily distinguishable from PM-localized mcherry.

“Merged images showed that the ER fluorescence was readily distinguishable from PM-localized mcherry (Fig. 1 A, first column).”

May combine two claims.

The footprint/ER ratio for α4-eGFPβ2wt-transfected cells is 3.42, and nicotine treatment reduces it to 2.19.

“the footprint/ER ratio for α4-eGFPβ2wt transfected cells was 3.42, and nicotine treatment reduced the footprint/ER ratio to 2.19”

May combine two claims.

Human and chicken β2 subunits possess an intact LFM motif, whereas chicken β2 subunits contain an RQTQ sequence in place of the RRQR motif.

“A cross-species comparison of mouse, human, and chicken β2 subunit sequences shows that, unlike mouse subunits, human and chicken β2 subunits possess an intact LFM motif, whereas chicken β2 subunits contain an RQTQ sequence in place of the RRQR motif (Fig. 2 D, 2).”

May combine two claims.

The mouse β4 M3-M4 large intracellular loop contains an LFM motif (amino acids 344-346), whereas mouse β2 subunits possess a leucine residue at position 349, resulting in an LFL sequence at the aligning positions.

“Sequence alignments showed that the mouse β4 M3–M4 large intracellular loop contains an LFM motif (amino acids 344–346), whereas mouse β2 subunits possess a leucine residue at position 349, resulting in an LFL sequence at the aligning positions (Fig. 2 D, 1).”

Probably not a substantial claim: may not pass S3 (load-bearing).

The β2 and β4 subunit sequences are strikingly similar in all other regions.

“The β2 and β4 sequences are strikingly similar in all other regions.”

May combine two claims.

The footprint/ER ratio for α4eGFPβ2 double-mutant nAChR-transfected cells is 8.18, a 2.4-fold increase over α4eGFPβ2wt-transfected cells.

“The footprint/ER ratio for α4eGFPβ2 double-mutant nAChR-transfected cells was 8.18, a 2.4-fold increase over the ratio observed for α4eGFPβ2wt-transfected cells.”

May combine two claims.

Compared with wild-type receptors, α4-eGFPβ2-365AAQA368 nAChRs show a 1.74-fold increase in PM intensity, almost equivalent to that observed for nicotine-treated α4-eGFPβ2wt nAChRs.

“When compared with wt receptors, α4-eGFPβ2-365AAQA368 nAChRs showed a 1.74-fold increase in the PM intensity, almost equivalent to that observed for nicotine-treated α4-eGFPβ2wt nAChRs.”

May combine two claims.

Unlike wild-type α4eGFPβ2 nAChRs, which show little or no vesicular dynamics, α4eGFPβ2-enhanced ER export double-mutant receptors display many 0.5-1-μM vesicles trafficking within the TIRFM footprint.

“Unlike wt α4eGFPβ2 nAChRs, which showed little or no vesicular dynamics (Video 1), α4eGFPβ2-enhanced ER export double-mutant receptors displayed many 0.5–1-µM vesicles trafficking within the TIRFM footprint (Video 3).”

May combine two claims.

β2-365AAQA368 subunits increase PM localization of nAChRs, and this effect is enhanced after exposure to nicotine.

“β2-365AAQA368 subunits increased PM localization of nAChRs, and this effect was enhanced after exposure to nicotine.”

May combine two claims.

50% of α4-eGFPβ2-L349M-transfected cells show mixed ER plus PM localization, and the remaining 50% show TG/TGN- and PM-localized nAChRs.

“50% of α4-eGFPβ2-L349M–transfected cells showed a mixed ER plus PM localization, and the remaining 50% showed TG/TGN- and PM-localized nAChRs (Fig. 4 A, second and third columns).”

May combine two claims.

The footprint/ER ratio for α4-eGFPβ2wt-transfected cells is 3.42, whereas nicotine treatment causes a significant reduction in peripheral ER pixels of α4-eGFPβ2wt receptors (footprint/ER ratio = 2.19).

“The footprint/ER ratio for α4-eGFPβ2wt–transfected cells was 3.42, whereas nicotine treatment caused a significant reduction in the peripheral ER pixels of α4-eGFPβ2wt receptors (footprint/ER ratio = 2.19).”

May combine two claims.

α4β2 nAChRs containing the β2-enhanced-ER-export double-mutant subunit localize strongly to the plasma membrane even in the absence of nicotine, and nicotine produces only a modest further up-regulation at the plasma membrane.

