Voltage sensors and pore determinants in cooperative gating of CaV1.2

Voltage gated L-type calcium channels (CaV1.2) open during a membrane depolarisation when voltage-sensing S4 segments move from their resting (down) towards their activated (up) position. Deactivation (closure) of open channel is initiated by downward movement of S4 segments during hyperpolarization and subsequent translocation of the S6 segments towards the resting position. CaV1.2 gate almost normally (close to wild type) after neutralisation of all five charges of segment IIS4. This indicates either a loose coupling between voltage sensors and the CaV1.2 pore or, alternatively, a more important role of other voltage sensors than IIS4 in CaV1.2 gating. My previous studies also revealed that a single S4 segment (IIS4) interacts not only with the S6 gate of domain II but also with segments IS6, IIIS6 and IVS6. It was proposed that this transmission occurs via cooperative interactions between gating structures of S6 segments (Beyl et al. 2012). Conserved small residues (GAGA ring) in segments IS6-IVS6 interacting with bulky hydrophobic residues in neighboring S6 segments are assumed to stabilize the closed channel pore in a cooperative manner (Depil, Beyl et al., 2011). The molecular mechanism of such cooperative gating in CaV1.2 is unknown and subject of the current application. In order to elucidate the underlying molecular base I will apply site-directed mutagenesis, fluorometry, biophysical methods and homology modelling. Potentially cooperative mechanisms in CaV1.2 closure will be investigated by introducing paired mutations into neighboring S6 residues. Systematic structural changes in the S4-S5 linker region will be induced to mimic the gating distortions induced by neutralization of charge in the corresponding S4 segment. The minimal voltage sensor charge required to open and to close CaV1.2 gates will be estimated by consecutively neutralising the charged S4 residues. Insights into the individual roles of S4 segments in CaV1.2 gating and corresponding interactions with the channel pore will be obtained by means of fluorometric approaches. The comparison of the fluorometric and biophysical data (activation/deactivation kinetics) will clarify how structural changes in S4 segments are transmitted to gate structures of neighbouring domains and thus help to understand the cooperative gating in CaV1.2.

Tuned calcium entry through voltage-gated calcium channels is a key requirement for many cellular functions. This is ensured by channel gates which open during membrane depolarizations and seal the pore at rest. The gating process is determined by distinct sub-processes: movement of voltage-sensing domains (charged S4 segments) as well as opening and closure of pore gates (pore forming S6 segments). Neutralization of S4 charges revealed that pore opening of voltage-gated calcium channel (CaV1.2) is triggered by a "gate releasing" movement of all four voltage-sensing segments with activation of IS4 (and IIIS4) being a rate-limiting stage. Segment IS4 additionally plays a crucial role in channel inactivation. Remarkably, S4 segments carrying only a single charged residue efficiently participate in gating. However, the complete set of S4 charges is required for stabilization of the open state. Making use of biophysical (patch clamp) and pharmacological studies, mathematical simulations (incl. the estimation of gating parameters) and recently published structural data enabled us to propose an novel gating scheme for voltage gated calcium channels. Our concept of channel gating provides novel interpretations of channelopathy mutations, in particular the pore region and on voltage sensing S4 segments.