Regulation of the L-type Ca2+Channel by Calmodulin

Voltage-gated L-type (class C-type=CaV1.2) Ca2+ channels are pore-forming membrane-bound proteins that govern diverse physiological functions. The activity of L-type Ca2+ channels is regulated by Ca2+ entry. This project aims at quantitative analysis of the molecular mechanism of calmodulin (CaM) action in mediating Ca2+- dependent inactivation of class C-type Ca2+ channels employing a combined approach of electrophysiology and atomic force microscopy. Whole-cell and particularly single channel current parameters will be correlated with single molecule interaction forces. While Ca2+-dependent binding of CaM to an IQ CaM binding motif in the carboxyl-tail of alpha1C has been sufficiently established, the functional involvement of the recently identified CaM tether site at resting cell Ca2+ is less documented. The functional importance of this CaM binding region (CBR) will be electrophysiologically studied by using either peptide sequences corresponding to CBR or alpha1C subunits with alterations in CBR both of which are expected to interfere with the Ca2+-dependent inactivation process. Results obtained with the CBR alpha1C mutants will be correlated with atomic force microscopy measurements on the respective carboxyl tail proteins. Here interaction forces of CaM with CBR and IQ motif in dependence of Ca2+/CaM concentrations will be determined. Furthermore, conformational changes of carboxyl tail proteins induced by Ca2+/CaM will be measured by force sensing, and studying protein mechanics will resolve additional structural details. Calmodulin mutants with intact EF hands but with altered structural coupling between or within N- and C-terminal lobes will be screened for a dominant negative effect on Ca2+-dependent inactivation. Interaction forces of these CaM mutants with CBR and IQ motif will be correlated with electrophysiological results to gain molecular insight in a potential inhibitory effect. Complementary, domains and subregions of CaM interacting with CBR and IQ motif will be further characterized and interaction forces determined. We will additionally test for intracellular domains of the Ca2+ channel, including also other CaM binding motifs, that may interact with the carboxyl tail, possibly via CaM in mediating Ca2+ channel inactivation. Another putative IQ CaM binding motif identified in the beta subunits will be investigated for its involvement in modulating Ca2+-dependent inactivation. Fluorescent-labeled components will be additionally employed to visualize in vivo interactions of alpha1C with CaM.

Voltage-gated (L-type) and non voltage-gated (TRPV6) Ca2+ channels are pore-forming membrane-bound proteins that govern diverse physiological functions. The activity of these Ca2+ channel types is regulated by Ca2+ entry. This project aimed at quantitative analysis and comparison of the molecular mechanisms of calmodulin (CaM) action in mediating Ca2+-dependent inactivation of both Ca2+ channels employing a combined approach of electrophysiology, atomic force as well as fluorescence microscopy and surface plasmon resonance (Biacore). The interaction of CaM with the L-type (Cav1.2) Ca2+ channel was initially probed by Biacore measurements and then extended to atomic force microscopy by establishing the methodology to measure CaM interaction with His6- tagged Ca2+ channel fragments. Here, a novel functional surface was developed that is based on covalently attached self-assembled monolayers (SAMs) on ultraflat gold containing NTA-thiols. This enabled specific, oriented and functional binding of the His6-tagged Ca2+ channel C-terminus and characterization of its Ca2+- dependent interaction with CaM at the single molecule level. This methodology was also successfully used to characterise the membrane interaction of beta 2-glycoprotein. Fluorescence Resonance Energy Transfer (FRET) methodology was carefully established employing expression of CFP/YFP-labeled proteins in living cells and revealed beta-type dependent modulation of L-type Ca2+ channel gating. This provided the basis for sophisticated comparison of Ca2+/CaM-dependent inactivation of L-type and TRPV6 Ca2+ channels. Moreover, it revealed a novel mechanism of Ca2+-dependent gating of the Cav1.4 L-type Ca2+ channel which is found in a a1-subunit mutation associated with human Congenital Stationary Night Blindness Type-2.