“The α4β2enhanced-ER-export nAChRs localize strongly to the PM, even in the absence of nicotine; nicotine produces only a modest further up-regulation at the PM.”

May combine two claims.

Compared with non-nicotine-treated wt α4-eGFPβ2 nAChRs, the PM fraction of α4-eGFPβ2-365AAQA368 receptors is increased by 2.4-fold (footprint/ER ratio = 5.33), whereas nicotine treatment results in a further 1.6-fold increase (footprint/ER ratio = 8.53).

“When compared with non-nicotine–treated wt α4-eGFPβ2 nAChRs, the PM fraction of α4-eGFPβ2-365AAQA368 receptors was increased by 2.4-fold (footprint/ER ratio = 5.33), whereas nicotine treatment resulted in a further 1.6-fold increase in PM-localized α4-eGFPβ2-365AAQA368 receptors (footprint/ER ratio = 8.53).”

May combine two claims.

α4-eGFPβ2-L349M nAChRs show a 1.9-fold increase in PM-integrated density over α4-eGFPβ2wt nAChRs, whereas α4L358A-eGFPβ2wt nAChRs show a 2.9-fold reduction in PM-integrated density compared with α4-eGFPβ2wt nAChRs.

“The α4-eGFPβ2-L349M nAChRs showed a 1.9-fold increase in the PM-integrated density over α4-eGFPβ2wt nAChRs, whereas α4L358A-eGFPβ2wt nAChRs showed a 2.9-fold reduction in PM-integrated density when compared with α4-eGFPβ2wt nAChRs.”

May combine two claims.

These motifs modulate the stoichiometry and ER exit of α4β2 nAChRs.

“Furthermore, we identify novel motifs governing the ER localization of α4β2 nAChRs within the β2 subunit M3–M4 large intracellular loop and show that these motifs modulate the stoichiometry and ER exit of α4β2 nAChRs.”

May combine two claims.

The β2-enhanced-ER-export double-mutant subunit contains a disrupted ER retention motif (AAQA) and a reconstituted ER export motif (LFM).

“the “enhanced ER export” mouse β2 double-mutant subunit (β2-365AAQA368 L349M) contains a disrupted ER retention motif (AAQA) and a reconstituted ER export motif (LFM).”

May combine two claims.

In the TG/TGN, A1/(A1 + A2) is 0.36 for α4β2wt, whereas α4β2-L349M and α4β2-365AAQA368 nAChRs yield 0.69 and 0.73, respectively.

“As noted above, in the TG/TGN, we observed A1/(A1 + A2) = 0.36 for α4β2wt; on the other hand, α4β2-L349M and α4β2-365AAQA368 nAChRs yielded 0.69 and 0.73, respectively.”

May combine two claims.

α4-eGFPβ2-365AAQA368-transfected cells show an average of ∼38 ERES per cell, significantly higher than α4-eGFPβ2wt-transfected cells (∼20 ERES per cell; P < 0.01).

“In parallel experiments, quantification of ERES number in α4-eGFPβ2-365AAQA368–transfected cells showed an average of ∼38 ERES per cell, which was significantly higher than α4-eGFPβ2wt–transfected cells (∼20 ERES per cell; P < 0.01; Fig. 6 D).”

May combine two claims.

nAChRs appear near ERES and move near, through, or past ERES.

“nAChRs do appear near ERES; furthermore, nAChRs seem to move near and, in some cases, through ERES or past ERES (Video 4).”

Probably not a substantial claim: may not pass S3 (load-bearing).

The time scale of the calculations is determined primarily by the rates of insertion and removal from the surface PM, k_Golgi→PM and k_S→L.

“The time scale of the calculations is determined primarily by the rates of insertion and removal from the surface PM, k_Golgi→PM and k_S→L.”

• AssumptionThe authors state this as an assumption: The mutation-sensitive step in ER exit has a limited flux capacity, so its effective rate constant decreases with increasing numbers of nAChR molecules.
• AssumptionThe authors state this as an assumption: The pool of export machinery can be depleted.
• AssumptionThe authors state this as an assumption: Several dozen export machinery–nAChR complexes presumably form each ERES or transport vesicle.
• AssumptionRests on an assumption: Nicotine binds at the subunit interface and thereby aids nAChR assembly.
• AssumptionThe authors state this as an assumption: The anterograde rate k_Folded→Golgi is zero.
• AssumptionThe authors state this as an assumption: All other steps in SePhaChARNS 2.0 follow first-order unidirectional kinetics, governed by rate constants with dimensions of s−1, as in the FOLDEX model.
• AssumptionThe authors state this as an assumption: The simulations force all molecular concentrations to scale with that of C1 unfolded, the initial constant source of unassembled, unfolded subunits.
• AssumptionThe authors state this as an assumption: The effect of nicotine is formalized as a binding step that helps nAChRs fold and assemble fully.
• AssumptionThe authors state this as an assumption: If further saturable steps do not occur, step 8 would govern the dose–response relation for up-regulation.

