Atrial fibrillation (AF), the most common sustained arrhythmia, is associated with abnormal intracellular Ca2+ handling. Understanding AF requires comprehensive understanding of ionic currents, Ca2+ handling, phosphorylation regulation and related signalling pathways, but appropriate models are limited. The aim of this thesis is to develop an ionic model of the mouse atrial myocyte to investigate the cellular proarrhythmic mechanisms. We have developed the first mouse atrial myocyte model that incorporates mathematically detailed ion channels, cellular Ca2+ and Na+ handling and their regulation by Ca2+-calmodulin-dependent protein kinase II (CaMKII) and protein kinase A. For the first time, the inositol 1,4,5-trisphosphate (IP3) production system and its effects on excitation-contraction coupling have also been described. The validated model predicted that: 1) hyperactivity of CaMKII and elevated intracellular Na+ concentration are the crucial factors that induce sarcoplasmic reticulum (SR) Ca2+ spontaneous release and delayed afterdepolarisations; 2) β-adrenergic stimulation may have proarrhythmic effects by exacerbating Ca2+ overload; and 3) enhanced activity in ryanodine receptors during IP3-induced Ca2+ release is the major cause of the arrhythmogenesis in IP3 signalling.

Membrane depolarization is an effective neurotrophic stimulus, with its trophic effect on spiral ganglion neurons (SGNs) even surpassing that of neurotrophins. Thus, SGN cultures are a favorable system to investigate pro-survival signal transduction downstream of depolarization. Depolarization promotes SGN survival by recruiting three distinct kinase pathways: cyclic AMP-dependent protein kinase (PKA), Ca2+/calmodulin-dependent protein kinase II (CaMKII) and CaMKIV. CaMKIV mediates the pro-survival effect of depolarization by activating CREB in nucleus. However, the mechanisms by which PKA and CaMKII promote survival are still not clear. By targeting constitutively active PKA or a PKA inhibitor (PKI) to the outer mitochondrial membrane (OMM), we showed that PKA activity at the OMM is sufficient to support SGN survival in the absence of other trophic factors and necessary for cAMP-dependent SGN survival. It has been suggested that PKA can promote survival by inactivating pro-apoptotic protein Bad. By cotransfection of SGNs with OMM-PKA and wild-type Bad, we showed that this was the case. We further showed that Ser112 and Ser136 in Bad, but not Ser155, a hypothetical PKA target, were necessary for functional inactivation of Bad by PKA. CaMKII mediates the third depolarization-dependent pro-survival pathway. A specific pro-survival target for CaMKII was identified through a separate investigation of the pro-apoptotic JNK-Jun signaling pathway, which we had identified as active in apoptotic SGNs in vivo. By measuring anti-phosphoJun immunofluorescence, we could quantify JNK-Jun activation in SGNs under different conditions. We showed that JNK inhibition or genetic deletion of JNK3 reduces SGN death after neurotrophic factor withdrawal. Neurotrophins have been shown to suppress JNK activation via their receptor protein tyrosine kinases (PTKs). By expressing constitutively active and dominant negative forms of candidate protein kinases, we identified a novel signaling pathway linking depolarization to JNK: Ca2+ entry - CaMKII - FAK/Pyk2 - PI-3-OH Kinase - Protein Kinase B - inhibition of MLKs (upstream activators of JNK). Thus, depolarization also recruits PTKs - the nonreceptor PTKs FAK and Pyk2 - to suppress JNK activation, implying a conserved PTK-PI3K-PKB pathway for suppression of pro-apoptotic JNK activation by neurotrophic stimuli.

