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Figure 1. Localizations of N-terminal variations to primary/secondary structure of nAChR hα6 subunits. (A) Degree of conservation of variant residues in nAChR hα6 subunit in relation to other human nAChR α subunits: Human nAChR α subunits (α1-α7, α9 and α10) are aligned using ClustalW. Indicated residues in nAChR hα6 subunits undergoing variation are fully (Asp92 and Ser156), strongly (Arg87 and Asn171) and weakly (Asp57, Asp199 and Asn203) conserved in human nAChR α subunits. Some of the variations in nAChR hα6 subunit are localized to indicated loop regions: Ala112Val (loop A), Ala184Asp (loop B), Ile226Thr (loop C), Asp92Glu (loop D), Asp199Tyr (loop F) and Asn203Thr (loop F) and Asn171Lys (cysteine-loop). (B) Degree of conservation of variant residues in nAChR hα6 subunit in relation to nAChR α6 subunits from other organisms: nAChR α6 protein sequences extracted from (GenBank) NP_067344.2 (Mouse), NP_004189.1 (Human), NP_990695.1 (Chicken), NP_476532.1 (Rat), NP_001029266.1 (Chimpanzee), XP_001099152.1 (Monkey), XP_584902.3 (Cow) and NP_001036149.1 (Zebrafish) are aligned by using ClustalW. For both (A) and (B); numbering begins at translation start methionine of nAChR hα6 subunit and is shown in the regions of interest. However, only segments of the alignment are presented to identify WT nAChR hα6 subunit AA residues (shaded, upward arrow mark and numbers above them) and their corresponding variations (noted above the numberings). Symbols below sequences indicate fully (*), strongly (:) or weakly (.) conserved residues: hα6 subunit AA residues at positions 87 (Arg), 92 (Asp), 156 (Ser) and 171 (Asn) are conserved in both human nAChR α subunits and nAChR α6 subunits of other organisms. Also shown (shaded) are the nAChR hα6 subunit residues including the loop E residue N143 that alone or in combination Met145 influences the function of hα6*-nAChRs [26].
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Figure 2. Localizations of AA variations to secondary structures and interfaces of nAChR hα6 subunit. [(A) and (B)] AA residues undergoing variation are identified in a 3D model of the nAChR hα6 subunit: A 3D homology model of the nAChR hα6 subunit was generated based on the crystal structure of Torpedo muscle nAChR α subunit (PDB code: 2BG9:A). Hence structural features are approximate and may deviate from what seen with α subunit of Torpedo muscle nAChR. β strands constitute inner β-sheet (strand β1-β3, β5, β6 and β8) and outer β-sheet (strand β4, β7, β9 and β10); and are connect to each other via loops. These loops constitute positive (+) (loops A, B and C) and negative face (loops D, E and F) of α6 subunit and contribute to subunit assembly, ligand binding, and formation of ligand binding pocket and/or coupling agonist binding to channel gating. Cysteine loop and other loop residues undergone variations are also identified. [(C)-(E)] Interfaces contributed by α6 subunit to formation of α6*-nAChR: Adhering to the canonical rule of pentamer formation, α6β2-nAChR would be formed out of three α6 and two β2 subunits [i.e., (i) (α6)3(β2)2-nAChR] or two α6 and three β2 subunits [i.e., (ii) (α6)2(β2)3-nAChR]. In the event β3 or gain-of-function β3 (i.e., β3V9’S) subunits to be integrated into α6β2*-nAChR complexes these subunits would take the position of 3rd α6 subunit in the 1st (i) configuration or 3rd β2 subunit in the 2nd (ii) configuration [i.e., (iii) (α6)2(β2)2(β3or β3V9’S)-nAChR]. Similarly, β4 subunit would substitute β2 subunit for formation human α6β4-[i.e., (α6)3(β4)2 and (α6)2(β4)3]-nAChR. For formation of (α6)2(β4)2β3- or (α6)2(β4)2β3V9’S-nAChR β3 or β3V9’S subunits would substitute one α6 subunit in (α6)3(β4)2 configuration or one β4 subunit in (α6)2(β4)3 configuration. Two presumed agonist (ACh or nicotine and others) binding sites in the interface of α6(+) and β2(−) subunits are identified as ovals. Variations in the structural loops in the (−)-ve face (loop D, E and F) and (+)-ve face (loop A, B and C) of the hα6 subunit are expected to affect the function of hα6*-nAChRs involving interfaces identified by arrow marks.