May combine two claims.

A1/(A1 + A2) for α4β2-L349M and α4β2-365AAQA368 receptors are greater (0.70 and 0.63, respectively) than for α4β2wt, indicating an increased proportion of nAChRs possessing (α4)2(β2-L349M)3 and (α4)2(β2-365AAQA368)3 stoichiometry.

“However A1/(A1 + A2) for α4β2-L349M and α4β2-365AAQA368 receptors were greater (0.70 and 0.63, respectively), indicating an increased proportion of nAChRs possessing a (α4)2(β2-L349M)3 and (α4)2(β2-365AAQA368)3 stoichiometry.”

May combine two claims.

In whole cell section ROI, nicotine increases A1/(A1 + A2) for (α4)2(β2-L349M)3 from 0.70 to 0.79 and for (α4)2(β2-365AAQA368)3 from 0.67 to 0.70.

“For (α4)2(β2-L349M)3 nAChR pixels, nicotine increased A1/(A1 + A2) from 0.70 to 0.79. The analogous effect for the (α4)2(β2-365AAQA368)3 nAChR pixels was a change from 0.67 to 0.70.”

Classified as probably not a substantial claim.

The exemplar parameters in Tables II and III provide a good fit to the six target measures.

“Tables II and III give the exemplar parameters that provide a good fit to the six target measures.”

• AssumptionThe authors state this as an assumption: The mutation-sensitive step in ER exit has a limited flux capacity, so its effective rate constant decreases with increasing numbers of nAChR molecules.
• AssumptionThe authors state this as an assumption: The pool of export machinery can be depleted.
• AssumptionThe authors state this as an assumption: Several dozen export machinery–nAChR complexes presumably form each ERES or transport vesicle.
• AssumptionRests on an assumption: Nicotine binds at the subunit interface and thereby aids nAChR assembly.
• AssumptionThe authors state this as an assumption: The anterograde rate k_Folded→Golgi is zero.
• AssumptionThe authors state this as an assumption: All other steps in SePhaChARNS 2.0 follow first-order unidirectional kinetics, governed by rate constants with dimensions of s−1, as in the FOLDEX model.
• AssumptionThe authors state this as an assumption: The simulations force all molecular concentrations to scale with that of C1 unfolded, the initial constant source of unassembled, unfolded subunits.
• AssumptionThe authors state this as an assumption: The effect of nicotine is formalized as a binding step that helps nAChRs fold and assemble fully.
• AssumptionThe authors state this as an assumption: If further saturable steps do not occur, step 8 would govern the dose–response relation for up-regulation.

May combine two claims.

∼17 ERES per cell are observed with α4L358A-eGFPβ2wt receptors, whereas α4-eGFPβ2wt receptors show ∼30 ERES per cell.

“∼17 ERES per cell were observed with α4L358A-eGFPβ2wt receptors, whereas α4-eGFPβ2wt receptors showed ∼30 ERES per cell.”

May combine two claims.

Both incubation in nicotine and specific trafficking motifs within the β2 M3-M4 loop can influence the subcellular localization and trafficking of assembled nAChRs.

“Our data show that both incubation in nicotine and specific trafficking motifs within the β2 M3–M4 loop can influence the subcellular localization and trafficking of assembled nAChRs.”

May combine two claims.

α4-eGFPβ2-L349M receptors show ∼52 ERES per cell, significantly different from α4-eGFPβ2wt receptors (P < 0.001).

“α4-eGFPβ2-L349M receptors showed ∼52 ERES per cell, which was significantly different from α4-eGFPβ2wt receptors (two-tailed t test; P < 0.001).”

May combine two claims.

Incubation in nicotine, or manipulation of ER export and ER retention/retrieval motifs in the M3-M4 loop of nAChRs, increases nAChR PM expression.

