The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from PDF of title page (University of Missouri--Columbia, viewed on March 10, 2010). Vita. Thesis advisor: Lorson, Christian L. "December 2008" Includes bibliographical references

The DExD/H-box protein Prp43 is essential for two biological processes: nucleoplasmic pre-mRNA splicing and nucleolar rRNA maturation. The biological basis for the temporal and spatial regulation of Prp43 remains elusive. The Spp382/Ntr1, Sqs1/Pfa1 and Pxr1/Gno1 G-patch proteins bind to and activate the Prp43 DExD/H box-helicase in pre-mRNA splicing (Spp382) and rRNA processing (Sqs1, Pxr1). These Prp43-interacting proteins each contain the G-patch domain, a conserved sequence of ~48 amino acids that includes 6 highly conserved glycine (G) residues. Five annotated G-patch proteins in baker’s yeast (i.e., Spp382, Pxr1, Spp2, Sqs1 and Ylr271) and with the possible exception of the uncharacterized Ylr271 protein, all are associated with ribonucleoprotein (RNP) complexes. Understanding the role of G-patch proteins in modulating the DExD/H box protein Prp43 biological function was the motivation of this thesis. The G-patch domain has been proposed as a protein-protein or a protein-RNA interaction module for RNP proteins. This study found that the three Prp43-associated G-patch domains interact with Prp43 in a yeast 2 hybrid (Y2H) assay but differ in apparent relative affinities. Using a systemic Y2H analysis, I identified the conserved Winged-helix (WH) domain in Prp43 as a major binding site for G-patch motif. Intriguingly, removal of the non-essential N-terminal domain (NTD) of Prp43 (amino acids 2-94), greatly improves G-patch binding, suggesting that the NTD may play a role in modulating enzyme activity by the G-patch effectors. I identify a second site within the Pxr1 that strongly binds Prp43 but, unlike the G-patch, is dispensable for Pxr1 function in vivo. By constructing chimeric proteins, I demonstrated that individual G-patch peptides differ in the ability to reconstitute Spp382 and Pxr1 function in support of pre-mRNA splicing and rRNA biogenesis, respectively. Through amino acid sequence comparisons and selective mutagenesis I identified several residues within the G-patch motif critical for Prp43-stimulated pre-mRNA splicing without greatly altering its ability to bind Prp43. These data lead me to propose that the G-patch motif is not a simple Prp43 binding interface but may contribute more directly to substrate selection or Prp43 enzyme activation in the biologically distinct pre-mRNA splicing and rRNA processing pathways.

Fasciola cause considerable monetary loss in the agriculture industry, while parasitism of humans is an emerging disease. Fasciola cathepsin L-like proteases are believed to aid parasite invasion and survival through a range of functions including feeding, immune evasion and modulation, tissue migration, egg production and excystment. As such these proteases are considered good targets for chemotherapies and vaccine development. Fasciola cathepsins are evolutionarily divided into clades that reflect function and life stage of expression. Analysis of F. gigantica genomic DNA and mRNA identified novel cathepsin L-like sequences which are incorporated into a phylogenetic analysis of the complete Fasciola cathepsin L-like protease family. Analysis of mRNA transcripts isolated in this study also points to trans-splicing occurring amongst cathepsin transcripts, the first time this has been identified in Fasciola species. S2 subsite specificity is important in determining substrate interactions with cathepsin L-like proteases. Previous work has shown that amino acid substitutions at this site can dramatically influence substrate specificity. A number of substitutions, specifically those that have been observed, or predicted to occur during the evolution of Fasciola cathepsins L-like proteases, were introduced into the S2 subsite of FhCatL5 at aa69 to determine their influence. The introduction of L69C and L69S substitutions resulted in low overall activity indicating their expression provides no functional advantage, thus explaining the absence of such variants amongst fluke. The L69F variant showed an increase in the ability to cleave substrates with P2 proline, indicating F69 variants expressed by fluke are also likely to have this ability, similar to that shown with L69Y and FhCatL2. The introduction of a L69W substitution leads to increased cleavage of substrates with P2 proline, along with a decrease in cleavage of substrates with P2 phenylalanine. FgCatL1G transcripts were isolated from F. gigantica metacercariae. This contrasts with FhCatL5 and FhCatL2 which have been isolated in adult F. hepatica. These cathepsins differ at aa69, possessing tryptophan, leucine and tyrosine respectively. The processing and substrate specificities of each recombinant enzyme was analysed and compared. While FhCatL5 and FhCatL2 process in vitro in a manner similar to that reported for FhCatL1, FgCatL1G requires different processing conditions, including neutral pH. Combined with FgCatL1G possessing increased stability at acidic pH, this reflects the different environment into which FgCatL1G is expressed by immature compared to the adult flukes. The substrate specificity of FgCatL1G also differed from previously reported cathepsins, with a preference for P2 proline and low activity against substrates with P2 phenylalanine. This is the first time recombinant expression and purification of a cathepsin L-like protease specific to the immature life stages of Fasciola has been undertaken and had enzyme specificity analysed. This work has expanded knowledge of the repertoire of cathepsin proteases expressed at various life-stages of the liver fluke. Vaccination and/or drug inhibition studies may in the future be targeted towards cathepsins that are expressed in either the adult or immature stage, or perhaps both in a multi-targeted approach. The knowledge gained in this study may allow such targets to be chosen.