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Figure 3. Variations in nAChR hα6 subunit influence the current responses of human α6β2β3V9’S-nAChRs expressed in oocytes using codon optimized nAChR hβ2 subunits. Mean (±SEM) peak inward current responses upon exposure to 100 μM nicotine (5 sec exposure; ordinate) are estimated from oocytes (n = 3-7) voltage clamped at −70 mV and heterologously expressing the indicated nAChR subunits. Current responses of hα6hβ2hβ3V9’S-nAChR are completely abolished (D57N or S156R), partially abolished (S43P, N46K, R87C, D92E or N171K), not changed (E101K, A112V, A184D, N203T or I226T) and increased (R96H, D199Y or S233C) as a result of the indicated variations in nAChR hα6 subunits. Oocytes coexpressing nAChR hα6(D199Y) subunits, codon optimized hβ2 subunits and hβ3V9’S subunits yield largest current responses to nicotine. Comparisons of peak current responses between control (hα6hβ2hβ3V9’S-nAChR) and variant nAChR groups were analyzed using one-way ANOVA with Dunnett’s multiple comparisons test (*, p < 0.05; and **, p < 0.01).
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Figure 4. Variations in nAChR hα6 subunit influence the current responses of human α6β2β3V9’S-nAChR expressed in oocytes using WT nAChR hβ2 subunits. Attempts were made to verify the results obtained for human α6β2β3V9’S-nAChR using WT nAChR hβ2 subunits instead of codon optimized hβ2 subunits. Mean (±SEM) peak inward current responses upon exposure to 100 μM nicotine (5 sec exposure; ordinate) are estimated from oocytes (n = 3-7) voltage clamped at −70 mV and heterologously expressing the indicated nAChR subunits. (A) Oocytes coexpressing nAChR hα6(R96H, D199Y or S233C), hβ2WT and hβ3V9’S subunits yield current responses to nicotine that are higher than those expressing nAChR hα6, hβ2WT and hβ3V9’S subunits. These results are in agreement with those obtained using codon optimized hβ2 subunits. (B) Initial recordings 3 days after cRNA injection indicated that oocytes expressing WT hα6 or variant hα6(E101K, A112V, A184D, N203T or I226T) subunits along with WT hβ2 and hβ3V9’S subunits yield ~10-30 nA of current in response to 100 μM nicotine [see the hα6hβ2hβ3V9’S-nAChR response in (A)]. However recordings done after 2 additional days of waiting indicated that nicotine elicited current responses from human α6E101Kβ2β3V9’S-, α6A112Vβ2β3V9’S-, α6A184Dβ2β3V9’S-, α6N203Tβ2β3V9’S- or α6I226Tβ2β3V9’S-nAChRs are equal (p > 0.05) to those obtained from human α6β2β3V9’S-nAChRs. These results are in agreement with those obtained using codon optimized hβ2 subunits. Comparisons between groups were analyzed using one-way ANOVA with Tukey’s post hoc comparison and only those differ from the control (hα6hβ2hβ3V9’S-nAChR) are shown with asterisks. *, p < 0.05; and **, p < 0.01.
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Figure 5. Variations in nAChR hα6 subunit influence the current responses of hα6hβ4-nAChRs. Mean (±SE) peak inward current responses upon exposure to 100 μM nicotine (5 sec exposure; ordinate) are estimated from oocytes (n = 6-9) voltage clamped at −70 mV and heterologously expressing the indicated nAChR subunits. Oocytes coexpressing hα6A184D (variation in loop D) and hβ4 subunits yield largest current responses to 100 μM nicotine. Comparisons of peak current responses between control (hα6hβ4-nAChR) and variant groups were analyzed using one-way ANOVA with Dunnett’s multiple comparisons test (*, p < 0.05; and **, p < 0.01).