“Our TIRFM experiments show that incubation in nicotine, or manipulation of ER export and ER retention/retrieval motifs in the M3–M4 loop of nAChRs, increases nAChR PM expression.”

May combine two claims.

In all cases, 48-h nicotine exposure increases the proportion of nAChRs with a (α4)2(β2)3 stoichiometry, most markedly with wt nAChRs.

“In all cases, 48-h nicotine exposure increased the proportion of nAChRs with a (α4)2(β2)3 stoichiometry. This increase was most marked with wt nAChRs, which show the weakest bias toward the (α4)2(β2)3 stoichiometry in the absence of nicotine”

May combine two claims.

Coexpression of Sec24D, even with wt nAChRs and in the absence of nicotine, produces additional dynamic features in the nAChR population, as though the addition of Sec24D increases the proportion of nAChRs exiting the ER.

“TIRF videos of Neuro-2a cells cotransfected with α4-mcherry plus β2wt plus Sec24D-eGFP showed that coexpression of Sec24D, even with wt nAChRs and in the absence of nicotine, produced additional dynamic features in the nAChR population, as though the addition of Sec24D increases the proportion of nAChRs exiting the ER.”

Classified as probably not a substantial claim. May combine two claims.

In the model, k_Golgi→PM is the slowest step (10−4 s−1) and governs the simulated time course of PM up-regulation.

“In our formulation, this is the slowest step (k_Golgi→PM = 10−4 s−1) and governs the simulated time course of PM up-regulation (shown in Fig. 7 B).”

• AssumptionThe authors state this as an assumption: The mutation-sensitive step in ER exit has a limited flux capacity, so its effective rate constant decreases with increasing numbers of nAChR molecules.
• AssumptionThe authors state this as an assumption: The pool of export machinery can be depleted.
• AssumptionThe authors state this as an assumption: Several dozen export machinery–nAChR complexes presumably form each ERES or transport vesicle.
• AssumptionRests on an assumption: Nicotine binds at the subunit interface and thereby aids nAChR assembly.
• AssumptionThe authors state this as an assumption: The anterograde rate k_Folded→Golgi is zero.
• AssumptionThe authors state this as an assumption: All other steps in SePhaChARNS 2.0 follow first-order unidirectional kinetics, governed by rate constants with dimensions of s−1, as in the FOLDEX model.
• AssumptionThe authors state this as an assumption: The simulations force all molecular concentrations to scale with that of C1 unfolded, the initial constant source of unassembled, unfolded subunits.
• AssumptionThe authors state this as an assumption: The effect of nicotine is formalized as a binding step that helps nAChRs fold and assemble fully.
• AssumptionThe authors state this as an assumption: If further saturable steps do not occur, step 8 would govern the dose–response relation for up-regulation.

May combine two claims.

α4β2wt assembles as (α4)2(β2wt)3 and (α4)3(β2wt)2 receptors with a nearly equal proportion of pixels containing the two stoichiometries (A1/(A1 + A2) = 0.51).

“As noted above, α4β2wt assembled as (α4)2(β2wt)3 and (α4)3(β2wt)2 receptors, with a nearly equal proportion of pixels containing the two stoichiometries: A1/(A1 + A2) = 0.51.”

May combine two claims.

Human β2 subunits contain the LFM ER export motif as well as the RRQR motif.

“Interestingly, human β2 subunits contain the LFM ER export motif as well as the RRQR motif, whereas chicken α4β2 nAChRs contain the LFM motif but do not possess the RRQR motif (Fig. 2 D, 2).”

May combine two claims.

Chicken α4β2 nAChRs contain the LFM motif but do not possess the RRQR motif.

“Interestingly, human β2 subunits contain the LFM ER export motif as well as the RRQR motif, whereas chicken α4β2 nAChRs contain the LFM motif but do not possess the RRQR motif (Fig. 2 D, 2).”

The quote only partly states this claim.

The model was unsuccessful in simulating the observed 1.86-fold increase in ERES sites in the enhanced ER export β2 double mutant in the absence of nicotine, unless k_F→ERES for wt nAChR was ∼10-fold lower than the exemplar parameter value.

“Here, the model was unsuccessful, simulating the observed increase in ERES numbers only if k_F→ERES for the wt nAChR was ∼10-fold lower than the exemplar parameter value.”

May combine two claims.

Nicotine up-regulated PM α4β2-wt nAChRs, despite markedly increasing the proportion of ER-localized α4β2-wt nAChRs.

