Tryptophan Substitutions Reveal the Role of Nicotinic Acetylcholine Receptor -TM3 Domain in Channel Gating: Differences between T
详细信息 查看全文
- 作者：Manuel Navedo ; Madeline Nieves ; Legier Rojas ; and Jose A. Lasalde-Dominicci
- 刊名：Biochemistry
- 出版年：2004
- 出版时间：January 13, 2004
- 年：2004
- 卷：43
- 期：1
- 页码：78 - 84
- 全文大小：225K
- 年卷期：v.43,no.1(January 13, 2004)
- ISSN：1520-4995
文摘
A recent tryptophan scanning of the 
-TM3 domain of the Torpedo californica AChRdemonstrated that this domain can modulate ion-channel gating [Guzman, G., Santiago, J., Ricardo, A.,Martí-Arbona, R., Rojas, L., Lasalde-Dominicci, J. (2003) Biochemistry 42, 12243-12250]. Here weextend the study of the -TM3 domain to the muscle-type AChR by examining functional consequencesof single tryptophan substitutions at five conserved positions (M282, F284, V285, A287, and I290)homologous to the -TM3 positions that were recently characterized in the Torpedo AChR. Similarly tothe Torpedo AChR, mutations M282W and V285W, which are presumed to face the interior of theprotein, did not exhibit functional channel activity. Nevertheless, significant expression levels of thesemutants were observed at the oocyte surface. In contrast to the Torpedo AChR, in the muscle-type AChR,tryptophan substitution at positions F284, A287, and I290 produces a significant increase in normalizedmacroscopic response. Single-channel recordings at low ACh concentration revealed that the increase inAChR sensitivity for the F284W, A287W, and I290W is due to an increase in the mean open duration.These results suggest that tryptophan substitution directly affects channel gating, primarily the channelclosing rate. Our results suggest that residues facing the interior of the protein (i.e., M282 and V285)may similarly affect channel gating in Torpedo and muscle-type AChR. However, equivalent mutations(i.e., F284W and I290W) presumably facing the lipid environment display a very different functionalresponse between these two AChR species.
-TM3 domain of the Torpedo californica AChRdemonstrated that this domain can modulate ion-channel gating [Guzman, G., Santiago, J., Ricardo, A.,Martí-Arbona, R., Rojas, L., Lasalde-Dominicci, J. (2003) Biochemistry 42, 12243-12250]. Here weextend the study of the -TM3 domain to the muscle-type AChR by examining functional consequencesof single tryptophan substitutions at five conserved positions (M282, F284, V285, A287, and I290)homologous to the -TM3 positions that were recently characterized in the Torpedo AChR. Similarly tothe Torpedo AChR, mutations M282W and V285W, which are presumed to face the interior of theprotein, did not exhibit functional channel activity. Nevertheless, significant expression levels of thesemutants were observed at the oocyte surface. In contrast to the Torpedo AChR, in the muscle-type AChR,tryptophan substitution at positions F284, A287, and I290 produces a significant increase in normalizedmacroscopic response. Single-channel recordings at low ACh concentration revealed that the increase inAChR sensitivity for the F284W, A287W, and I290W is due to an increase in the mean open duration.These results suggest that tryptophan substitution directly affects channel gating, primarily the channelclosing rate. Our results suggest that residues facing the interior of the protein (i.e., M282 and V285)may similarly affect channel gating in Torpedo and muscle-type AChR. However, equivalent mutations(i.e., F284W and I290W) presumably facing the lipid environment display a very different functionalresponse between these two AChR species.