Modulation Of Gain-Of-Function Î±6*-Nicotinic Acetylcholine Receptor By Î23 Subunits

Background: Function of physiologically important α6β3*-nicotinic receptor (nAChR) is differentially impacted by β3 subunits. Results: nAChR expressed in several novel ways indicates that β3 subunits mostly potentiate gain-of-function α6*-nAChR. Conclusion: Extracellular domain loop E region in α6 subunits governs effect of β3 subunit on gain-of-function α6*-nAChR. Significance: Novel α6β3*-nAChR reported could be used to assess and/or develop smoking cessation aids. We previously have shown that β3 subunits either eliminate (e.g. for all-human (h) or all-mouse (m) α6β4β3-nAChR) or potentiate (e.g. for hybrid mα6hβ4hβ3- or mα6mβ4hβ3-nAChR containing subunits from different species) function of α6*-nAChR expressed in Xenopus oocytes, and that nAChR hα6 subunit residues Asn-143 and Met-145 in N-terminal domain loop E are important for dominant-negative effects of nAChR hβ3 subunits on hα6*-nAChR function. Here, we tested the hypothesis that these effects of β3 subunits would be preserved even if nAChR α6 subunits harbored gain-of-function, leucine- or valine-to-serine mutations at 9′ or 13′ positions (L9′S or V13′S) in their second transmembrane domains, yielding receptors with heightened functional activity and more amenable to assessment of effects of β3 subunit incorporation. However, coexpression with β3 subunits potentiates rather than suppresses function of all-human, all-mouse, or hybrid α6(L9′S or V13′S)β4*- or α6(N143D+M145V)L9′Sβ2*-nAChR. This contrasts with the lack of consistent function when α6(L9′S or V13′S) and β2 subunits are expressed alone or in the presence of wild-type β3 subunits. These results provide evidence that gain-of-function hα6hβ2*-nAChR (i.e. hα6(N143D+M145V)L9′Shβ2hβ3 nAChR) could be produced in vitro. These studies also indicate that nAChR β3 subunits can be assembly partners in functional α6*-nAChR and that 9′ or 13′ mutations in the nAChR α6 subunit second transmembrane domain can act as gain-of-function and/or reporter mutations. Moreover, our findings suggest that β3 subunit coexpression promotes function of α6*-nAChR.

In vitro expression of functional, all-mouse (m) or all-human (h), wild-type ␣6␤3*-nAChR has been difficult to achieve despite strong evidence for expression of ␣6␤3*-nAChR in rodent brain (3,4,6,7,10,(12)(13)(14)(15). Functional expression of ␣6*-nAChR only has been achieved in Xenopus oocytes when using specific forms of mutant or chimeric subunits or in hybrid ␣6*-nAChR composed of subunits from different species (16 -20). For example, function is achieved when chimeric, h␣6/h␣3 subunits (composed of the N-terminal, first extracellular domain of the h␣6 subunit fused to the first transmembrane domain through to the C terminus of the h␣3 subunit) are coexpressed with h␤2 or h␤4 subunits alone or in the presence of h␤3 subunits (19). ␣6*-nAChR are functional when expressed as hybrids of mouse and human ␣6 and other subunits, and there is function of some complexes containing ␤3 subunits mutated at specific residues in their second transmembrane domains (leucine-or valine-to-serine mutations at 9Ј or 13Ј positions; L9ЈS or V13ЈS) to confer gain-of-function effects (4,15,21). Potentiation of function is sometimes seen when wildtype ␤3 subunits are incorporated into hybrid complexes, but this is in contrast to dominant-negative effects of coexpression with wild-type ␤3 subunits on function of ␣6␤4*-nAChR when all subunits are from the same species (4,21). There may be host cell specificity in some of these effects because nAChR h␤3 subunits promote expression and nicotine-induced up-regulation of h6*-nAChR in transfected cell lines (22).
We and others have taken advantage of gain-of-function mutations in the nAChR ␤3 subunit to produce functional nAChR, including those containing ␣6 subunits, in part to assess capabilities of subunits to coassemble, but also as a strategy to increase functional gain (signal:noise) to facilitate such assessments (4,15,21). For example, coexpression with ␤3 V9ЈS subunits increases agonist sensitivity and efficacy for ␣6*-nAChR. We hypothesized that similar mutations in nAChR ␣6 subunits would increase agonist sensitivity and efficacy of ␣6 (L9ЈS or V13ЈS) (␤4 or ␤2)*-nAChR to provide enough functional gain to facilitate evaluation of effects of wild-type ␤3 subunits on complexes and even to ensure that we can detect incorporation of wild-type ␤3 subunits into ␣6 (L9ЈS or V13ЈS) *-nAChR. We also hypothesized that wild-type ␤3 subunits would have the same effects, dominant-negative or potentiating, depending on the subunit combination investigated, on gain-of-function ␣6 (L9ЈS or V13ЈS) *-nAChR as they did on wildtype ␣6*-nAChR. This would help us assess whether any reduction or abolishment of function is due to altered open channel probability (21) or due to reduced surface expression of nAChR because ␤3 subunit incorporation facilitates formation of dead end intermediates (23). Our results indicated that whenever nAChR ␤3 subunits are incorporated into (␣6 or h␣6(N143DϩM145V)) (L9ЈS or V13ЈS) *-nAChR, function is potentiated (i.e. there is higher agonist potency and larger magnitude responses) irrespective of whether there are dominantnegative or potentiating effects of ␤3 subunits on wild-type ␣6*-nAChR.