“TIRFM experiments recapitulated that nicotine up-regulated PM α4β2-wt nAChRs, despite markedly increasing the proportion of ER-localized α4β2-wt nAChRs (Fig. 2, B and C).”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

With n = 10, the simulations were marginally acceptable (sum of squared residuals = 0.0127, with all predicted values in error by <7% difference).

“With n = 10 (an endogenous “effective nicotine concentration” 10-fold lower that the experimental value of 0.1 µM), the simulations were marginally acceptable (sum of squared residuals = 0.0127, with all predicted values in error by <7% difference).”

May combine two claims.

The data argue against roles for Ca2+-induced second messenger signaling cascades and transcriptional processes in nicotine-induced nAChR up-regulation.

“These data argue against roles for Ca2+-induced second messenger signaling cascades and transcriptional processes in nicotine-induced nAChR up-regulation.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

RRQR motifs are thought to prevent the exit of cargo from the ER and may additionally function as ER retrieval motifs via the binding of the RRXR domain to COPI proteins.

“RRQR motifs are thought to prevent the exit of cargo from the ER and may additionally function as ER retrieval motifs via the binding of the RRXR domain to COPI proteins (Scott et al., 2001, 2003; Brock et al., 2005; Michelsen et al., 2005).”

• cites Scott et al., 2001, 2003Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

• cites Brock et al., 2005 sourceNot checked yet

  Not checked in the free run.

• cites Michelsen et al., 2005 sourceNot checked yet

  Not checked in the free run.

May combine two claims.

LFM motifs in the M3-M4 loop of α4 and β2 subunits alter the ratio of receptors localized within the cellular PM versus the ER, and the PM nAChR number increases monotonically with the number of LFM export motifs present in receptor pentamers.

“Collectively, these results demonstrate that LFM motifs in the M3–M4 loop of α4 and β2 subunits alter the ratio of receptors localized within the cellular PM versus the ER, and that the PM nAChR number increases monotonically with the number of LFM export motifs present in receptor pentamers.”

May combine two claims.

Mutation of the β2 ER retention/retrieval motif interacts with nicotine treatment to further up-regulate nAChRs at the PM and increases the export of nAChRs from the ER, causing a net depletion of ER-localized receptors.

“Mutation of this motif interacts with nicotine treatment to further up-regulate nAChRs at the PM and increases the export of nAChRs from the ER, thus causing a net depletion of ER-localized receptors.”

May combine two claims. The quote only partly states this claim.

The TIRF data in Figs. 1-4 are robust to two alternative analyses: cell-by-cell calculations and pixel counting, yielding similar patterns and significance.

“Differences calculated with this method were also significant when the pixel-based analyses showed significance.”

• Also stated in: § Alternative analyses of the TIRFM data · plain

  “Again, we found patterns similar to those of Figs. 1–4. Thus, the data of Figs. 1–4 satisfy two additional tests.”

Probably not a substantial claim: may not pass S3 (load-bearing).

The time scale (48 h) is relevant to the initial stages of nicotine-induced up-regulation in the brains of mice.

“The time scale is also relevant to the initial stages of nicotine-induced up-regulation in the brains of mice (Marks et al., 1985).”

• cites Marks et al., 1985 sourceNot checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

The nicotine concentration used (0.1 μM) is somewhat less than that in the blood within a few minutes after a human smokes a cigarette and perhaps more appropriate to average values during a day of smoking.

“The experiments use a nicotine concentration (0.1 µM) somewhat less than that in the blood within a few min after a human smokes a cigarette, and perhaps more appropriate to the average values during a day of smoking (Matta et al., 2007).”

• cites Matta et al., 2007 sourceNot checked yet

  Not checked in the free run.

May combine two claims.

The β2 subunit possesses an ER export motif (LFM) and an ER retention/retrieval motif (RRQR) that can influence the anterograde trafficking of β2* nAChRs.

“the β2 subunit possesses an ER export motif (LFM) and an ER retention/retrieval motif (RRQR) that can influence the anterograde trafficking of β2* nAChRs”

May combine two claims.

β2-365AAQA368 subunits markedly increase the localization of receptors in the PM and reduce their localization in the ER, consistent with the presence of an ER retention/retrieval motif in the mouse β2 M3-M4 loop.

“These data show that β2-365AAQA368 subunits markedly increase the localization of receptors in the PM and reduce their localization in the ER, consistent with the presence of an ER retention/retrieval motif in the mouse β2 M3-M4 loop.”

