Brain function depends on the ability of neurons to sense and respond to electricity, which is mediated by small modules in the neuronal membrane called voltage-sensor domains (VSDs). Disruption of VSD function can cause neurological disease such as epilepsy. VSDs contain positively charged amino acids that move in response to changes in membrane potential. This movement transfer energy to other coupled effectors, such as the pore of a voltage-gated ion channel. In this thesis, I have studied the physiology and regulation of ion-channel VSDs, as well as their role in disease.

Voltage-gated ion channels are composed of four VSDs that controls the opening of a central ion-conducting pore. Voltage-gated potassium (K_V) channels are tetramers assembled by four subunits, where each subunit consists of a VSD and 1/4 of the pore. In contrast, voltage-gated sodium (Na_V) and voltage-gated calcium (Ca_V) channels are pseudotetramers composed of four non-identical, concatenated subunits (repeats I-IV). Our genes encode a broad repertoire of voltage-gated ion channels, promoting diversity and specialization of neuronal subtypes. Specifically, 40 K_V-, 9 Na_V-, and 10 Ca_V-channels have been identified. This thesis includes studies on i) VSD operation in the Ca_V2.2 channel, known for its role in pain transmission, ii) G-proteins Gβγ inhibition of Ca_V2.2 VSDs, a potential tool to control pain, and iii) characterization of two different epilepsy-associated mutations in the VSD of the K_V1.2 channel, important for repolarization of the action potential. To do this, the methods voltage-clamp fluorometry (VCF) under cut-open oocyte voltage clamp mode using Xenopus oocytes, or flow cytometry using a mammalian cell line (COS-7) were used.

VCF was implemented in the human Ca_V2.2 channel and VSD activation in relation to pore opening was characterized. The voltage dependence of VSD-I activation was found to correlate with pore opening, VSD II is likely immobile (it did not generate any VCF signals), VSD III activated at very negative potentials, and VSD IV activation had similar voltagedependence to that of pore opening. Next, Gβγ-inhibition of the VSDs was explored. VSD I was strongly and proportionally inhibited compared to pore opening, VSD III was unaffected and VSD IV was modestly inhibited. In the following studies, the role of the K_V1.2-VSD in disease was explored. Two different epilepsy-associated mutations in the VSD of K_V1.2 were characterized. The first mutation, F302L, facilitated channel activation and spontaneous closure (inactivation) without affecting surface trafficking. The second mutation, F233S, caused a severe surface trafficking deficiency, extending to WT-subunits and closely related K_V1.4 partner subunits. In conclusion, VSDs of ion channels are fundamental for the complexity of our nervous system, their regulation can be used to further diversify neurons or to control excitability, and their importance is revealed by disease-associated mutations that prevent normal function.