EXPERIMENTAL PROCEDURES
Chemicals-All chemicals for electrophysiology were obtained from Sigma. Fresh stock solutions of nicotine or mecamylamine were made daily in Ringer's solution and were diluted as needed.
Oocyte Preparation and cRNA Injection-Female Xenopus laevis (Xenopus I, Ann Arbor, MI) were anesthetized using 0.2% tricaine methanesulfonate (MS-222). The ovarian lobes were surgically removed from the frogs and placed in an incubation solution that consisted of (in mM) 82.5 NaCl, 2.5 KCl, 1 MgCl 2 , 1 CaCl 2 , 1 Na 2 HPO 4 , 0.6 theophylline, 2.5 sodium pyruvate, 5 HEPES supplemented with 50 mg/ml gentamycin, 50 units/ml penicillin, and 50 g/ml streptomycin; pH 7.5. The frogs were allowed to recover from surgery before being returned to the incubation tank. Ovarian lobes were cut into small pieces and digested with 0.08 Wünsch units/ml Liberase blendzyme 3 (Roche Applied Science) with constant stirring at room temperature for 1.5-2 h. The dispersed oocytes were thoroughly rinsed with incubation solution. Stage VI oocytes were selected and incubated at 16°C before injection. Micropipettes used for injection were pulled from borosilicate glass (Drummond Scientific, Broomall, PA) using a Sutter P87 horizontal puller, and the tips were broken with forceps to ϳ40 m in diameter. cRNA was drawn up into the micropipette and injected into oocytes using a Nanoject microinjection system (Drummond Scientific) at a total volume of ϳ60 nl. To express nAChR in oocytes, about 4 ng of cRNA corresponding to each subunit was injected; i.e. at ratios of 1:1 or 1:1:1 for binary or trinary receptors, respectively, with the exception that for coexpression of h␣6(N143DϩM145V)ϩh␤2*-nAChR in the presence or absence nAChR h␤3 subunit, about 10 ng of cRNA corresponding to each subunit including nAChR h␤2 opt subunit was injected.
Oocyte Electrophysiology-Two to seven days after injection, oocytes were placed in a small-volume chamber and continuously perfused with oocyte Ringer's solution, which consisted of (in mM) 92.5 NaCl, 2.5 KCl, 1 CaCl 2 , 1 MgCl 2 , and 5 HEPES; pH 7.5. The chamber was grounded through an agarose bridge. The oocytes were voltage-clamped at Ϫ70 mV (unless otherwise noted) to measure agonist-induced currents using an AxoClamp 900A and the pClamp 10.2 software (Axon Instruments, CA). The current signal was low pass-filtered at 10 Hz with the built-in low pass Bessel filter in the AxoClamp 900A and digitized at 20 Hz with an Axon Digidata1440A and the pClamp10.2 software. Electrodes contained 3 M KCl and had a resistance of 1-2 megaohms. Drugs (agonists and antagonists) were prepared daily in bath solution. Drug was applied using a Valvelink 8.2 perfusion system (Automate Scientific, Berkeley, CA). All electrophysiological measurements were conducted or checked in at least two batches of oocytes.
Experimental Controls-Injection of water or empty vector (used as two forms of negative controls) or of cRNA corresponding to one subunit alone or pairwise combinations of ␤3 subunits with either an ␣6 or a mutant ␣6 subunit or ␤2 or ␤4 subunits (8 -20 ng total of cRNA) did not result in the expression of functional nAChR. Current responses to 100 M nicotine were less than 5-10 nA (data not shown).
Data Analyses-Raw data were collected and processed in part using pClamp 10.2 (Molecular Devices, Sunnyvale, CA) and a spreadsheet (Excel; Microsoft, Bellevue, WA), using peak current amplitudes as measures of functional nAChR expression and results pooled across experiments (mean Ϯ S.E. for data from at least three oocytes). In some cases, mean peak current amplitudes in response to a single concentration of an agonist were compared across different subunit combinations. However, assessment of true I max values for different nAChR subunit combinations required assessment based on more complete concentration-response relationships, in which mean peak current amplitudes at specified ligand concentrations were fit to the Hill equation or its variants using Prism 4 (GraphPad Software, San Diego, CA). F-tests (p Ͻ 0.05 to define statistical significance) were carried out to compare the best fit values of log molar EC 50 values across specific nAChR subunit combinations.
