Mutation Causing Autosomal Dominant Nocturnal Frontal Lobe Epilepsy Alters Ca²⁺ Permeability, Conductance, and Gating of Human α4β2 Nicotinic Acetylcholine Receptors

Functional differences between wild-type and mutant α4β2 AChRs. Top, Use-dependent functional upregulation of the responses mediated by mutant α4β2 AChRs.Left, Currents induced by the first and fifth application of 3 μm ACh are shown for oocytes expressing wild-type and mutant α4β2 AChRs. Oocytes that did not have previous exposure to the agonists were held at −50 mV. ACh was applied at 2 min intervals. Right, Plot of the response peak amplitude on the initial five consecutive applications of 3 μm ACh on the oocytes expressing wild-type (open circles) or mutant α4β2 AChRs (filled circles). Currents were normalized to the peak amplitude of the first response.Bottom, S247F mutation causes significant changes in the desensitization of the α4β2 and α4β2α5 AChRs.Left, Comparison of the time course of the superimposed normalized averaged currents mediated by the wild-type (thin trace) and mutant (thick trace) α4β2 AChRs.Right, Comparison of time course for the wild-type and mutant α4β2α5 AChRs. Averaged currents were obtained from 15 to 22 oocytes by normalizing to the same current amplitude. Oocytes were held at −50 mV. Both perfusion and agonist solution contained no Cl⁻ ions to prevent contamination with endogenous Ca²⁺-dependent Cl⁻ current. Oocytes were preincubated in Cl⁻-free media for 4–16 hr.  

Comparison of pharmacological properties of human wild-type and mutant α4β2 AChRs. Top, Concentration/response curves for ACh and nicotine for wild-type and mutant α4β2 AChRs. Middle, Inhibition of mutant α4β2 AChR-mediated currents by DHβE, quinine, and amantadine. Equivalent inhibition of wild-type α4β2 AChRs was produced at these concentrations of DHβE and quinine. Control responses superimposed with responses induced by coapplication of ACh and antagonists, and responses induced after 5 min washout, are shown. Bottom left, Comparison of the voltage dependence of the inhibition of the mutant α4β2 AChRs by quinine (thin line) and amantadine (thick line) are shown. Voltage dependence is represented by plotting the amount of the inhibition (I_c −I_a)/I_c · 100% against holding potential, where I_c is the current in the presence of the ACh (100 μm), andI_a is the current induced by coapplication of the ACh (100 μm) and quinine (10 μm) or amantadine (100 μm). Currents were measured by the application of 2 sec voltage ramps from −100 to +50 mV to the oocytes before and during application of the agonist or coapplication of the agonist and antagonist. Passive currents induced before application of the agonist were subtracted. Bottom right, Concentration/response curves built for amantadine inhibition of wild-type (open circles) and the mutant (filled circles) α4β2 AChRs. Data obtained from four to six oocytes held at −50 mV were normalized to the control responses induced by 100 μm ACh, averaged, and fit by using the Hill equation (EC₅₀ and IC₅₀ = values, and nH values are listed in the text).  

S247F mutation of the α4 subunit eliminates Ca²⁺ permeability of the α4β2 AChRs, which was restored by coexpression of α5 subunits. Top, Shift of the reversal potential of wild-type (left) and mutant (right) α4β2 AChRs induced by a 10-fold increase of Ca²⁺ concentration from 1.8 to 18 mm. Representative currents induced by the application of voltage ramps on oocytes perfused by 100 μm ACh with 1.8 mm(dashed trace) or 18 mmCa²⁺ (solid trace) in the solution are plotted against membrane potential. Currents induced by the ramps in agonist-free solutions are subtracted. Recordings were performed in Cl⁻-free solutions on the oocytes preincubated in the Cl⁻-free media. Middle, Plot of the reversal potential shifts induced by a 10-fold increase of the extracellular Ca²⁺ concentration (from 1.8 to 18 mm) for wild-type (open bars) and mutant (filled bars) α4β2 and α4β2α5 AChRs. Averaged data obtained from 7 to 14 oocytes as described on thetop panel represent mean value ± SE.Bottom, Currents conducted only by Ca²⁺ ions through wild-type and mutant α4β2 AChRs. Left, Currents induced in normal ND-96 media and an equiosmotic solution containing 1.8 mmCa²⁺ as the only permanent ion were compared for both wild-type and mutant α4β2 AChRs. Currents were induced by 100 μm ACh in Cl⁻-free media.Right, Plot of the percentage of the inward current induced in the “Ca²⁺-only” (1.8 mm) solution as compared with the current in the normal ND-96 solution for wild-type (open bar) and mutant (shaded bar) α4β2 AChRs. Bar in the negative direction for the mutant indicates that in some experiments currents were outward in the “Ca²⁺-only” conditions. Data were obtained from seven oocytes preincubated in the Cl⁻-free media for each AChR; mean values and SE are plotted.

Single channel currents of the human wild-type and mutant α4β2 AChRs. Top, Records are from outside-out patches taken from oocytes that were injected with either human α4β2 or human αS247F mutant α4β2 AChRs. Currents were recorded at −80 mV by steady-state application of 50 nmACh, with the patch isolated in a continuously flowing stream of agonist diluted from stock into ND-96 solution. Channel activity was initiated by lateral displacement of the perfusion tube across the face of the recording electrode. Wild-type AChRs exhibited two conductance levels, as indicated by the dashed lines, whereas mutant AChRs exhibited predominantly a single conductance level. For display, records were sampled at 10 kHz and filtered at 2 kHz (eight-pole Bessel, −3 dB). Bottom, Both wild-type and mutant AChR single channel currents were blocked by 1 μm DHβE. Currents were activated originally by 50 nm ACh by rapid movement of the perfusion tube across the tip of the recording electrode. High levels of expression allowed robust inward currents to be observed because of massive simultaneous channel activations. After ∼1 min of wash, agonist was reapplied along with 1 μmDHβE. The initial channel activity was significantly lower as compared with the level in the absence of antagonist. After the first 3–5 sec, the channel activity was essentially gone because of inhibition by DHβE. The inhibition was reversible within 60 sec (time for solution change).