Orientador: Jörg Kobarg / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-25T14:15:32Z (GMT). No. of bitstreams: 1 Basei_FernandaLuisa_D.pdf: 43645148 bytes, checksum: 4cb7da11417bc57aea32c2db81dddc18 (MD5) Previous issue date: 2014 / Resumo: As Neks são proteínas quinases similares a NIMA, proteína que é indispensável para a entrada em mitose de células de Aspergillus nidulans. Em humanos foram identificadas 11 Neks (1-11) e, estudos crescentes vêm demonstrando a participação destas em diversas funções celulares além do controle do ciclo e divisão celular. A Nek4 é um dos maiores membros dessa família e sua relação com a manutenção ciliar e resposta ao DNA danificado já foi demonstrada. Contudo, seus parceiros de interação e substratos são ainda desconhecidos. Para melhor compreender o papel da Nek4 foi realizado um estudo de interatoma para identificar novos processos biológicos com os quais a Nek4 está envolvida. Inicialmente foi identificada uma nova isoforma para a Nek4 e assim, realizou-se o estudo de interatoma para as duas isoformas com a finalidade de comparar o perfil de interação das duas proteínas. As duas isoformas da Nek4 foram expressas em células humanas e após imunoprecipitação seguida de identificação por espectrometria de massas, foram identificadas 474 proteínas que interagem com a isoforma 1 da Nek4, Nek4.1 e 149 para a isoforma 2, Nek4.2. Dentre as proteínas identificadas, 102 interagem com ambas isoformas da Nek4. Nossos resultados confirmam o envolvimento da Nek4 com a resposta ao DNA danificado, função ciliar, estabilização dos microtúbulos e ainda sugerem o envolvimento da Nek4 em funções completamente novas, como processamento de RNAm, resposta ao estresse, controle de qualidade das proteínas e apoptose. Entre os parceiros de interação encontramos importantes proteínas como TRAP-1, Whirlin, PCNA, 14-3-3?, Btf, PARP-1, SRSF1, PAI-RBP1 e KAP-1. As duas isoformas compartilham funções que não foram ainda descritas para os membros da família Nek e a isoforma 1 ainda apresenta funções que já foram descritas para outros membros da família. Aliado ao resultado da imunoprecipitação ainda foram realizadas imunofluorescências que permitiram verificar a localização da Nek4 em diferentes estruturas celulares, como os speckles nucleares e a mitocôndria, corroborando com a função no processamento de RNAm e apoptose. O experimento de imunoprecipitação seguido de identificação por espectrometria de massas também apontou para a possibilidade de autofosforilação e dimerização da Nek4. Além disso, foi possível observar diferenças entre o perfil de interação das duas isoformas da Nek4, sendo que a isoforma 1 interage com proteínas que mantém funções biológicas similares a outras Neks, que a isoforma 2 não apresenta / Abstract: Neks are serine-threonine kinases that are similar to NIMA, a protein found in Aspergillus nidulans which is essential for cell division. In humans there are eleven Neks (1-11) which are involved in different biological functions besides the cell cycle control. Nek4 is one of largest members of the Neks family and has been related to the primary cilia formation and in DNA damage response. However, its substrates and interaction partners are still unknown. Thus in an attempt to better understand the role of Nek4 we performed an interactomics study to find new biological processes in which Nek4 is involved. Besides, we described here a novel Nek4 isoform and compared the interactomics profile of these two Nek4 proteins. Isoform 1 and isoform 2 of Nek4 were expressed in human cells and after an immunoprecipitation followed by mass spectrometry, 474 interacting proteins were identified for isoform 1 and 149 for isoform 2 of Nek4. 