A depolarization-initiated influx of Ca through voltage-gated Ca (CaV) channels gives rise to a plethora of physiological responses such as neurotransmitter release, muscle contraction and gene expression. Membrane depolarization is sensed by four transmembrane structures, the voltage sensor domains (VSDs), which surround and control the activation, deactivation and inactivation properties of a central Ca-selective pore governing the amount and timing of Ca influx. In contrast to homotetrameric KV channels, the CaV pore and four VSDs are encoded by a single long polypeptide chain (alpha1). Thus, each VSD has a unique primary amino acid sequence, suggesting distinct voltage-sensing properties. Critically, the voltage-sensing processes coupling membrane depolarization to Ca influx are still poorly understood and the molecular mechanisms by which auxiliary subunits, such as beta and alpha2delta, alter the voltage dependence of the channel, still need to be elucidated. This lack of knowledge persists in part because ionic and gating current measurements have not thus far captured the properties of individual VSDs in CaV channels. Using Voltage Clamp Fluorometry (VCF), we have resolved that the time- and voltage-dependent properties of each of the four VSDs of human CaV1.2 revealing their highly distinct functional properties. We now have the experimental tools and theoretical formulation to answer key unresolved questions on the operation of CaV1.2 channels, as delineated in four specific aims: (1) To establish the contribution of individual voltage sensing domains to CaV1.2 channel activation. (2) To establish the molecular mechanism by which accessory subunits regulate voltage-dependent activation of CaV1.2 channels. (2a) regulation by alpha2delta subunits (2b) regulation by beta subunits (3) To determine the role of each VSD in Voltage- and Ca-dependent Inactivation and (4) To develop a CaV1.2 model accounting for the operation and role of the four distinct VSDs. CaV1.2 channels specifically labeled at each VSD with small, environment-sensitive fluorophores will be voltage-clamped using the cut-open oocyte technique, so that voltage-evoked fluorescence changes will reflect local conformational rearrangements. A series of physically-relevant models consistent with the CaV1.2 structure and accounting for all experimentally- resolved aspects of CaV1.2 voltage-dependent operation, including the interactions governing excitation- evoked Ca influx. The innovative aspects of this proposal include (1) the experimental approach, unprecedented for the CaV superfamily; (2) the hypothesis that VSDs are the targets of regulation by modulatory subunits; (3) the premise, supported by striking preliminary results, that CaV1.2 VSDs are drivers and regulators for inactivation; (4) the theoretical approach proposes the first model consistent with the molecular architecture and asymmetry of CaV channels. Finally, this study will contribute to the understanding of the molecular mechanisms of pathological states caused by altered CaV1.2 voltage dependence, such as Timothy Syndrome.
The propagation of the electrical signals in nerves, and the contraction of the heart and muscles are vital physiological processes controlled by a highly specialized class of proteins, which regulate the flux of charged ions into and out of the cells: these proteins are called ion channels. The voltage-sensitive L-type Calcium channels let Calcium ions into the cells, triggering the rhythmic contraction of the heart or regulating the vascular tone; as such, they are targeted by important drugs used to control high blood pressure and cardiac arrhythmias. In this research proposal, we are implementing a cutting-edge experimental approach (voltage clamp fluorometry) to unravel the exquisite molecular mechanism by which these proteins sense and respond to electrical signals, to understand how these proteins work in health and disease, and help direct the design of novel therapies.
| John, Scott; Kim, Brian; Olcese, Riccardo et al. (2018) Molecular determinants of pH regulation in the cardiac Na+-Ca2+ exchanger. J Gen Physiol 150:245-257 |
| Savalli, Nicoletta; Pantazis, Antonios; Sigg, Daniel et al. (2016) The ?2?-1 subunit remodels CaV1.2 voltage sensors and allows Ca2+ influx at physiological membrane potentials. J Gen Physiol 148:147-59 |
| Pantazis, Antonios; Savalli, Nicoletta; Sigg, Daniel et al. (2014) Functional heterogeneity of the four voltage sensors of a human L-type calcium channel. Proc Natl Acad Sci U S A 111:18381-6 |
