Hg2+ binding test |
Binding of cocaine was inhibited. |
9, 10
|
Cocaine-binding site is near the extracellular side of TM domain of DAT. |
Epitope-specific immunoprecipitation studies |
Cocaine binding site should be near helix 6 of DAT |
35 |
Cocaine-binding site is formed by α-helices 1, 3, 6, and 8 of DAT |
Trp84Leu mutation |
Cocaine-binding affinity was increased (~8-fold decrease for the apparent dissociation constant). |
28, 39
|
The mutation provides more space to accommodate the cationic head of cocaine at the binding site. |
Phe154Ala and Phe155Ala mutations |
Cocaine-binding affinity was decreased. |
26 |
The mutation weakens the packing between the two residues and the benzoyl ester group of cocaine. |
Phe391Ala mutation |
Cocaine-binding affinity was decreased by ~6-fold. |
26 |
The mutation weakens packing between the residue and the cationic head of cocaine. |
Asp313Asn and Asp313Gln mutations |
Cocaine-binding affinity was increased (~3-fold decrease for the apparent dissociation constant). |
28, 39
|
Arg85-Asp476 salt bridge matches better around cationic head of cocaine after the mutation. |
Leu104Val/Phe105C ys/Ala109Val mutation |
The mutant was ~70-fold insensitive to cocaine inhibition compared to the wild-type DAT. |
4, 29
|
Hydrophobic interactions with Phe105 is destroyed and α-helix 6 stays away from the cocaine-binding site in the mutant. |
Asp79Glu mutation |
The mutation considerably decreased DAT binding with dopamine, but with little change in DAT-cocaine binding. |
30 |
The negatively charged side chain of the residue has direct contact with the cationic head of dopamine, but has not direct contact with that of cocaine. |