Our memories are fundamental components of who we are as individuals. They influence almost every aspect of our lives such as our decisions, our personalities, our emotions, and our purpose in life. Diseases that affect memory have devastating impacts on the individuals who bear them. Imagine not being able to recall pleasant memories or even the faces of close family members. It's important to understand the biology of memory formation not only because it's an intriguing scientific question, but because of its consequences when these processes are lost. N-methyl-D-aspartate-type glutamate receptors (NMDARs) and calcium/calmodulin-dependent kinase II (CaMKII) are essential molecules involved in learning and its physiological correlate, synaptic plasticity. Calcium influx through NMDARs activates CaMKII, which translocates to the postsynaptic signaling sites through its interactions with the NMDAR subunits NR1 and NR2B. The significance of CaMKII's translocation is not fully known, however we hypothesize that it is an early molecular event that is necessary for the expression of synaptic plasticity and learning. Our laboratory has developed two strains of mice with targeted mutations to NR1 and NR2B (NR1KI and NR2BKI) that are deficient in their ability to bind to CaMKII to test the role of CaMKII binding to NMDARs in synaptic plasticity and learning. We found that CaMKII binding to NR2B is necessary for long-term potentiation (LTP), the most commonly studied form of synaptic plasticity. NR2BKI mice are able to learn spatial and cued tasks normally, however they are unable to consolidate spatial tasks for long-term memory storage. On the other hand, we found that CaMKII binding to NR1 is not necessary for LTP. Furthermore NR1KI mice do not show impairments in contextual or cued learning. We found that NR1 mutations resulted in an age-dependent truncation of the intracellular domains of NR1 that reduced its activity leading to severe impairments in synaptic transmission, LTP, and learning. Our results suggest that CaMKII binding to NR2B is the more important for synaptic plasticity and memory formation than NR1. However, we found that the intracellular domains of NR1 are critical for NMDAR and synapse function.

The Sarco-endoplasmic Ca2+ ATPase (SERCA2a) plays a crucial role in sequestering cytosolic calcium into the sarco-endoplasmic reticulum (SR/ER) and is an important regulator of muscle contraction and relaxation. Recent findings suggest that a novel CAMKIIα splice variant, αKAP, that plays the role of a CAMKII anchoring protein in the myocardium, also directly interacts with SERCA2a. We examined the effects of αKAP on SERCA2a activity using transfection of HEK-293T cells as well as lentiviral infection of primary neonatal mouse cardiomyocytes (NMCM). Our data showed that αKAP reduced Ca2+ ATPase activity, and downregulated SERCA2a expression in both HEK-293T cells coexpressing αKAP and SERCA2a, as well as NMCM overexpressing αKAP. Interestingly in a rat model of myocardial infarction, αKAP expression was found to be elevated, alongside elevated CaMKIIδ, and depressed SERCA2a expression. These data suggest that αKAP may be a unique regulator of SERCA2a activity and cardiac function.

The kisspeptin receptor (KISS1R), functioning as a metastasis suppressor and gatekeeper of GnRH neurons, is a potent activator of intracellular Ca2+. The surge in cytoplasmic Ca2+ mediates the exocytosis of GnRH from GnRH neurons. However, the regulatory processes which enable KISS1R to sense increasing intracellular Ca2+ and avoid Ca2+ excitotoxicity via a signalling off-switch mechanism remain unclear. This thesis provides evidence for the interaction between KISS1R and the Ca2+ regulated proteins of calmodulin (CaM), and αCa2+/CaM-dependent-protein kinase II (α-CaMKII). Binding of CaM to KISS1R was shown with three independent approaches. Firstly, cell-free spectrofluorimeter assays showed that CaM selectively binds to intracellular loop (IL) 2 and IL3 of the KISS1R. Secondly, KISS1R co-immunoprecipitation experiments identified ligand/Ca2+-dependent binding of KISS1R to HEK-293 endogenous CaM. Thirdly, confocal experiments showed CFPCaM co-localises with YFP-KISS1R. The functional relevance of CaM binding was examined with alanine substitution of critical residues of the CaM binding motifs in IL2 and IL3 of KISS1R. This approach revealed that the receptor activity (relative maximum responsiveness) was increased in the mutated residues of the juxtamembrane regions of IL3 and the N-terminus of IL2 relative to wild-type KISS1R. The Ca2+/CaM regulated αCaMKII was also found to interact with KISS1R by selectively phosphorylating T77 of IL1. Phosphomimetic mutations of T77 into E or D created a receptor that was unable to elicit inositol phosphate production upon ligand stimulation. Finally, in vivo studies using ovariectomised rats that were intracerebroventricularly administered with a cell-permeable αCaMKII inhibitor augmented the effects of kisspeptin ligand stimulation of plasma luteinizing hormone levels. Taken together, this thesis demonstrates that the KISS1R-G protein coupling is regulated by Ca2+-dependent CaM binding and αCaMKII-mediated KISS1R phosphorylation.

