Low-voltage-activated (T-type) calcium channels play a crucial role in a number of physiological processes, including neuronal and cardiac pacemaker activity and nociception. Therefore, finding specific modulators and/or blockers of T-type channels has become an important field of drug discovery. The aim of this thesis is to look at how different molecules block T-type calcium channels and to look at developing new molecules to expand the limited arsenal of drugs known to modulate this important ion channel family.
First, I examined similarities between T-type channels and their better characterized cousins, the voltage-gated sodium channels (Nav). T-types are thought to be evolutionary related to Navs as they share similar membrane topology and gating kinetics. Simple Multiple Sequence Alignment also revealed several highly conserved regions between T-type and Nav channels that corresponded to drug binding sites known to alter voltage-dependent gating kinetics. I thus reasoned that certain drugs acting on Nav may also modulate T-type channels.
My first experiments determined that a selective Nav1.8 drug (A803467) also bound selectively and potently to T-types in an area of the channel corresponding to the local anesthetic binding site found in Nav channels.
I next examined another well-characterized Nav blocker, spider toxins. Two tarantula toxins, Protoxin I and Protoxin II, had been shown to be potent and selective blockers of Nav channels and my research confirmed that these two toxins also potently and selectively blocked both Cav 3.1 and Cav 3.2 respectively.
The second part of my thesis looked at developing new compounds to block or modulate T-type channels. Studies have suggested that certain dihydropyridines (DHPs), a well known class of L-type blocking drugs, may also block T-type. We therefore synthesized a series of novel DHP derivatives and our results indicated modifying the ester substituent dramatically increased our compounds’ ability to block L-type and T-type calcium channels and furthermore, substituting the ester group with 3-pyridylmethyl, conferred approximately 30-fold selectivity to T-type versus L-type calcium channels. We then took our most effective blocker and structurally similar compounds and demonstrated in-vivo that they were efficacious in reducing pain responses in mice subjected to peripheral inflammation or nerve injury.
Finally, in the last section of my thesis, I examine some novel organic molecules that have structures similar to lipoamino acids, which are endogenous molecules known to interact with T-type calcium channels. We tested their inhibitory effects on T-type channels and then took the most effective inhibitor and demonstrated that it was also efficacious in animal models of pain.