The carboxyterminus of the alpha1C subunit, a key structure for L-Type Ca2+ channel regulation by calpastatin and Ca2+

The L-type Ca2+ channel of heart muscle is essential for muscle contraction. Ca2+ entry through this channel initiates Ca2+ release from sarcoplasmic reticulum resulting in muscle contraction. The understanding of the molecular mechanisms involved in Ca2+ channel regulation is therefore a prerequisite for therapeutic (pharmacological) interventions. The Ca2+ channel consists of four subunits, o which the alpha1 subunit is regarded as the most important. Its structure provides all features essential to ac voltage-dependent channel such as activation, permeation (pore) and inactivation. In addition, Ca2+ channel activity is modulated by several intracellular processes of which phosphorylation is best characterized. Less is known on L-type Ca2+ channel regulation by calpastatin and Ca2+. Calpastatin maintains the Ca2+ channel in an active state, while Ca2+ induces channel inactivation. The hypothesis to be tested in this project proposes the carboxyterminus of the alpha1 subunit of the L-type Ca2+ channel as target for both calpastatin and Ca2+. Thus, it should be demonstrated that Ca2+ channel activity is indeed related to an interaction with calpastatin, and in extension of this result it will be examined whether Ca2+ inactivates Ca2+ channels by disturbing this interaction. Several methods will be employed in an attempt to elucidate the proposed interaction of calpastatin with the Ca2+ channel. The patch-clamp technique will be initially employed to study the dependece of Ca2+ channel activity on calpastatin interaction. Then it will be combined with novel microscopies such as single molecule microscopy and molecular recognition force microscopy to visualize this interaction and to correlate it with Ca2+ channel activity. Further, molecular biology and biochemical methods will be employed to detect in vitro interaction of calpastatin and the carboxyterminus of the alpha1 subunit. These techniques will be also applied to test for the postulated mechanism of Ca2+-induced inactivation. Furthermore, mutations in the carboxyterminus of the alpha1c subunit will be performed to locate domains essential for the inaction of calpastatin and Ca2+ with Ca2+ channel. Identification of calpastatin as an endogenous agonist of L-type Ca2+ channels would reveal a novel pathway of cellular regulation of this widely distributed channel type. Conformation of a direct interaction with the Ca2+ channel as well as localization of the domains essential for this interaction will significantly enhance our knowledge on structure-function relationships of the Ca2+ channel. Moreover, involvement of this regulatory mechanism in the Ca2+-induced inactivation process might provide a simple and effective mechanism for transient down-regulation of Ca2+ channel activity.

2+ This project aimed at resolving cellular mechanisms of L-type Ca channel regulation employing a combination of electrophysiology, biochemical and molecular biology techniques as well as fluorescence microscopy. A sequence (aa 1572-1651) in the carboxyterminus of the a 1C subunit was identified as molecular determinant for targeting, conductance, kinetics and run-down of L-type Ca2+ channels. The role of calmodulin in the Ca2+-induced inactivation process was analyzed at the single channel level by using a calmodulin mutant deficient in Ca2+ binding. A binding site for CaM at resting cell Ca2+ concentrations was identifed functionally in the a 1C subunit and the interaction with calmodulin biochemically characterized. An in vivo modulatory effect on L-type Ca2+ channel activity was observed by manipulating endogenous calpastatin levels with calpastatin sense and antisense cDNA. Additionally, we found that the ß 2a subunit controls fast gating of alpha1C-b channels and proposed a model consistent with multiple interaction sites, including AID as a determinant of the affinity of the alpha1-beta interaction (in collab. with K. Groschner). We further succeeded in resolving fluorescent (YFP)-labeled single Ca2+ channels in life cells (in collab. with T. Schmidt). Recently, we suggested S-nitrosation as an important determinant of Ca2+ channel gating and conductance (in collab with K. Groschner).