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Figure 6. Variations in nAChR hα6 subunit influence the current responses of hα6hβ4hβ3-nAChRs. Mean (±SE) peak inward current responses upon exposure to 100 μM nicotine (5 sec exposure; ordinate) are estimated from oocytes (n = 6-11) voltage clamped at −70 mV and heterologously expressing the indicated nAChR subunits. Oocytes coexpressing hα6(D57N, R87C, S156R or N171K) subunits plus hβ4 and hβ3 subunits do not yield current responses to nicotine though those coexpressing hα6, hβ4 and hβ3 subunit yield fairly robust current responses. hα6 subunit variation Asp92Glu (in loop D) partially abolishes the peak current responses of hα6hβ4hβ3-nAChRs. hα6 subunit variations Asn46Lys, Arg96His, Glu101Lys, Ala112Val, Ala184Asp, Asn203Thr, Ile226Thr or Ser233Cys do not affect nicotine elicited peak current responses of hα6hβ4hβ3-nAChRs. Comparisons between control (hα6hβ4hβ3) and variant groups were analyzed using one-way ANOVA with Dunnett’s multiple comparisons test (*, p < 0.05; and **, p < 0.01).
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Figure 7. Nicotinic agonists act as antagonists at hα6(D57N, S156R or N171K)hβ4hβ3V9’S-nAChRs. (A) Representative traces are shown for inward or outward current responses from oocytes (voltage clamped at -70 mV) responding to the application of indicated concentrations of nicotine or atropine (shown with the duration of drug exposure as black bars above or below the traces) and expressing hα6hβ4hβ3V9’S- [(A) (i) and (v)], hα6D57Nhβ4hβ3V9’S-[(A) (ii) and (vi)], hα6S156Rhβ4hβ3V9’S[(A) (iii) and (vii)] or hα6N171Khβ4hβ3V9’S- [(A) (iv) and (viii)] nAChRs. Calibration bars are for 200 (i), 40 [(ii), (iii) and (iv)] or 100 [(v), (vi), (vii) and (viii)] nA currents (vertical) or 5 sec (horizontal). Results for these and other studies were used to estimate mean (±SE) peak outward current responses to 100 μM nicotine (B), 100 μM ACh (C) or 1000 μM atropine (D) from oocytes (n=3-6) heterologously expressing the indicated nAChR subunits. hα6(D57N,S156RorN171K)hβ4hβ3V9’S-nAChRs elicit outward (positive) current in response to nicotine (B) or ACh (C) which is completely absent from hα6hβ4hβ3V9’S-nAChRs [(B) and (C)]. hα6(D57N,S156RorN171K)hβ4hβ3V9’S-nAChRs in rare occasion display minuscule inward currents in response to ACh or nicotine (data not shown). Comparisons between groups were analyzed using one-way ANOVA with Tukey’s post hoc comparison and only those differ from the control (hα6hβ4hβ3V9’S-nAChR) are marked with asterisks. ***; p < 0.001.
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Figure 8. Variations in nAChR hα6 subunit influence the ACh sensitivity of hα6hβ4*-nAChRs. (A) Representative traces are shown for current responses from oocytes (voltage clamped at −70 mV) responding to the application of indicated concentrations of ACh (shown with the duration of drug exposure as black bars above the traces) and expressing indicated nAChR (i.e., R1: hα6A184Dhβ4-nAChR, R2: hα6A184Dhβ4hβ3-nAChR, R3: hα6hβ4hβ3-nAChR]. (B) Results averaged across experiments were used to produce concentration-response (CR) curves (ordinate-mean normalized current ± SEM; abscissa - ligand concentration in log μM) for inward current responses to ACh as indicated for the nAChR expressed in oocytes and voltage clamped at −70 mV. Current amplitudes are represented as a fraction of the peak inward current amplitude in response to the most efficacious concentration of ACh. Leftward shifts in ACh CR curves for hα6R96Hhβ4hβ3-(●) [(B) (i)], hα6A184Dhβ4hβ3-(●) [(B) (ii)], hα6D199Yhβ4hβ3-(●) [(B) (iii)], or hα6S233Chβ4hβ3-(●) [(B) (iv)] nAChR are evident relative to that of hα6hβ4hβ3-nAChR (○). Furthermore ACh curves for hα6A184Dhβ4-(■) [(B) (ii)], hα6D199Yhβ4-(■) [(B) (iii)], or hα6S233Chβ4-(■) [(B) (iv)] nAChR are shifted leftward relative to those nAChR containing the same subunits but in the additional presence of hβ3 subunits. See Table 3 for parameters of ACh action.
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