May combine two claims.

The data support the mechanism that (α4)2(β2)3 stoichiometry would languish in the ER, but nicotine acts as a pharmacological chaperone to stabilize (α4)2(β2)3 nAChRs and presumably, as a consequence, ERES.

“The data strongly support the following mechanism: the (α4)2(β2)3 stoichiometry would languish in the ER, but nicotine acts as a pharmacological chaperone to stabilize (α4)2(β2)3 nAChRs and presumably, as a consequence, ERES. The result: nAChRs reach the PM in greater numbers.”

• AssumptionThe authors state this as an assumption: One or more mechanisms common to all cellular systems govern nicotine-induced nAChR up-regulation.

Classified as probably not a substantial claim. May combine two claims.

I/LXM-packaging motifs in ER cargo such as membrin and syntaxin 5 bind to a surface groove of Sec24D, a major component of COPII vesicles, and this binding partially enables packaging of ER cargo into COPII vesicles at ERES.

“Previous x-ray crystallographic and biochemical studies show that I/LXM (where X is any amino acid)-packaging motifs in ER cargo such as membrin and syntaxin 5 bind to a surface groove of Sec24D, which is a major component of COPII vesicles (Mancias and Goldberg, 2008).”

• cites Mancias and Goldberg, 2008Not checked yet

  Not checked in the free run.

May combine two claims.

Under the transient transfection conditions used in Neuro-2a cells, membrane protein expression levels have not reached maximum, rendering the optical assays sensitive to mutations or pharmacological agents.

“Previous studies using both fluorescent gamma amino butyric acid transporters and fluorescent nAChRs established that, under the conditions used, membrane protein expression levels have not reached maximum (Drenan et al., 2008; Imoukhuede et al., 2009; Moss et al., 2009; Son et al., 2009).”

• Also stated in: § α4β2 nAChR up-regulation monitored optically · plain

  “These conditions render the optical assays appropriately sensitive to mutations or pharmacological agents.”

• cites Drenan et al., 2008 sourceNot checked yet

  Not checked in the free run.

• cites Imoukhuede et al., 2009 sourceNot checked yet

  Not checked in the free run.

• cites Moss et al., 2009; Son et al., 2009Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

Probably not a substantial claim: may not pass S3 (load-bearing).

TIRFM enables the visualization of fluorescently labeled intracellular molecules within ∼300 nm of the cell-coverslip interface.

“TIRFM enables the visualization of fluorescently labeled intracellular molecules within ∼300 nm of the cell–coverslip interface (Oancea et al., 2006; Fish, 2009; Khiroug et al., 2009).”

• cites Oancea et al., 2006 sourceNot checked yet

  Not checked in the free run.

• cites Fish, 2009Not checked yet

  Not checked in the free run.

• cites Khiroug et al., 2009 sourceNot checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing).

The ER forms the primary intracellular compartment of Neuro-2a cells.

“Our previous work used confocal microscopy to show that the ER forms the primary intracellular compartment of Neuro-2a cells (Moss et al., 2009).”

• cites Moss et al., 2009 sourceNot checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing).

NFRET could not be systematically studied in unlabeled COPI/COPII structures, peripheral trafficking vesicles, endosomes, and lysosomes.

“It has not been possible to systematically study NFRET in the additional structures present in our optical sections: unlabeled COPI/COPII structures, peripheral trafficking vesicles, endosomes, and lysosomes.”

May combine two claims.

Both nAChR stoichiometries traffic to the TG/TGN, but nAChRs with the mutant β2 subunits studied here demonstrate a relatively high proportion of (α4)2(β2)3 nAChRs.

“Both nAChR stoichiometries traffic to the TG/TGN, but nAChRs with the mutant β2 subunits studied here demonstrate a relatively high proportion of (α4)2(β2)3 nAChRs.”

May combine two claims.

In cells expressing equal numbers of α4 subunits tagged with donor or acceptor FPs plus untagged β2 subunits, 50% of nAChRs with the (α4)3(β2)2 stoichiometry possess adjacent donor and acceptor α4-FP subunits, and these nAChRs show greater NFRET values than the (α4)2(β2)3 stoichiometry.