There are limitations in the ability to compare levels of functional nAChR expression, although we injected similar amounts of RNA for all constructs. This is because expression levels assessed as peak current amplitudes are affected by batch-to-batch variation in oocytes, time between cRNA injection and recording, and subunit combination-specific parameters, such as open probability (influenced by gating rate constants, rates, and extents of desensitization), single channel conductance, assembly efficiency, and efficiency of receptor trafficking to the cell surface (24). We made no attempt to measure or control for subunit combination-specific effects, but whenever preliminary studies revealed possible differences in peak current amplitudes, findings were further confirmed across different subunit combinations using the same batch of oocytes and the same time between cRNA injection and recording. Peak current amplitudes shown from representative traces in some figures presented below, pooled data from limited sets of studies, and mean peak current amplitudes across all studies for a given combination of subunits given in tables sometimes differ. However, when we make statements about results comparing ligand potencies and peak current amplitudes across subunit combinations, we do so for studies done under the same or very similar conditions, and the observations are clear, statistically significant, and in agreement whether for pooled data or for results from smaller sets of studies (one-way anal-yses of variance followed by Tukey's multiple comparison tests).

Human nAChR ␣6 L9ЈS Subunits Form Functional Receptors in Association with nAChR h␤4 and h␤3 Subunits with
Increased Receptor Agonist Sensitivity and Efficacy-Earlier, we observed that oocytes coinjected with nAChR h␣6 and h␤4 subunit cRNAs produce functional h␣6h␤4-nAChR in only a few out of many injected oocytes and then only have minimal responses to nicotinic agonists (4). Although we could measure a peak current of 22 Ϯ 3 nA for h␣6h␤4-nAChR in response to 100 M acetylcholine, we were unable to measure reliable and reproducible functional responses to nicotine. Also, oocytes injected with nAChR h␣6, h␤4, and h␤3 subunit cRNAs do not produce reliable and reproducible functional h␣6h␤4h␤3-nAChR, suggesting that the small amount of function seen for h6h␤4-nAChR is either reduced or completely eliminated, probably due to ␤3 subunits exerting a negative effect on function of h␣6h␤4*-nAChR. We replicated those findings in the current work, and we also found that oocytes coexpressing nAChR h␣6 L9ЈS and h␤4 subunit cRNAs have marginally increased, but more reproducible, responses to nicotine (peak current of 32 Ϯ 7 nA for h␣6 L9ЈS h␤4-nAChR in response to 100 M nicotine; Fig. 1; Table 1). Thus, replacement of h␣6 L9ЈS for h␣6 subunits does not have as great of a gain-of-function effect on ␣6␤4*-nAChR as does introduction of h␤3 V9ЈS subunits (4) into otherwise wild-type h␣6h␤4*-nAChR.
Consistent with our previous observations regarding introduction of gain-of-function ␤3 subunits into ␣6*-nAChR (4), oocytes coexpressing nAChR h␣6 L9ЈS and h␤4 subunits and exposed to the nAChR noncompetitive antagonist and open channel blocker, mecamylamine, respond with an apparent outward peak current of 12 Ϯ 5 nA (Table 1). Because mecamylamine coexposure more than blocks inward currents produced by nicotinic agonists, also leading under those conditions to production of apparent outward current responses, and does so in a concentration-dependent manner, we again interpret these effects as showing the ability of mecamylamine to block spontaneous opening of ␣6 L9ЈS h␤4-nAChR channels (Table 1). Given the magnitudes of peak current responses to nicotine alone and to mecamylamine alone, about 27% of h␣6 L9ЈS h␤4-nAChR appear to be spontaneously open at any given time (12/ (12 ϩ 32) ϭ 0.27).
When nAChR h␣6 L9ЈS and h␤4 were coexpressed with h␤3 subunits instead of alone, oocyte responsiveness to nicotine (EC 50 value of 0.9 M) increases over 10-fold (to a peak current response of 350 Ϯ 52 nA; Fig. 1, Table 1). This suggests that wild-type ␤3 subunits incorporate into h␣6 L9ЈS h␤4*-nAChR and strongly potentiate receptor function. However, this does not occur with a change in agonist potency upon h␤3 subunit incorporation into h␣6 L9ЈS h␤4*-nAChR because there is not a significant change in nicotine EC 50 values (Table 1). Outward current production in the same oocytes (9.5 Ϯ 1.5 nA) in response to 1000 M mecamylamine indicates that there is spontaneous opening of h␣6 L9ЈS h␤4h␤3-nAChR, but levels of spontaneous opening are comparable with those for h␣6 L9ЈS h␤4-nAChR in the absence of h␤3 subunits, indicating that a smaller proportion of h␣6 L9ЈS h␤4h␤3-nAChR is spontaneously open at any time (9.5/(9.5 ϩ 350) ϭ 0.026; Table 1) than for h␣6 L9ЈS h␤4-nAChR. No function was observed in response to nicotine or mecamylamine in oocytes coexpressing nAChR h␣6 or h␣6 L9ЈS subunits plus h␤2 subunits with or without h␤3 subunits.
