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PLoS One
2016 Jan 01;116:e0158032. doi: 10.1371/journal.pone.0158032.
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Mutation Linked to Autosomal Dominant Nocturnal Frontal Lobe Epilepsy Reduces Low-Sensitivity α4β2, and Increases α5α4β2, Nicotinic Receptor Surface Expression.
Nichols WA
,
Henderson BJ
,
Marotta CB
,
Yu CY
,
Richards C
,
Dougherty DA
,
Lester HA
,
Cohen BN
.
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A number of mutations in α4β2-containing (α4β2*) nicotinic acetylcholine (ACh) receptors (nAChRs) are linked to autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE), including one in the β2 subunit called β2V287L. Two α4β2* subtypes with different subunit stoichiometries and ACh sensitivities co-exist in the brain, a high-sensitivity subtype with (α4)2(β2)3 subunit stoichiometry and a low-sensitivity subtype with (α4)3(β2)2 stoichiometry. The α5 nicotinic subunit also co-assembles with α4β2 to form a high-sensitivity α5α4β2 nAChR. Previous studies suggest that the β2V287L mutation suppresses low-sensitivity α4β2* nAChR expression in a knock-in mouse model and also that α5 co-expression improves the surface expression of ADNFLE mutant nAChRs in a cell line. To test these hypotheses further, we expressed mutant and wild-type (WT) nAChRs in oocytes and mammalian cell lines, and measured the effects of the β2V287L mutation on surface receptor expression and the ACh response using electrophysiology, a voltage-sensitive fluorescent dye, and superecliptic pHluorin (SEP). The β2V287L mutation reduced the EC50 values of high- and low-sensitivity α4β2 nAChRs expressed in Xenopus oocytes for ACh by a similar factor and suppressed low-sensitivity α4β2 expression. In contrast, it did not affect the EC50 of α5α4β2 nAChRs for ACh. Measurements of the ACh responses of WT and mutant nAChRs expressed in mammalian cell lines using a voltage-sensitive fluorescent dye and whole-cell patch-clamping confirm the oocyte data. They also show that, despite reducing the maximum response, β2V287L increased the α4β2 response to a sub-saturating ACh concentration (1 μM). Finally, imaging SEP-tagged α5, α4, β2, and β2V287L subunits showed that β2V287L reduced total α4β2 nAChR surface expression, increased the number of β2 subunits per α4β2 receptor, and increased surface α5α4β2 nAChR expression. Thus, the β2V287L mutation alters the subunit composition and sensitivity of α4β2 nAChRs, and increases α5α4β2 surface expression.
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27336596
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Fig 1. Transfection of N2a cells with just α4 or β2 fails to produce significant surface expression.
Transfection of just α4 or β2 cDNA without the complementary subunit failed to produce significant α4 or β2 protein expression in the plasma membrane (PM). The α4 and β2 subunits were tagged with superecliptic pHluorin (SEP) and imaged with total internal reflection fluorescent (TIRF) microscopy. a. TIRF images of N2a cells expressing SEP-tagged α4 (α4-SEP) (top row) and β2 subunits (β2-SEP) (bottom row) at an extracellular pH of 7.4 (left column) and 5.5 (right column). The absence of a significant effect of extracellular pH on cellular fluorescence shows that SEP-tagged protein expression on the PM was negligible. Scale bars are 10 μm. b. The bars are the internal (INT), total (TOT), and PM fluorescent intensity measured in arbitrary units (A.U.) for N2a cells transfected with α4-SEP or β2-SEP cDNA. Internal and total fluorescence was not significantly different (N.S.) for the α4-SEP, or β2-SEP, subunit.
http://dx.doi.org/10.1371/journal.pone.0158032.g001
Fig 2. β2V287L alters the surface expression and subunit stoichiometry of SEP-labeled α4β2 nAChRs.
a. TIRF images of α4-SEP fluorescence in N2a cells transfected with α4-SEPβ2 (top row), or α4-SEPβ2V287L, cDNA (bottom row) at an extracellular pH of 7.4 (left column) and pH 5.5 (right column). Reducing the pH from 7.4 to 5.5 dimmed cellular fluorescence because surface SEP-tagged subunits do not fluoresce at pH 5.5. Scale bars are 10 μm. b. The bars (from left to right) are the PM, INT, and TOT fluorescent intensities for cells transfected with α4-SEPβ2 (WT) or α4-SEPβ2V287L (VL) cDNA. The β2V287L mutation significantly (p < 0.001,***) reduced the TOT, INT, and PM α4-SEP fluorescence. c. Images of β2-SEP fluorescence in cells transfected with α4β2-SEP (top row), or α4β2V287L-SEP, cDNA (bottom row) at an extracellular pH of 7.4 (left column) and pH 5.5 (right column). d. The bars are the PM, INT, and TOT fluorescent intensities for cells transfected with α4β2-SEP (WT) or α4β2V287L-SEP (VL) cDNA. The β2V287L mutation significantly (p < 0.001) reduced PM β2-SEP fluorescence, but did not affect TOT fluorescence. It also significantly (p < 0.01,**) increased INT fluorescence.
