TABLE 1.
Summary of ACh concentration-response data for wild-type and mutant α4β2-nAChR in HS (1:5) and LS (5:1) isoform preparations
The loop location for each mutation is shown, and the cRNA injection ratio indicates the biased receptor population (HS 1:5 and LS 5:1 α4:β2, respectively). In the LS isoform preparation (5:1 α4:β2), the ACh concentration-response data were best fit using either monophasic or biphasic fits. Specifically, mutants β2(W176A), β2(D179A), β2(D217A), and β2(Y127A) were best fit using a biphasic logistic equation (5:1 comparison of fits test results, where P < 0.05 resulted in rejection of the data being best fit using a monophasic logistic equation: α4β2 F3,37 = 0.5804, P = 0.6316; α4β2(W176A) F3,104 = 2.863, P = 0.0404; α4β2(T177A) F3,38 = 0.03193, P = 0.9922; α4β2(D179A) F3,32 = 5.328, P = 0.0043; α4β2(D217A) F3,42 = 2.937, P = 0.0442; α4(D218A)β2 F3,47 = 0.5280, P = 0.6652; α4β2(D116A) F3,28 = 2.797, P = 0.0585; α4β2(Y120A) F3,38 = 2.542, P = 0.0707l; α4β2(Y127A) F3,33 = 4.517, P = 0.0092). Imax is the maximal ACh-induced current amplitude for each of the receptors. pEC50_HS and pEC50_LS is the apparent ACh potency for each receptor. The nH values are the calculated Hill coefficients. Data are the mean ± S.E.M. Differences between groups were analyzed using one-way analysis of variance with Tukey’s post hoc comparison (see Materials and Methods).
| Subunits | Mutation Loop Location | Ratio (α4:β2) | ACh |
|||||
|---|---|---|---|---|---|---|---|---|
|
Imax ± S.E.M. |
pEC50_HS ± S.E.M. |
nH_HS ± S.E.M. |
pEC50_LS ± S.E.M. |
nH_LS ± S.E.M. |
HS-Phase Function |
|||
| nA | % | |||||||
| High sensitivity | ||||||||
| α4:β2 | 1:5 | 310 ± 60 | 5.2 ± 0.1 | 1.9 ± 0.3 | N/A | N/A | N/A | |
| α4:β2(W176A) | B | 1:5 | 240 ± 30 | 6.0 ± 0.1** | 0.8 ± 0.1 | N/A | N/A | N/A |
| α4:β2(T177A) | B | 1:5 | 3290 ± 1050** | 5.7 ± 0.2 | 5.7 ± 0.2 | N/A | N/A | N/A |
| α4:β2(D179A) | B | 1:5 | 630 ± 190 | 6.0 ± 0.1** | 0.7 ± 0.1 | N/A | N/A | N/A |
| α4:β2(D217A) | C | 1:5 | 120 ± 8 | 5.8 ± 0.1* | 1.1 ± 0.3 | N/A | N/A | N/A |
| α4:β2(D218A) | C | 1:5 | 67 ± 8 | 5.5 ± 0.1 | 1.2 ± 0.1 | N/A | N/A | N/A |
| α4:β2(D116A) | A | 1:5 | 460 ± 160 | 5.2 ± 0.1 | 0.9 ± 0.2 | N/A | N/A | N/A |
| α4:β2(Y120A) | A | 1:5 | 370 ± 84 | 5.7 ± 0.2** | 0.4 ± 0.1 | N/A | N/A | N/A |
| α4:β2(Y127A) | A | 1:5 | 32 ± 1 | 6.4 ± 0.1*** | 0.5 ± 0.1 | N/A | N/A | N/A |
| Low sensitivity | ||||||||
| α4:β2 | 5:1 | 4900 ± 1400 | N/A | N/A | 4.5 ± 0.1 | 1.4 ± 0.3 | N/A | |
| α4:β2(W176A) | B | 5:1 | 30 ± 3* | 5.9 ± 0.2 | 1.1 ± 0.5 | 3.0 ± 1.0 | ∼1 | 0.6 ± 0.4 |
| α4:β2(T177A) | B | 5:1 | 24,200 ± 860*** | N/A | N/A | 3.6 ± 0.7 | 0.5 ± 0.2 | N/A |
| α4:β2(D179A) | B | 5:1 | 7800 ± 1100 | 4.7 ± 0.8 | 1.2 ± 0.7 | 3.9 ± 0.1 | 2.0 ± 0.6 | 0.4 ± 0.5 |
| α4:β2(D217A) | C | 5:1 | 3020 ± 990 | 5.6 ± 0.6 | 1.2 ± 1.4 | 4.5 ± 0.1 | 2.6 ± 1.3 | 0.3 ± 0.3 |
| α4:β2(D218A) | C | 5:1 | 1820 ± 450 | N/A | N/A | 4.4 ± 0.1 | 0.8 ± 0.2 | N/A |
| α4:β2(D116A) | A | 5:1 | 1200 ± 320 | N/A | N/A | 4.2 ± 0.1 | 1.1 ± 0.1 | N/A |
| α4:β2(Y120A) | A | 5:1 | 1940 ± 370 | N/A | N/A | 4.0 ± 0.1 | 1.3 ± 0.2 | N/A |
| α4:β2(Y127A) | A | 5:1 | 180 ± 26* | 5.3 ± 0.3 | ∼1 | 3.7 ± 0.1 | 3.4 ± 1.6 | 0.4 ± 0.2 |
P < 0.05; **P < 0.01; ***P < 0.0001.