102 proteins are common interactors between both isoforms. Our results confirm Nek4 involvement in the DNA damage response, cilia maintenance and microtubules stabilization, and raise the possibility of new functional contexts including mRNA processing, apoptosis signaling, stress response, translation and protein quality control. Among the interaction partners, we found important proteins such as TRAP-1, Whirlin, PCNA, 14-3-3?, Btf, PARP-1, SRSF1, PAI-RBP1 and KAP-1. We could observe that both isoforms share functions that are new to the Nek family, and isoform 1 apparently has also maintained functions which have already been established to other Nek family members. From our immunoprecipitation followed by mass spectrometry experiment a possible site for Nek4 autophosphorylation and dimerization was identified. This study provides new insights into Nek4 functions, identifying new interaction partners, localization to new cellular compartment and further suggests an interesting difference between isoform 1 and the novel isoform 2 of Nek4. Nek4 isoform 1 may have maintained similar roles compared to other Neks and these roles are not related to isoform 2 / Doutorado / Bioquimica / Doutora em Biologia Funcional e Molecular

Stem cells, with the ability to self-renew and differentiate into intended cell types, are a valuable tool for disease modeling and mechanistic study. CRISPR-Cas9 has been widely used for genome editing due to its high efficiency and convenience. However, CRISPR-Cas9 has large-deletion safety issues that dramatically restrict its applications. Wiskott-Aldrich syndrome (WAS) is an inborn immunological disorder caused by WASP deficiency. WASP functions in the nucleus, which may help to understand WAS pathology, are poorly defined. Pannexin 1 (PANX1) forms large plasma membrane pores to exchange intracellular small molecules with the extracellular environment and functions in inflammatory processes. The regulatory mechanisms of the PANX1 channel remain obscure. In this dissertation, I focused on mechanistic studies of CRISPR-Cas9 genome editing, and two immune disease relevant genes, WASP and PANX1 using stem cell-derived immune cells. We first found that CRISPR-induced large deletions (LDs) are predominantly mediated by the MMEJ repair pathway through statistical studies. Further, we found POLQ and RPA play vital roles in CRISPR-induced LDs. Modulation of POLQ and RPA can decrease CRISPR-induced LDs and increase HDR efficiency. Using three isogenic WAS iPSC models generated via gene editing, we successfully recapitulated WAS phenotypes, and for the first time, revealed that WASP regulates RNA splicing via epigenetically controlling the transcription of splicing factors and directly participating in the splicing machinery through a liquid-liquid phase separation process. We established a full-length human PANX1 (hPANX1) channel model via cryo-electron microscopy experiments and molecular dynamics simulation study, and found that hPANX1 channel is a homo-heptamer with both the N- and C-termini stretching deeply into the pore funnel. Functional studies of three selected residues support the new hPANX1 channel model and suggest the potential regulatory role of hPANX1 in pyroptosis upon immune responses. Overall, the mechanistic studies of WASP, PANX1 and CRISPR genome editing revealed new roles of WASP in regulating RNA splicing, new functional insights of PANX1 in pyroptosis, and uncovered two critical players POLQ and RPA in CRISPR-induced LDs.

