Human nACh b2 -- Val 287

rVal262Leu

The V262L mutation reduced the effect Ca²⁺ had on the effect of the 30mM ACh response.

This mutation significantly increased steady-state desensitization. The fast desensitization time constant was not affected. The slow desensitization time constant was reduced 44% in 0mM Ca²⁺, but there was no significant difference in 2mM Ca²⁺. The mean fractional amplitude for the fast desensitization was increased approximately 2-fold in 2mM Ca²⁺ and no effect was seen in 0mM Ca²⁺.

The ratio of the peak ACh responses in 2 and 0 mM Ca²⁺ (I_Ca²⁺/I₀) was significantly reduced at 30mM ACh. At 10nM and 50nM ACh, this ratio was not significantly affected.

The V262L mutation significantly reduced the high and low affinity EC50 of ACh. However, for low (>30mM) levels of ACh, the fractional response was increased.

Also studied: a4 S252F, a4 +L264, a4 S256L and b 2 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

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rVal262Met

The V262M mutation reduced the effect Ca²⁺ had on the effect of the 30mM ACh response.

This mutation significantly increased steady-state desensitization. The fast desensitization time constant was not affected. The slow desensitization time constant was not affected.

The ratio of the peak ACh responses in 2 and 0 mM Ca²⁺ (I_Ca²⁺/I₀) was significantly reduced at 30mM ACh. At 10nM and 50nM ACh, this ratio was not significantly affected.

When WT a4 cRNA was injected along with V262Ma4, the I_Ca²⁺/I₀ was not significantly different from when the V262Ma4 cRNA was injected without the WT.

The V262L mutation did not significantly affect the high- affinity EC50 of ACh, but the concentration-response relationship lacked a noticeable low-affinity component. For low (>30mM) levels of ACh, the fractional response was increased.

When V262M was the co-expressed with E180Q, the Ca²⁺ blocked the 30 mM ACh response. The I_Ca²⁺/I₀ of the E180Q 30 mM ACh response was 0.85 +/- 0.05, but for E180Q/V262M, it was 0.60 +/- 0.01.

Also studied: a4 S252F, a4 +L264, a4 S256L and b2 V262L.

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

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Incubation with 320 mM Carbamazepine inhibited the ACh-evoked currents. The effect on the V287M mutant was greater than on the S252L mutant. Choline evoked a current with an EC50 value of 0.43 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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