“Our previous work exploited the idea that in cells expressing equal numbers of α4 subunits tagged with donor or acceptor FPs (α4-FP subunits plus untagged β2 subunits), 50% of nAChRs with the (α4)3(β2)2 stoichiometry possess adjacent donor and acceptor α4-FP subunits. These nAChRs show greater NFRET values than the (α4)2(β2)3 nAChR stoichiometry, which has nonadjacent donors and acceptors (Son et”

• cites Son et al., 2009 sourceNot checked yet

  Not checked in the free run.

Classified as probably not a substantial claim.

Nicotine is thought to increase the assembly of receptors with a (α4)2(β2)3 stoichiometry.

“Because nicotine is thought to increase the assembly of receptors with a (α4)2(β2)3 stoichiometry (Nelson et al., 2003; Son et al., 2009)”

• cites Nelson et al., 2003 sourceNot checked yet

  Not checked in the free run.

• cites Son et al., 2009 sourceNot checked yet

  Not checked in the free run.

May combine two claims.

Treatment with nicotine and mutations enhancing ER exit have similar effects of enhancing (α4)2(β2)3 stoichiometry, and these effects partially occlude each other, reaching a common maximal value in which 0.75-0.8 of the nAChRs possess the (α4)2(β2)3 stoichiometry.

“as though (a) treatment with nicotine and mutations enhancing ER exit have similar effects of enhancing (α4)2(β2)3 stoichiometry; and (b) these effects partially occlude each other, reaching a common maximal value in which 0.75–0.8 of the nAChRs possess the (α4)2(β2)3 stoichiometry.”

Probably not a substantial claim: may not pass S2 (checkable), S3 (load-bearing), S4 (based).

The exemplar parameters are physically plausible and expected to fall within threefold of actual values.

“We consider these parameters physically plausible and expect that they fall within threefold of actual values.”

• AssumptionThe authors state this as an assumption: The mutation-sensitive step in ER exit has a limited flux capacity, so its effective rate constant decreases with increasing numbers of nAChR molecules.
• AssumptionThe authors state this as an assumption: The pool of export machinery can be depleted.
• AssumptionThe authors state this as an assumption: Several dozen export machinery–nAChR complexes presumably form each ERES or transport vesicle.
• AssumptionRests on an assumption: Nicotine binds at the subunit interface and thereby aids nAChR assembly.
• AssumptionThe authors state this as an assumption: The anterograde rate k_Folded→Golgi is zero.
• AssumptionThe authors state this as an assumption: All other steps in SePhaChARNS 2.0 follow first-order unidirectional kinetics, governed by rate constants with dimensions of s−1, as in the FOLDEX model.
• AssumptionThe authors state this as an assumption: The simulations force all molecular concentrations to scale with that of C1 unfolded, the initial constant source of unassembled, unfolded subunits.
• AssumptionThe authors state this as an assumption: The effect of nicotine is formalized as a binding step that helps nAChRs fold and assemble fully.
• AssumptionThe authors state this as an assumption: If further saturable steps do not occur, step 8 would govern the dose–response relation for up-regulation.

May combine two claims.

Sec24D binds to IXM motifs of vesicular proteins syntaxin 5 and membrin, so ERES formation is expected to increase with the number of Sec24D molecules recruited by LFM motifs within assembled nAChRs.

“Because a component of the COPII ERES complex, Sec24D, binds to IXM motifs of vesicular proteins syntaxin 5 and membrin (Mancias and Goldberg, 2008), one expects the formation of ERES to increase with the number of Sec24D molecules recruited by LFM motifs within assembled nAChRs.”

• cites Mancias and Goldberg, 2008Not checked yet

  Not checked in the free run.

May combine two claims.

Mutations in the M3-M4 loop of α4 and β2 subunits can influence ERES numbers and the rate of ER exit of nAChRs, either by affecting Sec24D binding to LFM motifs or by preventing ER retention through an RRQR motif in the mouse β2 M3-M4 loop.

“Collectively, these data show that mutations in the M3–M4 loop of α4 and β2 subunits can influence ERES numbers and the rate of ER exit of nAChRs, either by affecting Sec24D binding to LFM motifs or by preventing the ER retention of nAChRs through an RRQR motif in the mouse β2 M3–M4 loop.”

May combine two claims.

Increasing the number of Sec24-binding sites in the nAChR protein stabilizes a pentamer that binds Sec24D, and the Sec24D-containing ERES complex is in turn stabilized by binding to nAChR pentamers.

“Thus, increasing the number of Sec24-binding sites in the nAChR protein stabilizes a pentamer that binds Sec24D, and the Sec24D-containing ERES complex is in turn stabilized by binding to nAChR pentamers.”

