There is strong pharmacological, biological, and genetic evidence supporting the 
role of N-type calcium channels (CaV2.2) in nociception. There is also human 
validation data from ziconotide, the CaV2.2-selective peptidyl inhibitor used 
clinically to treat refractory pain. Unfortunately, ziconotide utility is 
limited by its narrow therapeutic window and required intrathecal route of 
administration. A major focus has been placed on identifying state-dependent 
CaV2.2 inhibitors to improve safety margins. Much less attention, however, has 
been given to characterizing the kinetics of CaV2.2 inhibitors as a means to 
further differentiate compounds and maximize therapeutic potential. Here we 
provide a detailed characterization of the CaV2.2 inhibitor T4 in terms of its 
state-dependence, use-dependence, kinetics, and mechanism of inhibition. 
Compound T4 displayed a >20-fold difference in potency when measured under 
inactivating conditions (IC50=1.1 μM) as compared to closed-state conditions 
(IC50=25 μM). At 3 μM, T4 produced a 15-fold hyperpolarizing shift in the 
inactivation curve for CaV2.2 while having no effect on channel activation. To 
assess the kinetic properties of T4 in a more physiological manner, its 
inhibition kinetics were assessed at 32°C using 2 mM Ca(2+) as the charge 
carrier. Surprisingly, the repriming rate for CaV2.2 channels at hyperpolarized 
potentials was similar in both the presence and absence of T4. This was in 
contrast to other compounds which markedly delayed repriming. Furthermore, T4 
inhibited CaV2.2 channels more potently when channel inactivation was driven 
through a tonic sub-threshold depolarization rather than through a use-dependent 
protocol, despite similar levels of inactivation.