Dynamik der cGMP-Synthese in Sehzellen - Regulation membranständiger Guanzylatzkyklasen durch modifizierte Kalzium-Sensor-Proteine

Membrane bound guanylate cyclases GC1 and GC2 are important for the phototransduction in photoreceptors. They regulate, in an interplay with a phosphodiesterase, the concentration of the intracellular messenger cyclic guanosine monophosphate (cGMP). At low calciumconcentrations both GCs are activated by guanylate cyclase activating proteins (GCAP1 and GCAP2). The cGMP-concentration increases and the photoreceptor adapts. If GCs become activated a GC-dimer forms a complex with an unidentified number of GCAPs. The stoichiometry and calcium dependent conformational changes within this complex are unknown. GCAP2 has three cysteine residues, interestingly one in the first and one in the third calcium-binding-motif (EF-hand-motif). In this work cysteine mutants of GCAP2 were generated, heterologously expressed, and purified. All cysteine mutants exhibited EC50- and IC$_{50}$-values comparable to GCAP2-wildtype. However, mutants with no cysteine residue in the first EF-Hand-motif activated GCs weaker compared to GCAP2-wildtype. This indicates an important function of this cysteine residue in GC-regulation. In further experiments I investigated the accessibility of cysteine residues for the thiolreactive substance 5,5'-Dithiobis(2-nitrobenzoic acid). The cysteine residues within the first and third EF-hand-motif were only accessible at low calcium concentrations. This was surprising because the first EF-hand-motif is assumed to bind calcium with only very low affinity. Thus no calcium induced conformational change was expected. By determining the calcium sensitivities of the DTNB-reaction, which could be interpreted as apparent calcium affinities of the EF-hand-motifs, I was able to develop following model: calcium dissociation from the third EF-hand-motif in GCAP2 induces a conformational change that causes GC-activation. Furthermore I demonstrated that magnesium increases the apparent calcium affinities of the first EF-hand-motif in GCAP2 and of the first and third EF-hand-motif in GCAP1. I coupled thiolreactive dyes to cysteine mutants of GCAP2. Thereby I wanted to detect conformational changes in the vicinity of the dye. Furthermore, by recording Förster Resonance Energy Transfer between dye labeled GCAP1- and GCAP2-mutants I wanted to show the simultaneous binding of both GCAP isoforms to GC1. Both experimental approaches gave negative results. Using a soluble, enzymatic active GC1-construct instead of wildtype GC1 produced also not the expected results