Classified as probably not a substantial claim. May combine two claims.

ER exit codes include LL motifs, bound by the Sec24A isoform, and I/LXM motifs, bound by Sec24D.

“ER exit codes include LL motifs, bound by the Sec24A isoform, and I/LXM motifs, bound by Sec24D (Wendeler et al., 2007; Mancias and Goldberg, 2008).”

• cites Wendeler et al., 2007 sourceNot checked yet

  Not checked in the free run.

• cites Mancias and Goldberg, 2008Not checked yet

  Not checked in the free run.

May combine two claims.

Human and chicken receptors would still be up-regulated by chronic nicotine because they harbor the RRQR motif.

“Thus, our data suggest that human and chicken receptors might exit the ER more efficiently than mouse and rat receptors, but human and chicken receptors also harbor the RRQR motif and would therefore still be up-regulated by chronic nicotine.”

Classified as probably not a substantial claim. May combine two claims.

Four Sec24 isoforms (A, B, C, and D) recognize distinct ER exit codes in cargo proteins and participate in COPII vesicle assembly.

“Four Sec24 isoforms (A, B, C, and D) recognize distinct ER exit codes in cargo proteins and participate in COPII vesicle assembly (Russell and Stagg, 2010).”

• cites Russell and Stagg, 2010Not checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing).

Additional mechanisms may be required to explain the 1.86-fold increased ERES sites in the enhanced ER export β2 double mutant in the absence of nicotine.

“The Discussion notes that additional mechanisms may be required to explain this datum.”

Classified as probably not a substantial claim.

Sec24D-binding sites allow Sec24D to stabilize the (α4)2(β2)3 stoichiometry.

“Reciprocally, increasing the number of LFM motifs also biased the receptors toward the (α4)2(β2)3 stoichiometry (Fig. 5 B and Table I); possibly, these Sec24D-binding sites allow Sec24D to stabilize this stoichiometry.”

May combine two claims.

The β2-365AAQA368 mutation may increase ERES numbers by preventing the ER retention of assembled nAChRs and/or by decreasing the likelihood that COPII is displaced by a COPI-mediated ER retrieval of exported receptors.

“The β2-365AAQA368 mutation may increase ERES numbers by preventing the ER retention of assembled nAChRs and/or by decreasing the likelihood that COPII is displaced by a COPI-mediated ER retrieval of exported receptors.”

Probably not a substantial claim: may not pass S3 (load-bearing).

The LL ER export motifs were previously identified in the M3-M4 loop of nAChR subunits.

“The LL ER export motifs were previously identified in the M3–M4 loop of nAChR subunits (Ren et al., 2005).”

• cites Ren et al., 2005 sourceNot checked yet

  Not checked in the free run.

May combine two claims.

The nicotine-favored state is both more capable of export and less susceptible to misfolding/degradation than other states within the ER.

“The key common concept is that the nicotine-favored state is both more capable of export and less susceptible to misfolding/degradation than other states within the ER.”

May combine two claims.

Newly assembled nicotine-bound nAChRs show increased ER exit; therefore, greater numbers of nAChRs undergo anterograde trafficking and insertion into the PM.

“Newly assembled nicotine-bound nAChRs show increased ER exit; therefore, greater numbers of nAChRs undergo anterograde trafficking and insertion into the PM.”

May combine two claims.

wt (α4)3(β2)2 receptors contain a 3:2 ratio of LFM (ER export motifs) to RRQR (ER retention/retrieval) motifs and traffic efficiently to the PM.

“wt (α4)3(β2)2 receptors contain a 3:2 ratio of LFM (ER export motifs) to RRQR (ER retention/retrieval) motifs and traffic efficiently to the PM, whereas (α4)2(β2)3 nAChRs with a 2:3 ratio of ER export to retention/retrieval motifs largely localize to the ER.”

May combine two claims.

Nicotine enters the ER and stabilizes nAChRs with (α4)2(β2)3 stoichiometry.

“Nicotine enters the ER and stabilizes nAChRs with (α4)2(β2)3 stoichiometry (see Fig. 7; step 8).”

May combine two claims.

At the PM, smoking-relevant nicotine concentrations only partially occupy nAChRs, revealing enhanced function of up-regulated α4β2 nAChRs.

“At the PM, smoking-relevant nicotine concentrations only partially occupy nAChRs, revealing enhanced function of up-regulated α4β2 nAChRs.”