The Interaction between DIV Pore loop and DIV S6 in the Na+ Channel
| Autor: | Jui-Yi Hsieh, 謝瑞頤 |
|---|---|
| Rok vydání: | 2006 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 94 Anticonvulsants and local anesthetic drugs block voltage gated sodium channels in a similar use-dependent manner, and are found to bind to a common receptor in the channels. Some researchers believe that this common receptor is located at the internal pore of sodium channels, whereas others believe that the receptor is located at the external pore of sodium channels. Both W1716 at DIV P-loop and F1764 at mid DIV S6 segment have been implicated as the putative receptor site for anticonvulsants and local anesthetics. However, according to the traditional view of sodium channel structure, W1716 is located at the external pore mouth and F1764 is located in the internal pore. In this present study, we tried to explore why and how external and internal pore could affect drug binding affinities simultaneously. To reach this goal, we tested the affinity of carbamazepine (a typical anticonvulsant, CBZ), lidocaine (a typical local anesthetic), or diclofenac (a non-steroid anti-inflammatary drug structurally similar to carbamazepine) to different mutant channels. The affinity changes showed the following pattern for all mutants: lidocaine > CBZ > diclofenac. The attenuation of drug affinity is more significant in mutating W1716 or F1764 to A or C than to E or R. While W1716C/F1764A showed no additive effect on drug binding as compared to corresponding single mutants, W1716E/F1764R showed supra-additive effects. We also characterized the sensitivity of different mutant channels to cadmium ion, and demonstrating an interesting order: W1716C > F1764C > WT ~ W1716C/F1764C. This order strongly implied that the sensitivity of W1716C and that of F1764C were cancelled mutually in double mutant channel W1716C/F1764C. Moreover, while W1716C showed its sensitivity to external MTSET in a state dependent manner, WT and F1764C had no significant modification by external MTSET. Interestingly, as in the case of cadmium sensitivity, we found that F1764C attenuated the sensitivity of W1716C to external MTSET, especially at the inactivated channel. That is to say, W1716C/F1764C mutant channel thus was less rather than more modified than W1716C mutant channel. Although F1764C can not be modified by external MTSET, it can be modified by external MTSEA. Modification of W1716C by MTSET attenuated peak current. In contrast, MTSEA blocked sustain current of F1764C and shifted its inactivation curve negatively. On the other hand, we use RE mutant to strengthen the interaction between W1716 and F1764. Both single mutant channels (W1716E and F1764R) showed little effects on channel gating or ion selectivity. However, W1716E/F1764R double mutation caused changes in the slope factor of the steady state inactivation curve and significant shift of reversal potential. These results supported that W1716 and F1764 may be located in a common region which allows state dependent interaction between these two residues. The traditional view of sodium channel pore structure thus may have to be modified to incorporate our data. From the pharmacological point of view, this state-dependent interaction may constitute the molecular basis of use-dependent drugs, such as anticonvulsants or local anesthetics. W1716 and the external pore mouth may not only be involved in ion selectivity but also serve as a key linkage between the gating machinery and the ion permeation pathway in sodium channels. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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