Activation gating in L-type calcium channels

This project is initiated by our recent finding that a novel retinal disorder is caused by a point mutation in an L- type Ca2+ channel that is predominantly expressed in the retina. A single point mutation in segment IIS6 (I745T) of the pore forming a1-subunit of these channels (subtype CaV 1.4) shifts the voltage-dependence of CaV 1.4 channel activation by about -30 mV (Hemara-Wahanui, 2005). Subsequent studies of our group in Cav1.2 highlighted the importance of this isoleucine and neighbouring residues in the pore-lining segment IIS6. A unique kinetic phenotype was observed for residues 779-782 (LAIA) located in the lower third of segment IIS6: i) a shift in the voltage dependence of activation of these mutants that was accompanied by ii) a deceleration of activation at hyperpolarized potentials; iii) a deceleration of deactivation at all potentials (I781P and I781T) and iv) decreased inactivation (Hohaus, 2005). These four residues are completely conserved in high voltage-activated calcium channels suggesting that these channels may share a common mechanism of gating. We have hypothesised that, in analogy to potassium channels (Jiang, 2002) and a bacterial sodium channel (NaChBac; Zhao, 2004) there may be a flexible center of helix bending at positions 779-782 in Cav1.2. The molecular mechanism of L-type channel opening and the disturbances in gating causing the retinal disorder are, however, not understood. One of the key questions is the localisation of the activation gate in Cav1.2 and if this gate is affected by I781T or substitution of neighbouring residues. In order to contribute to an answer we will perform systematic mutational and functional studies on heterologeously expressed CaV 1.2 channels. First we focus on identification of further determinants of "pore stability" in S6 segments of Cav1.2. We will analyze the different gating phenotypes of S6 mutants in terms of a mathematical model of channel gating accounting for steady state activation as well as for kinetic changes. This approach will help us to judge about the respective impact of a given mutation on closed and open state stability. A second aim is to understand the interplay between the pore and the voltage sensor regions. We particularly focus on the S4-S5 linker interaction with pore determinants. A structural model for interpreting mutational data has already been developed. This model will be validated in functional studies and be used for molecular modelling studies to determine the role of channel activation gating in Ca2+ channel inhibition by phenylalkylamines, diltiazem and 1,4 dihydropyridines (evaluation of receptor guarding and drug trapping mechanisms). Understanding of the molecular mechanism of CaV 1.2 activation will help to explain the differences in voltage-dependence of activation between L-channel isoforms and clarify the molecular basis of L-type channelopathies accociated changes in the voltage-dependence of activation (e.g. our previous studies in Hemara-Wahanui, 2005; Hohaus, 2005).

Understanding the structure and functional mechanisms of voltage-gated calcium channels is a major task in membrane biophysics and structural biology. When project P19614 was started in 2007 we focused on homology modeling techniques to interpret functional changes in channel gating caused by channelopathy mutations in pore forming S6 segments (Stary et al. 2008 a,b). In a review published in the same year we speculated that voltage sensors and the pore in L-type channels are essentially independent structural units with the voltage sensing machinery as robust "all-or-non" device while the varieties of voltage sensitivities of different channel types was accomplished by shaping pore stability (Hering et al. 2008). In order to quantify the structural changes caused by channelopathy mutations we proposed a circular four-state model accounting for an activation R-A-O and a deactivation O-D-R pathway with transitions between resting-closed (R) and activated-closed (A) states (rate constants x(V) and y(V)) and open (O) and deactivated-open (D) states (u(V) and w(V)) describe voltage-dependent sensor movements (Beyl et al. 2009). In the same paper we reported that a pore mutation (A780P) is likely to affect the movement of the voltage sensors. Subsequently we developed an approach (mutation correlation analysis) to analyze how physicochemical properties of amino acids in pore forming S6 segments affect activation gating of Ca(V)1.2 (Beyl et al. 2011). Homology modeling revealed that Gly-432 forms part of a highly conserved structure motif (G/A/G/A) of small residues in homologous positions of all four domains (Gly-432 (IS6), Ala-780 (IIS6), Gly-1193 (IIIS6), Ala-1503 (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 Ca(V)1.2 in the resting closed state (Depil et l. 2011). Very recently we observed that gating distortions in segments IS6-IVS6 can be rescued (reversed) by replacing the charged residues in IIS4 by glutamines. 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 neutralisation 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. (2013a). In Beyl et al. (2013b) 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 (Beyl et al. 2013b, in press). Further investigations during this project concerned structure-activity studies on Cav3.1 (performed in collaboration with the group of Dr. Laccinova [Karmazinova et al. 2010]) and the location of the diltiazem binding site on Cav1.2 (Shabbir et al. 2011).