Mouse nAChR ␣6 V13ЈS Subunits Form Functional Receptors in Association with nAChR m␤4 and m␤3 Subunits with Increased Receptor Agonist Sensitivity and Efficacy-We have shown earlier that oocytes coinjected with m␣6 and m␤4 nAChR subunit cRNAs form functional nAChR, but with minimal responses to nicotinic agonists, and function is further reduced in the presence of nAChR m␤3 subunits, indicating that nAChR m␤3 subunits exert dominant-negative effects on the function of m␣6m␤4*-nAChR (4). Here, we observed that oocytes coexpressing either wild-type m␣6 or mutant m␣6 L9ЈS along with m␤4 subunits give comparably modest peak current responses to 100 M nicotine (I max ϭ 27 Ϯ 7 or 29 Ϯ 1 nA, respectively; Table 1). Thus, although oocytes expressing m␣6 L9ЈS and m␤4 subunits give outward current responses to mecamylamine, consistent with spontaneous channel opening, the 9Ј mutation in the nAChR m␣6 subunit does not significantly increase the magnitude of functional responsiveness (Table 1). Similarly, there is no increase in functional responsiveness to nicotine for oocytes coexpressing nAChR m␣6 L9ЈS , m␤4, and m␤3 subunits (peak current ϭ 26 Ϯ 4 nA), although FIGURE 1. Functional properties of h␣6*-nAChR. A, representative traces are shown for inward currents in oocytes held at Ϫ70 mV, responding to application at the indicated concentrations of nicotine (shown with the duration of drug exposure as black bars above the traces), and expressing nAChR h␣6 L9ЈS and h␤4 subunits (i) or nAChR h␣6 L9ЈS , h␤4, and h␤3 subunits (ii). B, results for these and other studies averaged across experiments were used to produce concentration-response curves (ordinate, mean normalized current Ϯ S.E.; abscissa, ligand concentration in log M) for inward current responses to nicotine as indicated for oocytes expressing nAChR h␣6 L9ЈS and h␤4 subunits alone (f) or with h␤3 subunits (Ⅺ), where current amplitudes are represented as a fraction of the peak inward current amplitude in response to the most efficacious concentration of nicotine. Much higher levels of evoked currents are evident for functional nAChR containing h␣6 L9ЈS , h␤4, and h␤3 subunits when compared with receptors lacking h␤3 subunits. See Table 1 for parameters.

TABLE 1 Parameters for agonist or antagonist action at nAChR containing gain-of-function ␣6 mutant subunits
Potencies (micromolar EC 50 or IC 50 values with 95% confidence intervals), Hill coefficients (n H Ϯ S.E.), mean Ϯ S.E. efficacies (two-electrode voltage-clamp peak responses, I max , in nanoamperes), and concentrations where maximal peak current amplitudes (I max concentration in micromolar) are achieved (M) are provided for nicotine as an agonist or mecamylamine as an antagonist acting at nAChR composed of the indicated subunits derived from the specified species and from the indicated number of independent experiments (n) based on studies as shown the figures. Closed up arrows or closed down arrows indicate a significant (p Ͻ 0.05) increase or decrease, respectively, in potency or efficacy of the indicated agent at the indicated nAChR subtype relative to nAChR containing the wild type ␣6 subunit. Filled triangle indicates a significant increase in indicated agonist or antagonist potency or efficacy at the indicated nAChR containing ␣6 L9ЈS or ␣6 V13ЈS subunits relative to the same complex but lacking ␤3 subunits. Open up arrows or open down arrows indicate a significant increase or decrease, respectively, in potency or efficacy of the indicated agonist at the indicated nAChR containing ␣6 V13ЈS subunits relative to nAChR containing ␣6 L9ЈS subunits. Note that no or very rare and then small responses to nicotine were seen for the following subunit combinations (n ϭ 6 -9 each): h␣6 or h␣6 L9ЈS plus h␤2 alone or with h␤3; m␣6 or m␣6 L9ЈS or m␣6 V13ЈS plus m␤2 alone or with m␤3 or h␤3; and m␣6 or m␣6 L9ЈS plus h␤2 alone or with h␤3. -indicates that absent or inconsistent functional responses in two-electrode voltage-clamp studies precluded determination of the parameter of interest; # indicates data from (4).
to mecamylamine, again an indication that functional and spontaneously opening m␣6 L9ЈS h␤4h␤3-nAChR are formed ( Table 1). The kinetics of traces generated in response to application of nicotine differs between m␣6 L9ЈS h␤4-and m␣6 L9ЈS h␤4h␤3-nAChR. m␣6 L9ЈS h␤4h␤3-nAChR in response to activation by 1000 M nicotine exhibit a tail current that is significantly reduced or absent in m␣6 L9ЈS h␤4-nAChR when activated by the same concentration of nicotine. Precisely, in the presence of h␤3 subunits, there is a pronounced functional block of the receptor at higher concentration of nicotine. Removal of the functional block imposed by nicotine (probably acting as an open channel blocker), as a result of switching out to buffer application, leads to activation of the receptor by the residual nicotine that results in formation of a tail current. This is not unusual given that incorporation of accessory subunits into nAChR differentially affects various functional characteristics of the receptor (25).
