As a highly versatile signal, Ca2+ operates over a wide temporal range to regulate many different cellular processes, impacting nearly every aspect of cellular life including excitability, exocytosis, motility, apoptosis, and transcription. While it has been well recognized that Ca2+ acts as both a second messenger to regulate cell-cell communication upon external stimuli and as a first messenger to integrate extracellular with intracellular signaling in various cell types. Molecular bases for such regulation and related human diseases are largely hampered by the challenges related to key membrane proteins. In the present study, we first investigated the regulatory role of intracellular Ca2+ ([Ca2+]i) on Connexin45 (Cx45) gap junction through a ubiquitous Ca2+ sensor protein-Calmodulin (CaM). Using bioluminescence resonance energy transfer assay, this study provides the first evidence of direct association of Cx45 and CaM in a Ca2+-dependent manner in cells. Complementary approaches including bioinformatics analysis and various biophysical methods identified a putative CaM-binding site in the intracellular loop of Cx45 with high Ca2+/CaM-binding affinity and Ca2+-dependent binding mode that is different from alpha family of connexins. To understand the role of extracellular calcium in regulation of gap junction hemichannels, we would like to prove a possible Ca2+-binding site predicted by our computational algorithm MUGSR in Connexin 26 (Cx26) through mutagenesis study, metal binding affinity measurement, conformational changes examination of purified Cx26 protein from Sf9; however, we failed to achieve this goal due to either the limitation of available methods or lethal effect of mutating the predicted Ca2+-binding ligand. Additionally, in this study, we identified a putative Ca2+-binding site in metabotropic glutamate receptor 5 (mGluR5) and demonstrated the importance of this Ca2+-binding site in activation of mGluR5 and modulating the actions of other orthosteric ligands on mGluR5. In addition, we successfully solved the first crystal structure of the extracellular domain of Ca2+-sensing receptor (CaSR) bound with Mg2+ and an unexpected Trp derivative. The extensive study of mechanism of CaSR function specifically through Mg2+-binding site and the unexpected ligand-binding site was done using several cell-based assays in wild type CaSR and mutants. Studies in this dissertation provides more information on how Ca2+ regulates gap junction channels, modulates mGluR5 activities and structural basis for regulation of CaSR by Mg2+ and an unexpected Trp derivative co-agonist.

Durante o desenvolvimento pós-natal do hipocampo, as junções comunicantes (JC) formadas por conexinas (Cxs) neuronais participam na maturação da circuitaria hipocampal promovendo a regulação da atividade espontânea sincronizada neuronal. Neste estudo investigamos as duas Cxs neuronais mais abundantes no hipocampo, a Cx36 e Cx45, durante o desenvolvimento pós-natal. Identificamos mudanças nos níveis de transcritos e proteicos da Cx36 e Cx45 ao longo deste período. Nossos resultados revelaram que ambas as Cxs neuronais estão presentes nas sub-regiões do hipocampo e que sua distribuição é modulada em função da progressão do desenvolvimento. Atráves da avaliação dos níveis de atividade neuronal identificamos diferenças exercidas pelo bloqueio das JCs em hipocampo de neonatos e na segunda semana de vida. Nossos resultados mostram que as Cxs neuronais são reguladas durante o desenvolvimento pós-natal do hipocampo, assim como sua ação sobre sua excitabilidade, mostrando que as Cxs podem contribuir de forma distinta em periodo específicos do desenvolvimento hipocampal. / Gap junctions (GJ) composed of neuronal connexins (Cx) play a significant role in the activity-dependent circuitry maturation promoting modulation of coherent spontaneous neuronal activity. Herein, we evaluate two major hippocampal neuronal Cxs, Cx36 and Cx45 during postnatal development. We identified changes in Cx36 and Cx45 transcript and protein levels during these developmental periods. Interestingly, immunofluorescence analyses showed that Cx36 and Cx45 are located in all hippocampal subregions. Also, neuronal Cx distribution in these sub-regions is modulated throughout postnatal development. Using electrophysiological recording, we identified changes imposed by GJ blocker over the hippocampal activity in neonates and two-week-old rats. Our finds demonstrate a regulation of neuronal Cxs and distinct GJ role in activity modulation during postnatal hippocampal development, which might be essential to physiological processes that govern proper hippocampal development.

Recently, we demonstrated the beneficial effects of engineered heart tissues for the treatment of dilated cardiomyopathy in rats. For further development of this technique we started to produce engineered tissue (ET) from mesenchymal stem cells. Interestingly, we observed a malignant tumor invading the heart with an inverse relationship between proliferation markers and connexin expression.