Human nACh a4 -- Ser 248

Species Original Mutated to Mutation
Human Ser 248 Phe


Rat Equivalent Ser 252    
Mouse Equivalent      


Oocytes expressing wildtype a4b2 exhibited similar magnitudes in current to oocytes expressing a4S248Fb2. The initial response of the mutant receptor was much faster than the current response of the wildtype at 1mM ACh. After two minutes additional ACh was applied, and the mutant receptors showed a 25% decrease in current response. The mutant receptors showed an accelerated desensitization to ACh.

The wildtype cells responsed to 30 second ACh pulse intervals without desensitization; the current amplitude was similar for both agonist pulses. However, the mutant displayed a 60% reduced response to the second ACh pulse. The recovery rate from desensitization was different for the mutant receptor than the wildtype. The time constant for recovery for the wildtype was 6.4s; for the mutant was 58.8s.

The EC50 values of the mutant and the wild-type were the same.

Weiland S, Witzemann V, Villarroel A, Propping P, Steinlein O (1996) An Amino Acid Exchange in the Second Transmembrane Segment of a Neuronal Nicotinic Receptor Causes Partial Epilepsy by Altering Its Desnsitization Kinetics. Federation of European Biochemical Society Letters 398:91-96


Surface binding of 125I-mAb290 was equivalent for a4b2a5, a4b2, mutant a4b2a5 and mutant a4b2 AChRs. When these receptors were tested for their net charge during response of ACh, the mutant a4b2 AChRs showed 60% less charge than the wildtype. The mutant a4b2a5 AChRs showed 30% less charge than this wildtype.

The mutant receptors did not exhibit any increase of response to applications of an agonist after the initial activation. The wildtype receptors showed a response with a fairly consistent amplitude during consecutive application of an agonist.

Wildtype a4b2 did not show significant desensitization over 30 seconds, whereas the mutant's current decreased rapidly by 50 percent and then continued to decay in the presence of ACh. When a5 was added to the wild-type, the current did decrease to a level at about 65%. When it was added to the mutant, the desensitization was still faster than the wildtype a4b2a5.

ACh EC50 mM ACh Hill Coefficient Nicotine EC50 mM Nictoine Hill Coefficient
a4b2 2.2 +/- 0.1 2.1 0.3 +/- 0.04 1.8
a4S248Fb2 11.6 +/- 1.2 0.9 1.8 +/- 0.6 1.4

The wildtype and mutant receptors showed simliar inhibition by a competitive ACh binding site antagonist and by quinine. The noncompetitive ion channel blocker amantadine had a lower effect on the mutant receptors. The block by amantadine was voltage-dependent, where the inhibition was much greater at more negative potential.

Reversal potential for the a4b2 AChR currents depended on the concentration of Ca2+, but the mutant receptors did not show much change with changing concentrations of Ca2+. However, when a5 subunits were added, the mutant receptors a4b2a5 did reverse and the Ca2+ had a greater effect than the wildtype a4b2a5 receptors.

The channel activity for the wildtype a4b2 AChR lasted for 3-5 minutes before desensitizing under the presence of 50nM ACh inj outside-out patches, whereas the mutant receptor channel activity lasted for only 2-3 minutes. The wildtype channels could be reactivated after a period of recovery, but the mutant channels were inactive after the initial response.

When 1mM DHbE was coapplied with the 50nM ACh, the DHbE effectively antaginzed the channels that were activated by the ACh. When the DHbE was removed and the ACh was applied again, the channels behaved similar to before the presence of the antagonist. In the mutant, the channels did not recover after the removal of the DHbE.

The wildtype receptors had two amplitudes of 1.4 +/- 0.1 and 2.3 +\- 0.1pA, and the mutant showed a single conductance channel type with an amplitude of 0.90 +/- 0.01pA at a holding potential of -80mV.

Kuryatov A, Gerzanich V, Nelson M, Olale F, Lindstrom J (1997) Mutation Causing Autosomal Dominant Nocturnal Frontal Lobe Epilepsy Alters Ca2+ Permeability, Conductance, and Gating of Human a4b2 Nictonic Acetylcholine Receptors. The Journal of Neuroscience 17(23):9035-9047


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The apparent affinity for ACh of this mutant was found to be 19.8 mM, compared to 2mM for the control. Its desensitization is shifted toward lower ACh concentrations by a factor of 3000 or more.

EC50 ACh (mM) nH IC50 ACh (mM) nH EC50 cytisine (mM) nH IMac ACh (nA)
ha4(S248F)b2 19.8 +/- 7.0 0.70 +/- 0.09 0.00028 0.8 1.4 0.9 357 +/- 276

3.4 +/- 2.7

0.70 +/- 0.05 0.11 +/- 0.02 1.4 +/- 0.15 2.12 +/- 1.7 0.79 +/- 0.14 1762 +/- 439


Log intercept
Log intercept
Log intercept
ha4(S248F)b2 1.25 +/- 0.78 -3.70 +/- 0.8 1.05 +/- 0.38 -2.1 +/- 0.8 0.71 +/- 0.3 -3.12 +/- 1.48
control 1.21 +/- 0.17 -5.13 +/- 0.32 1.2 +/- 0.27 -3.16 +/- 0.47 1.24 +/- 0.27 -3.10 +/- 0.62

In the control receptor cytisine evoked about 24.77% of ACh currents, but with this mutation it evoked about 78.2%. The EC50 for cytisine in the control was 2.12 mM, but in the mutant receptors, it was 0.042 mM.

Bertrand S, Weiland S, Berkovic SF, Steinlein OK, Bertrand D (1998) Properties of Neuronal Nictoinic Acetylcholine Receptor Mutants from Humans Suffering from Autosomal Dominant Nocturnal Frontal Lobe Epilepsy. British Journal of Pharmacology 125:751-760

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Receptors with S248F showed ACh-evoked currents of lower amplitude. These receptors showed higher sensitivity to ACh.
Incubation with 320 mM Carbamazepine inhibited the ACh-evoked currents. The effect on the V287M mutant was greater than on the S252L mutant. a4S248F, a4L776ins3 and b2V287M are all more sensitive to CBZ than a4S252L. Choline evoked a current with an EC50 value of 1.5 mM.

Bertrand D, Picard F, Hellard SL, Weiland S, Favre I, Phillips H, Bertrand S, Berkovic SF, Malafosse A, Mulley J (2002) How Mutations in the nAChRs Can Cause ADNFLE Epilepsy. Epilepsia 43(Suppl. 5):112-122

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