Minimal and inconsistent nAChR function was observed when m␣6 L9ЈS subunits were coexpressed with h␤2 and h␤3 subunits. This is in contrast to our earlier observation that nAChR m␣6 subunits along with h␤2 subunits and gain-offunction h␤3 subunits (h␤3 V9ЈS ; h␤3 V273S ) form functional m␣6h␤2h␤3 V9ЈS -nAChR (4). This suggests that the gain-offunction mutation in the m␣6 L9ЈS subunit is inadequate to overcome what seems to be a strong, dominant-negative effect of nAChR ␤3 subunits in the presence of ␤2 subunits.

Mouse nAChR ␣6 L9ЈS Subunits Form Functional Receptors in Association with nAChR m␤4 and h␤3 Subunits with Increased
Receptor Agonist Sensitivity and Efficacy-Oocytes coinjected with nAChR m␣6, m␤4, and h␤3 subunit. cRNAs give Ͼ10fold larger and ϳ2-fold more sensitive responses to nicotinic agonists than do oocytes coinjected m␣6 and m␤4 subunit cRNAs and in stark contrast to the elimination of functional responses in oocytes coexpressing nAChR m␣6, m␤4, and m␤3 subunits (Table 1). Interestingly, here we found that oocytes coexpressing nAChR m␣6 L9ЈS , m␤4, and h␤3 subunits responded to nicotine with an EC 50 value of 0.48 M and with large peak currents 680 Ϯ 32 nA; Fig. 4; Table 1) and gave outward current responses (8.5 Ϯ 3.3 nA) when exposed to 1000 M mecamylamine (Table 1). No function was observed when m␣6 L9ЈS subunits were coexpressed with m␤2 and h␤3 subunits.

Human nAChR ␣6(N143DϩM145V) L9ЈS Subunits Form Functional Receptors in Association with nAChR h␤2 and h␤3 Subunits with Increased Receptor Agonist Sensitivity and
Efficacy-Earlier (4), we had shown that mutations in the N-terminal domain of the nAChR h␣6 subunit enable nAChR h␤3 V9ЈS subunits to exert a gain-of-function effect at h␣6(N143DϩM145V)h␤2*-nAChR (i.e. h␣6(N143Dϩ M145V)h␤2h␤3 V9ЈS -nAChR are functional). This finding led us to explore effects of incorporation of h␤3 subunits into h␣6(N143DϩM145V) L9ЈS h␤2*-nAChR. Functional properties of m␣6*-nAChR. A, representative traces are shown for inward currents in oocytes held at Ϫ70 mV, responding to application at the indicated concentrations of nicotine (shown with the duration of drug exposure as black bars above the traces), and expressing nAChR m␣6 V13ЈS , m␤4, and m␤3 subunits. B, results for these and other studies averaged across experiments were used to produce concentration-response curves (ordinate, mean normalized current Ϯ S.E.; abscissa, ligand concentration in log M) for inward current responses to nicotine as indicated for oocytes expressing nAChR m␣6 and m␤4 subunits (f) or m␣6 V13ЈS and m␤4 and m␤3 subunits (Ⅺ), where current amplitudes are represented as a fraction of the peak inward current amplitude in response to the most efficacious concentration of nicotine. Much higher levels of evoked currents are evident for functional nAChR containing m␣6 V13ЈS , m␤4, and m␤3 subunits when compared with receptors lacking m␣6 V13ЈS subunits. See Table 1 for parameters.

DISCUSSION
Recent studies have investigated how nAChR ␤3 subunits might incorporate as accessory partners into nAChR subtypes, specifically into ␣6*-nAChR (4). To further understand how ␤3 subunits might incorporate into ␣6*-nAChR, we exploited the gain-of-function/reporter mutant strategy (4,15,26) to reveal whether ␤3 subunits integrate into ␣6*-nAChR complexes that are on the cell surface and functional. This approach allows us to focus on cell surface, functional receptors without complications due to ambiguities of protein chemical or immunochemical studies confounded by the prevalent expression of intracellular and perhaps partially assembled receptor complexes and the unreliable quality and/or availability of most anti-nAChR antibodies for use in immunoprecipitation and/or immunoblot studies (15). In addition, we based the current studies on our findings (4) that (i) incorporation of nAChR ␤3 subunits into ␣6*-nAChR, mouse or human, has a dominant-negative effect; (ii) incorporation of nAChR h␤3 subunits into m␣6h␤4*-or m␣6m␤4*-nAChR leads to formation of functional nAChR; and (iii) mutations in the E1 N-terminal domain of the nAChR h␣6 subunit are essential for successful assembly and formation of functional h␣6(N143DϩM145V)-h␤2h␤3 V9ЈS -nAChR. , and expressing nAChR m␣6 L9ЈS and h␤4 subunits (i) or nAChR m␣6 L9ЈS , h␤4, and h␤3 subunits (ii). B, results for these and other studies averaged across experiments were used to produce concentration-response curves (ordinate, mean normalized current Ϯ S.E.; abscissa, ligand concentration in log M) for inward current responses to nicotine as indicated for oocytes expressing nAChR m␣6 L9ЈS and h␤4 subunits alone (f) or with h␤3 subunits (Ⅺ) or expressing m␣6 and h␤4 and h␤3 subunits (ϫ; data from Ref. 4), where current amplitudes are represented as a fraction of the peak inward current amplitude in response to the most efficacious concentration of nicotine. Much higher levels of evoked currents are evident for functional nAChR containing m␣6 L9ЈS , h␤4, and h␤3 subunits when compared with receptors lacking h␤3 subunits. See Table 1 for parameters.