http://dx.doi.org/10.1371/journal.pone.0158032.g002
Fig 3. WT and mutant ACh concentration-response relations using unbiased and α4-biased subunit injection ratios.The α4β2 and α4β2V287L receptors were expressed in Xenopus oocytes using either a 1:1 (a-b, e) or 10:1 α4:β2 mRNA injection ratio (c-d, f). a-b. the traces are voltage-clamped ACh responses of oocytes injected with α4β2 (a) or α4β2V287L (b) in a 1:1 α4:β2 stoichiometric ratio (w/w). The ACh concentrations (in μM) are listed on the left of, or below, the traces. The bars above the traces show the timing and duration of the ACh application. The downward deflections of the trace are ACh-induced inward currents. For clarity, only a subset of the 18 responses recorded from each oocyte is shown. c-d, Voltage-clamped α4β2 (c) and α4β2V287L responses (d) using a 10:1 α4:β2 injection ratio. e-f. Normalized ACh concentration-relations for the α4β2 (filled circles) and α4β2V287L nAChRs (open circles) using 1:1 (e) and 10:1 (f) α4:β2 injection ratios. Lines are fits to the sum of two hyperbolic binding components using non-linear least-squares regression, subject to the constraints described in the text (Results). The data or individual oocytes were normalized to the observed maximum response for each oocyte, pooled across oocytes, and re-normalized to the fitted maximum response (Methods). Symbols are the means of 7â17 oocytes. Error bars are SEMs in this, and subsequent, figures (obscured by the symbols at some ACh concentrations). Holding potential = -60 mV.
Fig 4. ACh concentration-response relations for α5α4β2 and α5α4β2V287L nAChRs.The α5α4β2 and α5α4β2V287L receptors were expressed in Xenopus oocytes using a large excess of α5 mRNA (α5:α4:β2 mRNA injection ratio of 10:1:1 w/w/w) to ensure that α5-containing (α5*) nAChRs were the predominantly expressed subtype. a-b. Voltage-clamped ACh responses of oocytes expressing α5α4β2 (a) or α5α4β2V287L receptors (b). c. Normalized ACh concentration-relations for the WT α5α4β2 (filled circles), and mutant α5α4β2V287L (open circles), nAChRs superimpose. The lines are fits to a single hyperbolic binding component using non-linear least-squares regression. The data for both receptors were normalized to the fitted maximum response to facilitate comparison of their ACh sensitivities. Symbols are the means of 4â8 oocytes. Holding potential = -60 mV.
Fig 5. Fluorescent responses of α4β2, α4β2V287L, α5α4β2, and α5α4β2V287L nAChRs to 5 and 300 μM ACh.ACh responses were measured using a membrane-potential-sensitive fluorescent dye. a. The bars are peak responses to 5 and 300 μM ACh of α4β2 (WT) and α4β2V287L (mutant) receptors (measured in relative fluorescent units (RFU)). HEK cells were transfected with α4 and β2 cDNA in a 1:1 stoichiometric ratio (w/w). The WT and mutant responses were measured in matched groups of cells transfected on the same day, incubated for the same time, and tested on the same day. All responses were normalized to the peak WT 300 μM ACh response. Sample sizes (n = number of culture wells) are given in parentheses inside the bars. Connecting lines above the bars in this, and subsequent, figures indicate statistical comparisons between various groups. Asterisks give the significance levels for post hoc comparisons between the groups. Not significant (N.S.) b. Similar analysis for HEK cells transfected with α5α4β2 (WT), or α5α4β2V287L (mutant), cDNA in a 1:1:1 α5:α4:β2 stoichiometric ratio.
Fig 6. ACh concentration-response relations for α4β2, α4β2V287L, α5α4β2, and α5α4β2V287L nAChRs using a membrane-potential dye.a. β2V287L shifted the α4β2 ACh concentration-response to the left and reduced the maximum response. The data were normalized to the 1 mM ACh α4β2 response. The filled circles are α4β2 responses, and the open circles, α4β2V287L responses. The lines are fits to the three-parameter Hill equation (see Table 2 for the fitted parameters). b. Co-expression with α5 increased the maximum response of cells transfected with the mutant receptor cDNA (α5α4β2V287L) relative to the WT control (α5α4β2). The data were normalized to the 1 mM ACh α5α4β2 response. The α5:α4:β2 cDNA transfection ratio was 1:1:1 (w/w). The filled squares are α5α4β2 responses, and the open squares, α5α4β2V287L responses. All else was the same as in a.
Fig 7. β2V287L increased the ACh sensitivity of α4β2 nAChRs in N2a cells but reduced maximum response.The α4 subunit was labeled with an enhanced green fluorescent protein (eGFP) tag to facilitate the identification of cells expressing α4β2 nAChRs. The cells were voltage-clamped at -60 mV in whole-cell mode. a. Normalized ACh concentration-response relations for the α4-eGFPβ2 (filled circles) or α4-eGFPβ2V287L receptors (open circles). The α4-eGFPβ2 and α4-eGFPβ2V287L ACh responses were normalized to the peak 100 μM response. The α4-eGFPβ2 symbol at 100 μM ACh is obscured by that for α4-eGFPβ2V287L. The dashed (α4-eGFPβ2V287L) and solid lines (α4-eGFPβ2) are fits to the three-parameter Hill equation (see text for values of fitted parameters). b-c. Superimposed traces of α4-eGFPβ2 (black) and α4-eGFPβ2V287L (red) responses to 300 ms applications of 1 (b) and 100 μM ACh (c). At 1 μM ACh, the α4-eGFPβ2V287L response was larger than α4-eGFPβ2 response. At 100 μM ACh, it was smaller. Downward arrows denote the onset of a 300 ms ACh application.
Fig 8. β2V287L increased α5 incorporation into surface α4β2* nAChRs.a. TIRF images of α5-SEP fluorescence in N2a cells co-transfected with α5-SEPα4β2 (top row), or α5-SEPα4β2V287L, cDNA (bottom row) at an extracellular pH of 7.4 (left column) and pH 5.5 (right column). b. Bar graphs of the PM, INT, and TOT fluorescent intensities of cells transfected with α5-SEPα4β2 (WT) or α5-SEPα4β2V287L (VL). The β2V287L mutation significantly (p < 0.001,***) increased TOT and PM α5-SEP fluorescence but did not affect INT fluorescence.
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