Domain specific contributions to CaV1.2 gating

L-type calcium channels (Cav1.2) channels open upon changes in the membrane potential (a process called activation) before returning either to the closed (resting) state or entering an inactivated (closed) state. Unlike potassium channels (Kv) that are composed of four identical domains Cav1.2 are composed of four concatenated structurally different domains. In my previous studies I have shown that mutations in a gating sensitive region of segment IIS6 (LAIA motif, Hohaus et al. 2005, Beyl et al. 2007, Stary et al. 2008) produce a very strong effect on channel activation while comparable mutations in segment IS6 have only minor effects or result in non conducting channel constructs (Kudrnac et al. 2009). These and other data suggest asymmetric contributions of different domains to channel activation and a particular role of domain II in opening of the channel. Mutations in segment IVS6 of Cav are apparently more important for inactivation (see Hering et al. 2000 for review). The molecular basis of domain specific effects on channel gating is currently unknown. It may reflect for instance interactions in the pore region (segments IS6-IVS6), differences in the voltage sensor movements in different domains or both. It is even feasible that current flow through Cav1.2 is triggered predominantly by a single voltage sensor of a particular domain. It is also not clear if the conformational changes during activation in different domains are independent from each other or energetically coupled. A major aim of this project is to analyse how structural differences in domains I - IV contribute to Cav1.2 gating and how different domains interact with each other. I aim to identify novel gating determinants in pore lining and adjacent channel regions. I will also investigate potential interactions between these residues in different domains by means of mutant cycling analysis of paired mutations (see for example Kudrnac et al. 2009). Charge neutralisation and charge reversal in IS4 - IVS6 segments, gating current measurements, fluorescent imaging and homology modelling will be employed to understanding domain- specific contributions of voltage sensors to activation and inactivation gating. Kinetic changes induced by point mutations in gating sensitive regions will be analysed in terms of a novel Cav1.2 gating model (Beyl et al. 2009). The calculated rate constants of Cav1.2 gating enable the evaluation of mutational effects on the voltage sensitive gating machinery or voltage independent conformational changes during pore opening (Beyl et al. 2009). These studies are expected to contribute to better understanding of the mechanism of Cav1.2 activation.

Understanding the structure and functional mechanisms of voltage-gated calcium channels is a major task in membrane biophysics and structural biology. When project P22600 was started we focused on mutational studies in the pore forming S6 segments to identify potentially distinct role of four domains in channel gating. During those studies we identified the new activation determinants in pore forming IIIS6 segment (G1193, see Beyl et al. 2011) and later in other S6 segments (Depil et al. 2011). At the same time we developed an approach (mutation correlation analysis) to analyze how physicochemical properties of amino acids in pore forming S6 segments affect activation gating of CaV1.2 (Beyl et al. 2011). To interpret functional changes in channel gating caused by mutations in pore forming S6 segments homology modeling techniques were applied. Homology modeling revealed that G1193 forms part of a highly conserved structure motif (G/A/G/A) of small residues in homologous positions of all four domains (G432 (IS6), A780 (IIS6), G1193 (IIIS6), A1503 (IVS6)). A hypothesis was formulated that in all four domains residues G/A/G/A are in close contact with larger bulky amino acids from neighboring S6 helices. These interactions apparently provide adhesion points for tight sealing of the activation gate of CaV1.2 in the resting closed state (Depil et al. 2011). Other functionally important unit of the voltage-gated channel is voltage sensor. Voltage sensors trigger the closedopen transitions in the pore of voltage-gated ion channels. To probe the transmission of voltage sensor signalling to the channel pore of CaV1.2, we investigated how elimination of positive charges in the S4 segments affects channel gating. Neutralization of all positively charged residues in IIS4 produced a functional channel, while replacement of the charged residues in IS4, IIIS4 and IVS4 segments resulted in nonfunctional channels. Interestingly, neutralization of IIS4 had no detectable effects on channel kinetics (Beyl et al. 2012). However, we observed that replacing the charged residues in IIS4 can rescue (reverse) gating distortions in pore-forming S6 segments (Beyl et al. 2012). Thermodynamic cycle analysis supports the hypothesis that IIS4 is energetically coupled with the distantly located G/A/G/A residues. This finding significantly extended our understanding of CaV1.2 gating. We speculate that conformational changes caused by neutralization of IIS4 are not restricted to domain II (IIS6) but are transmitted to gating structures in domains I, III and IV via the G/A/G/A ring. A novel (cooperative) gating model was formulated in Beyl et al. (2012). In Beyl et al. (2013) we developed our four state gating model and provide an algorithm enabling for the first time the estimation of gating parameters from macroscopic current kinetics off individual channel constructs. Further investigations during this project concerned the location of the diltiazem binding site on CaV1.2 (Shabbir et al. 2011).