The principal findings of this study, whenever functional expression levels are adequate to allow comparisons, and with exceptions that could be informative as discussed below, are: (i) that introduction of 9Ј or 13Ј mutations into the second transmembrane domain of m␣6 or h␣6 subunits typically has a gain-of-function effect, leading to production of (␣6 or ␣6(N143DϩM145V)) (L9ЈS or V13ЈS) (␤2 or ␤4)*-nAChR that have 6 -34-fold higher sensitivity to nicotine and much higher levels of function than do nAChR containing the same subunit combinations but with wild-type ␣6 subunits; (ii) that incorporation of ␤3 subunits into (␣6 or ␣6(N143DϩM145V)) (L9ЈS or V13ЈS) (␤2 or ␤4)*-nAChR typically increases levels of receptor function with or without concomitant increase in agonist potency; and (iii) that gain-of-function mutations in ␣6 or ␣6(N143DϩM145V) subunits still do not allow for formation of functional ␣6 (L9ЈS or V13ЈS) ␤2-nAChR complexes, thus continuing to confound assessments of roles played by ␤3 subunits in modulation of ␣6␤2*-nAChR.
The amount of functional expression for h␣6 L9ЈS h␤4-, m␣6 L9ЈS m␤4-, or m␣6 L9ЈS h␤4-nAChR is modest in absolute terms (27-80-nA peak current). However, with the exception of the insignificant difference in the magnitude of function seen for all-mouse m␣6 L9ЈS m␤4-and m␣6m␤4-nAChR, the increase in function upon expression with the ␣6 subunit 9Ј mutants is remarkable because of the lack of reliable function for wild-type, all human h␣6h␤4-, or hybrid m␣6h␤4-nAChR. The little-if-any function for all-wild-type ␣6␤4-nAChR complicates quantitative assessment of effects of ␣6 subunit gain-of-function mutations on agonist potency, although qualitatively, nicotine EC 50 values are over 10 M for ␣6␤4-nAChR and never higher than 3.1 M for ␣6 (L9ЈS or V13ЈS) ␤4-nAChR. However, gain-of-function effects manifest as increases in agonist potency and in peak current magnitudes are very clear based on comparisons of h␣6h␤4h␤3with h␣6 L9ЈS h␤4h␤3-nAChR and comparisons of m␣6h␤4h␤3-with m␣6 L9ЈS h␤4h␤3-nAChR. A difference in agonist potency is also clear for comparison of m␣6m␤4h␤3with m␣6 L9ЈS m␤4h␤3-nAChR, although there is only a 2-fold difference in peak current response across these receptors, partly due to the relatively high absolute levels of function for the hybrid m␣6m␤4h␤3-nAChR. Once again, however, all-mouse ␣6␤4␤3-nAChR are outliers because there is only modest function for m␣6 L9ЈS m␤4m␤3-nAChR, although there is no reliable function for the all-wild-type analog, m␣6m␤4m␤3-nAChR.
