Human nACh a4 -- Ser 252
|Rat Equivalent||Ser 256||Leu||S256L|
When the a4S252L was injected with b2, functional receptors were produced. Receptors with the muttaion were more sensitive to ACh than cells without it. When only the mutated receptors were injected, the ACh-evoked currents were lower than the control. But, when a4S252L and a4 were injected together, the maximal current was the same as the control.
The dependence of reversal potential versus extracellular calcium concentration and ACh-evoked currents in calcium concentrations were similar for both the a4S252L mutant and the control.
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 0.49 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
back to top
back to nACh a4
When 100nM ACh was applied to this mutation ten times for 150ms, spaced 5 seconds apart; it did not exhibit use-dependent potentiation. It appeared to accelerate the rising phase of the 10nM ACh response. The S256L mutation reduced the effect Ca2+ had on the effect of the 30mM ACh response.
This mutation significantly increased steady-state desensitization. In particular it affected the fast desensitization time constant by reducing it 74% in 0mM Ca2+ and by 72% in 2mM Ca2+, compared to the wild-type. The slow desensitization time constant was reduced 88% in 0mM Ca2+ and 87% in 2mM Ca2+. The mean fractional amplitude for the fast desensitization was increased approximately 2-fold in 0mM Ca2+ and no effect was seen in 2mM Ca2+.
The ratio of the peak ACh responses in 2 and 0 mM Ca2+ (ICa2+/I0) was significantly reduced at 30mM ACh. At 10nM and 50nM ACh, this ratio was not significantly affected.
In the WT, Ba2+ increased the peak amplitude of the 30mM ACh response, but in the S256L mutation, 2mM Ba2+ reduced the response by 16 +/- 3%.
When WT a4 cRNA was injected along with S256L a4, the ICa2+/I0 was not significantly different from when the S256L a4 cRNA was injected without the WT.
The S256L mutation did not significantly affect the EC50 of ACh. However, for low (>30mM) levels of ACh, the fractional response was increased. This mutation had little effect on [3H]epibatidine affinity or surface receptor expression.
Also studied: a4 S252F, a4 +L264, b2 V262L and b2 V262M.
Rodrigues-Pinguet N, Jia L, Li M, Figl A, Klaassen A, Truong A Lester HA, Cohen BN (2003) Five ADNFLE Mutations Reduce the Ca2+ Dependence of the Mammalian a4b2 Acetylcholine Response. Journal of Physiology 550(1):11-26
back to top
back to nACh a4