The Sarco-endoplasmic Ca2+ ATPase (SERCA2a) plays a crucial role in sequestering cytosolic calcium into the sarco-endoplasmic reticulum (SR/ER) and is an important regulator of muscle contraction and relaxation. Recent findings suggest that a novel CAMKIIα splice variant, αKAP, that plays the role of a CAMKII anchoring protein in the myocardium, also directly interacts with SERCA2a. We examined the effects of αKAP on SERCA2a activity using transfection of HEK-293T cells as well as lentiviral infection of primary neonatal mouse cardiomyocytes (NMCM). Our data showed that αKAP reduced Ca2+ ATPase activity, and downregulated SERCA2a expression in both HEK-293T cells coexpressing αKAP and SERCA2a, as well as NMCM overexpressing αKAP. Interestingly in a rat model of myocardial infarction, αKAP expression was found to be elevated, alongside elevated CaMKIIδ, and depressed SERCA2a expression. These data suggest that αKAP may be a unique regulator of SERCA2a activity and cardiac function.

No description available.

Indiana University-Purdue University Indianapolis (IUPUI) / Voltage-gated sodium channels (Navs) undergo remarkably complex modes of modulation to fine tune membrane excitability and neuronal firing properties. In neurons, the isoform Nav1.6 is highly enriched at the axon initial segment and nodes, making it critical for the initiation and propagation of neuronal impulses. Thus, Nav1.6 modulation and dysfunction may profoundly impact the input-output properties of neurons in normal and pathological conditions. Phosphorylation is a powerful and reversible mechanism that exquisitely modulates ion channels. To this end, the multifunctional calcium/calmodulin-dependent protein kinase II (CaMKII) can transduce neuronal activity through phosphorylation of diverse substrates to serve as a master regulator of neuronal function. Because Nav1.6 and CaMKII are independently linked to excitability disorders, I sought to investigate modulation of Nav1.6 function by CaMKII signaling to reveal an important mechanism underlying neuronal excitability. Multiple biochemical approaches show Nav1.6 is a novel substrate for CaMKII and reveal multi-site phosphorylation within the L1 domain; a hotspot for post-translational regulation in other Nav isoforms. Consistent with these findings, pharmacological inhibition of CaMKII reduces transient and persistent sodium currents in Purkinje neurons. Because Nav1.6 is the predominant sodium current observed in Purkinje neurons, these data suggest that Nav1.6 may be modulated through CaMKII signaling. In support of this, my studies demonstrate that CaMKII inhibition significantly attenuates Nav1.6 transient and persistent sodium currents and shifts the voltage-dependence of activation to more depolarizing potentials in heterologous cells. Interestingly, I show that these functional effects are likely mediated by CaMKII phosphorylation of Nav1.6 at S561 and T642, and that each phosphorylation site regulates distinct biophysical characteristics of the channel. These findings are further extended to investigate CaMKII modulation of disease-linked mutant Nav1.6 channels. I show that different Nav1.6 mutants display distinct responses to CaMKII modulation and reveal that acute CaMKII inhibition attenuates gain-of-function effects produced by mutant channels. Importantly, computational simulations modeling the effects of CaMKII inhibition on WT and mutant Nav1.6 channels demonstrate dramatic reductions in neuronal excitability in Purkinje and cortical pyramidal cell models. Together, these findings suggest that CaMKII modulation of Nav1.6 may be a powerful mechanism to regulate physiological and pathological neuronal excitability. / 2022-02-02

No description available.

No description available.