Nevertheless, and very interestingly, for all-mouse ␣6*-receptors, although there is not reproducible function for m␣6 V13ЈS m␤4-nAChR, there are increases both in agonist potency and in response magnitude for m␣6 V13ЈS m␤4m␤3-nAChR when compared with those parameters for any form of m␣6m␤4-nAChR or for m␣6m␤4m␤3-or m␣6 L9ЈS m␤4m␤3-nAChR. Our initial studies of mouse ␣6*-nAChR were prompted because of the reported difficulties in heterologous expression of all-human ␣6*-nAChR and because so many data on naturally expressed ␣6*-nAChR function came from studies using rodents, but we have found all-mouse ␣6*-nAChR no easier to express than human ␣6*-nAChR. Expression of hybrid FIGURE 4. Functional properties of gain-of-function hybrid m␣6h␤3*-nAChR. A, representative traces are shown for inward currents in oocytes held at Ϫ70 mV, responding to application at the indicated concentrations of nicotine (shown with the duration of drug exposure as black bars above the traces), and expressing nAChR m␣6 L9ЈS , m␤4 and h␤3 subunits. B, results for these and other studies averaged across experiments were used to produce concentrationresponse curves (ordinate, mean normalized current Ϯ S.E.; abscissa, ligand concentration in log M) for inward current responses to nicotine as indicated for oocytes expressing nAChR m␣6 and m␤4 subunits alone (32 ϫ) or with h␤3 subunits (f) or expressing m␣6 L9ЈS and m␤4 and h␤3 subunits (Ⅺ), where current amplitudes are represented as a fraction of the peak inward current amplitude in response to the most efficacious concentration of nicotine. Much higher levels of evoked currents are evident for functional nAChR containing m␣6 L9ЈS , h␤4, and h␤3 subunits when compared with receptors lacking h␤3 subunits. See Table  1 for parameters. nAChR made up of subunits from different species has been more productive, suggesting that subtle differences for a given subunit across species in amino acid sequences in N-terminal, extracellular domains, but also in cytoplasmic and perhaps transmembrane domains, and at what must be at subunit interfaces not heretofore recognized as being functionally important, can strongly influence whether functional ␣6*-nAChR can be produced (4). The fact that m␣6 L9ЈS and V13ЈS mutations differing in position by just one turn in the second transmembrane domain ␣-helix can have such a large difference in their impact on m␣6m␤4*-nAChR function indicates unexpectedly important roles for this channel-lining region in ␣6*-nAChR function. More work is warranted to more thoroughly characterize the bases for these influences.
Our findings demonstrate that ␣6 subunit L9ЈS or V13ЈS modifications can function as reporter and/or gain-of-function mutations, leading to production of receptors with heightened sensitivity to agonists, thus confirming the presence of ␣6 subunits in functional receptor complexes, as expected. These studies also further affirm and recapture the strategy applied to exploit gain-of-function ␣6 subunit mutations expressed in vivo to enhance sensitivity to agonists and thus to help reveal roles played by ␣6*-nAChR in dopaminergic pathways relevant to movement disorders and nicotine dependence (13).
This study was also initiated largely to assess whether effects previously described of nAChR ␤3 subunit incorporation into ␣6*-nAChR would be preserved when receptor functional levels at baseline were intentionally elevated by using reporter mutation/gain-of-function ␣6 subunits as coexpression part-ners. By contrast to earlier work by others (21), in which ␤3 subunits were coexpressed in excess over other subunits, we chose to introduce equal amounts of subunit cRNAs into oocytes for the current work, anticipating that approximately equal amounts of subunit proteins would be made and that this more closely approximates conditions in vivo. We confirmed our previous observations (4,15) that h␤3 subunit incorporation into h␣6h␤4*-nAChR has an uncertain effect on functional expression, that m␤3 subunit incorporation into m␣6m␤4*-nAChR has a dominant-negative effect on receptor function, that h␤3 subunit incorporation into hybrid m␣6h␤4*-nAChR potentiates function, but that there is even larger potentiation of function when h␤3 subunits are incorporated into hybrid m␣6m␤4*-nAChR. However, with the exception of the lack of an obvious effect of m␤3 subunit incorporation on low functioning m␣6 L9ЈS m␤4*-nAChR, wild-type ␤3 subunit incorporation into any of the tested ␣6 (L9ЈS or V13ЈS) ␤4-nAChR potentiated levels of function by Ͼ11-fold, notably including effects of h␤3 subunits on low functioning m␣6 L9ЈS m␤4*-nAChR and effects of m␤3 subunits on m␣6 V13ЈS m␤4*-nAChR. These findings indicate that ␤3 subunits do not always have dominantnegative effects on ␣6*-nAChR function as suggested earlier (21) and do not always promote formation of dead end, ␣6␤4*-nAChR intermediates as suggested previously (23). Instead, based on our results shown here, we can hypothesize that ␤3 subunits seem to promote assembly, cell surface expression, and/or functional responsiveness of ␣6␤4*-nAChR, at least when there is enough function for ␣6␤4(non-␤3)-nAChR to allow assessment of effects of ␤3 subunit incorporation. Our FIGURE 5. Functional properties of gain-of-function h␣6(N143D؉M145V) L9ЈS h␤3*-nAChR. A, representative traces are shown for inward currents in oocytes held at Ϫ80 mV, responding to application at the indicated concentrations of nicotine (shown with the duration of drug exposure as black bars above the traces), and expressing nAChR h␣6(N143DϩM145V) L9ЈS , h␤2, and h␤3 subunits. B, results for these and other studies averaged across experiments were used to produce concentration-response curves (ordinate, mean normalized current Ϯ S.E.; abscissa, ligand concentration in log M) for inward current responses to nicotine for oocytes expressing nAChR h␣6(N143DϩM145V) L9ЈS , h␤2, and h␤3 subunits (E), where current amplitudes are represented as a fraction of the peak inward current amplitude in response to the most efficacious concentration of nicotine. Much higher levels of evoked currents are evident for functional nAChR containing h␣6(N143DϩM145V) L9ЈS , h␤2, and h␤3 subunits when compared with receptors lacking h␤3 subunits. See Table 1 for parameters. findings using the oocyte expression system are in line with observations made regarding ␤3 subunit effects on ␣6*-nAChR functional expression in cell lines (22), suggesting that successful, functional ␣6␤4*-nAChR expression in oocytes does not require coexpression with chaperones missing from oocytes but present in neurons or selected cell lines. Notably, although peak current potentiation upon substitution of gain-of-function ␣6 subunits (or ␤3 subunits; see Refs. 4 and 15) occurs along with an increase in agonist potency, wild-type ␤3 incorporation into complexes increases peak current responses without affecting agonist potency.
