As demonstrated previously cocaine can use a wide variety of molecular events to create its disease state. After cocaine binds to its key targets, the body experiences an increase in monoamines, allows for the stimulation of the sympathetic system, leading to vasoconstriction, ischemia and decreased activity in sodium gated channels as they are stuck in the inactivated phase, creating cardiac arrhymias. While a wide variety of wonderful research has been accomplished inspecting these subjects, it is necessary to look beyond the current literature towards what can and should be performed. By assessing the next areas of research this project aims to improve our understanding and treatment of acute cocaine toxicity.
As previously discussed one of the primary mechanisms of acute cocaine toxicity is the use-dependant inhibition of voltage-gated sodium channels by cocaine. Previous research has demonstrated that mutations to the conserved aromatic residue Y1767 weakened both inactivation-dependent and the pore-blocking components of the cocaine inhibition, suggesting that cocaine binds to this common site; however, no further information is presently available regarding how cocaine binds to the inactivated sodium channel, or the changes in conformation associated with this inhibition(O’Leary and Chahine, 2002). Recently a crystal structure of a voltage-gated sodium channel was developed at a resolution of 2.7 angstroms, revealing potential interaction sites of pore blockers such as cocaine. I propose that the next step in understanding the mechanism of cocaine toxicity is to develop a crystal structure of a voltage-gated sodium channel bound to the pore blocker, cocaine. This crystal structure would reveal if there is a specific binding site on the S6 fragment which cocaine binds to and what form of conformational change the channel undergoes when bound to cocaine. From here I would propose using this developed crystal structure to develop a small molecule which could target cocaine-bound inactivated sodium channels, forcing the cocaine out of the channel, creating an antidote to the cardiac arrhymias caused by the local anesthetic properties of cocaine.
Fig 1. C These represent the site of S6 interaction where cocaine binds. Seen in D’s representation of the pore. The first project imagines creating a small molecule to bind beneath S6, causing the cocain to be pushed out and the channel to return to an open position. (O’Leary and Chahine, 2002)
While benzodiazepines effectively stop catecholamine activity, this area could use more specific treatments. To this extent I would like to develop a crystal structure of the norepinephrine reuptake protein bound to cocaine. As this crystal structure has not been developed previously it could be useful to know how cocaine binds to the membrane protein, to characterize whether there are consistently binding proteins which cocaine is situated around. From here I would like to develop a method of dislodging cocaine bound NRI, using something like a hairpin RNA to bind to both cocaine and the receptor. Once bound the hairpin would push on both the receptor and inhibitor, causing cocaine to separate. While there are some issues, such as creating a molecule that will be as specific for the section of cocaine present as the bound receptor, a similar process might allow for the decrease in sympathetic system without binding Alpha receptors.
Fig 2. A and B offer a top and side view of the hydrophobic regions of the sodium channel with the yellow Phe residues representing potential binding sites for cocaine. By completing further mutations and obtaining crystal structures the exact specificity of these Phe residues will be known. (O’Leary and Chahine, 2002)
Finally I would like to further understand how sodium channel inhibitors, known as local anesthetics, bind to the S6 subunit. While previous studies have demonstrated that mutations to S6 limit cocaine binding; this doesn’t inform us whether this binding pocket universally interacts with hydrophobic inhibitors (O’Leary and Chahine, 2002). To this extent I would like to repeat the set of experiments used to mutate S6 and test these mutants on a variety of local anesthetics to elucidate the specificity of Cocaine’s binding to the sodium channel. The results of this study will coincide with the importance of my first proposed experiment, broadening the scope of both experiments.