Depolarization-evoked opening of CaV2.1 (P/Q-type) Ca2+-channels triggers neurotransmitter release, while voltage-dependent inactivation (VDI) limits channel availability to open, contributing to synaptic plasticity. The mechanism of CaV2.1 response to voltage is unclear. Using voltage-clamp fluorometry and kinetic modeling, we optically track and physically characterize the structural dynamics of the four CaV2.1 voltage-sensor domains (VSDs). The VSDs are differentially sensitive to voltage changes, both brief and long-lived. VSD-I seems to directly drive opening and convert between two modes of function, associated with VDI. VSD-II is apparently voltage-insensitive. VSD-III and VSD-IV sense more negative voltages and undergo voltage-dependent conversion uncorrelated with VDI. Auxiliary β-subunits regulate VSD-I-to-pore coupling and VSD conversion kinetics. Hence, the central role of CaV2.1 channels in synaptic release, and their contribution to plasticity, memory formation and learning, can arise from the voltage-dependent conformational changes of VSD-I.

The present study aimed to characterize the SCN5A variant I1333V, found in five families with a history of suspected catecholaminergic polymorphic ventricular tachycardia (CPVT). SCN5A encodes the pore-forming subunit of the cardiac voltage-gated sodium channel NaV1.5. Gain of SCN5A function causes long QT syndrome type 3 (LQT3), but its involvement in CPVT is disputed. Nineteen patients harboring the I1333V variant were identified across five families, commonly presenting with exercise-induced arrhythmia, including polymorphic premature ventricular contractions, ventricular bigeminy, couplets, and ventricular tachycardias. Prolonged QT interval was a less consistent finding, and structural myocardial changes were absent. Human NaV1.5/β1 complexes were expressed in Xenopus laevis oocytes, using RNA combinations to emulate homozygous wild-type, heterozygous and homozygous I1333V-mutant conditions. Cells were studied using the cut-open oocyte Vaseline gap voltage-clamp to evaluate effects of I1333V on NaV1.5 function. NaV1.5(I1333V) channels required less depolarization to activate, classifying this variant as gain-of-function. Fast inactivation was unaffected, and action-potential (AP) clamp showed no significant differences in late Na+ current. A computational model of human ventricular myocyte excitability predicted no effect of I1333V on AP duration; instead, it showed stronger Na+ influx during the AP upstroke, concurrent with elevated Ca2+ import via the sodium‑calcium exchanger. Finally, NaV1.5(I1333V) channels exhibited a diminished response to cAMP (emulating adrenergic stimulation), which also likely contributes to arrhythmogenesis. In conclusion, I1333V is a gain-of-function variant of SCN5A with a unique set of functional consequences. It is associated with cardiac arrhythmia disease characterized by overlapping CPVT-like and LQT3 features. Our findings support that SCN5A should be considered in genetic screening of suspected CPVT.

How G proteins inhibit N-type, voltage-gated, calcium-selective channels (Ca_V2.2) during presynaptic inhibition is a decades-old question. G proteins Gβγ bind to intracellular Ca_V2.2 regions, but the inhibition is voltage dependent. Using the hybrid electrophysiological and optical approach voltage-clamp fluorometry, we show that Gβγ acts by selectively inhibiting a subset of the four different Ca_V2.2 voltage-sensor domains (VSDs I to IV). During regular “willing” gating, VSD-I and -IV activations resemble pore opening, VSD III activation is hyperpolarized, and VSD II appears unresponsive to depolarization. In the presence of Gβγ, Ca_V2.2 gating is “reluctant”: pore opening and VSD I activation are strongly and proportionally inhibited, VSD IV is modestly inhibited, while VSD III is not. We propose that Gβγ inhibition of VSDs I and IV underlies reluctant Ca_V2.2 gating and subsequent presynaptic inhibition.

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.