In almost every case, ␣6 (L9ЈS or V13ЈS) *-nAChR spend a finite amount of time in a spontaneously open channel state, as judged by the ability of mecamylamine to block those open channels, giving the appearance of production of outward currents. This is a common feature for nAChR containing subunits with second transmembrane domain mutations that give gainof-function effects (27,28). Interestingly, the absolute magnitudes of responses to mecamylamine generally are quite similar across all the ␣6*-nAChR studied (7.8 -12 nA), even when magnitudes of agonist-induced inward currents varied much more widely (26 -800 nA). The only exceptions are for m␣6 V13ЈS m␤4-nAChR, which curiously have no reproducible responses to nicotine or to mecamylamine, despite there being strong responses upon incorporation of m␤3 subunits to form m␣6 V13ЈS m␤4m␤3-nAChR, and for m␣6 L9ЈS h␤4h␤3-nAChR, which have slightly larger responses to mecamylamine (41 nA) but also have the largest responses to nicotine (870 nA).
Although the current findings support a role for ␤3 subunits in potentiating function of ␣6␤4*-nAChR with at least a modicum of baseline functional activity, we were confounded in our studies of ␣6␤2*-nAChR by a general lack of function. This made it impossible to assess effects of ␤3 subunit incorporation on ␣6␤2*-nAChR, but the results indicate that any gain-offunction earned by incorporation of ␣6 (L9ЈS or V13ЈS) subunits into complexes is inadequate to reveal effects of ␤3 subunits, perhaps due to the surprising incompatibilities (illuminated in Refs. 4 and 15) that often occur in attempts to use ␣6, ␤2, and ␤3 subunits to form functional receptors. In order for us to show that in fact a variant of gain-of-function h␣6 subunit can partner with h␤2 and h␤3 subunit to form functional nAChR, we took advantage of our site-directed mutagenesis work (4,15), which has implicated ␣6 residues 143 and 145 in the ability of ␤3 subunits to affect ␣6␤2*-nAChR function. The h␣6(N143DϩM145V) mutations change the indicated residues to those that are in the m␣6 subunit and permit mutated h␣6 subunits to show function when coexpressed with h␤2 and h␤3 subunits when wild-type h␣6 subunits do not. Human nAChR ␣6 subunit residues 143 and 145 are in the E1 domain, in loop E, on the (Ϫ) or complementary face of the subunit. This suggests that interactions between the ␣6 subunit (Ϫ) face with the (ϩ) face from either ␤2 subunits or ␤3 subunits are important for functional ␣6*-nAChR expression. In order for us to prove that the nAChR ␤3 subunit does affect the function of ␣6␤2*-nAChR, a 9Ј mutation was introduced into the h␣6(N143DϩM145V) subunit. Although coexpression of h␣6(N143DϩM145V) L9ЈS and h␤2 subunits did not yield receptors with reliable function, upon inclusion of the h␤3 sub-unit, function was evident in all oocytes coexpressing the three subunits together. These h␣6(N143DϩM145V) L9ЈS h␤2*-nAChR mimic the gain-of-function, high-affinity m␣6*-nAChR artificially expressed in mouse midbrain dopamine neurons (13).
We conclude, based on the current and previous findings, that gain-of-function/reporter mutations introduced into ␣6 subunits in ␣6(␤2 or ␤4)␤3-nAChR are effective in potentiating receptor function. This potentiation yields receptors with higher agonist potency and larger magnitude responses to agonists, and also a finite likelihood of existing in a spontaneously open channel state. We also conclude from the present studies that wild-type ␤3 subunit incorporation into functionally competent (␣6 or ␣6(N143DϩM145V)) (L9ЈS or V13ЈS) (␤4 or ␤2)*-nAChR has a potentiating effect irrespective of whether there are dominant-negative, null, or potentiating effects of ␤3 subunits on wild-type ␣6(␤2 or ␤4)*-nAChR. In fact, reliable expression of functional gain-of-function ␣6*-nAChR is achieved only in the presence of nAChR ␤3 subunits. These results suggest that wild-type ␤3 subunit coexpression is at least permissive for cell surface expression of ␣6␤4*-nAChR and very likely promotes function of these receptors. The strategies and results demonstrated here to increase function of ␣6*-nAChR to levels compatible with drug screening could facilitate the development of new drugs selective for ␣6*-nAChR. This is of increasing importance given the potentially important roles for ␣6*-nAChR in movement and movement disorders, mood disorders, and drug reinforcement (5, 13, 29 -31).