Acetylation is emerging as an important posttranslational modification, which is found in thousands of proteins in eukaryotes, as well as prokaryotes. Global proteomic studies implicated acetylation in regulation of various processes like metabolism, gene expression, cell cycle or aging to name a few. In this work I set out to investigate the role of acetylation of a splicing regulatory protein SRSF5 by creating mutations in its acetylation site. I tested the hypothesis that acetylation influences SRSF5 interaction with RNA. I expressed acetylation-mimicking (Q) or non-acetylable (R) mutant of SRSF5 in HeLa cells and measured their interaction with RNA by RNA immunoprecipitation or in vitro by fluorescence anisotropy. Both approaches agreed that mutants interact with RNA less than the wild type protein and Q mutant bound RNA weaker than R mutant. I did not detect further difference in localization or dynamics among the proteins in vivo, which suggests that difference caused by weakened interaction of mutants with RNA was outweighed by other factors influencing SRSF5 behaviour, probably protein-protein interactions. I also found out that mutant SRSF5 proteins do not have a dominant effect on splicing of fibronectin alternative EDB exon. The data obtained give an indirect evidence for the hypothesis that...

Les papillomavirus humains (HPV) sont des virus à ADN double brin qui infectent la peau et les muqueuses. Les infections par les HPV, bien que majoritairement asymptomatique, provoquent des défauts de prolifération cellulaire pouvant parfois générer des cancers. Selon leur pouvoir carcinogène, on distingue les HPV à bas risque oncogène (HPV-BR) provoquant des lésions bénignes, et les HPV à haut risque (HPV-HR) responsables de l’apparition de nombreux cancers ano-génitaux et de certains cancers des voies aéro-digestives supérieures. Parmi les HPV-HR, HPV16 est le plus prévalent. La carcinogenèse induite par les HPV-HR est corrélée à l’expression des protéines virales E6 et E7, qui dérégulent de nombreux processus cellulaires. L’expression des gènes viraux, réalisée par la machinerie de la cellule hôte, est finement régulée particulièrement au niveau posttranscriptionnel. En outre, l’épissage alternatif génère une vingtaine de transcrits viraux, permettant l’expression des protéines virales. L’épissage au sein de la région codante E6 permettant de former l’isoforme E6*I est présent uniquement chez les HPV-HR, mais pas chez les HPV-BR, ce qui suggère son implication dans la carcinogenèse induite par les HPV-HR. Toutefois, le rôle biologique de la protéine E6*I produite par les HPV-HR est encore controversé.Afin de mieux appréhender les mécanismes de la carcinogenèse induite par les HPV-HR, nous nous sommes intéressés à : (i) l’étude des fonctions biologiques de l’isoforme E6*I, et (ii) aux mécanismes impliqués dans la régulation de l’expression de E6 et E7.Pour appréhender le rôle biologique d’E6*I d’HPV16, nous avons utilisé le séquençage de l’ARN afin d’identifier des cibles dérégulées par son expression ectopique. L’expression des isoformes E6 et E6*I d’HPV16 dans des cellules HPV négatives dérégule des transcrits impliqués dans des processus biologiques relatifs à l’expression des gènes viraux, la carcinogenèse virale, la transduction du signal et la traduction. L’expression d’E6*I seule, dérégule des transcrits impliqués dans l’organisation de la matrice extracellulaire, des voies de signalisation et d’adhérence cellulaire. De façon intéressante, il a été montré que ces gènes dérégulés par l’expression d’E6*I sont communément affecté par le niveau intracellulaire de ROS (espèces réactives de l’oxygène). Cela corrobore le rôle d’E6*I dans l’augmentation de la production de ROS. Le stress oxydatif associé aux ROS pourrait favoriser l’intégration du génome viral à celui de la cellule hôte, caractéristique de carcinogenèse associée aux HPV-HR. En somme, E6*I pourrait avoir un rôle oncogénique indépendant de celui d’E6, et interviendrait dans la carcinogenèse associée aux HPV-HR.Nous avons aussi étudié le rôle du complexe de jonction des exons (EJC), dans la régulation posttranscriptionnelle de l’expression d’E6 et E7. L’EJC est un complexe multiprotéique déposé sur les ARNm via l’épissage influençant ainsi leur devenir. Nous avons montré qu’un facteur de l’EJC, eukaryotic initiation factor 4A3 (eIF4A3), se liait aux ARNm viraux. Par ailleurs, nous avons observé que les composants de l’EJC affectent, certes de différentes façons, l’expression d’E6 et E7. Enfin, nous avons aussi étudié l’effet du nonsense-mediated mRNA decay (NMD), un mécanisme lié à l’EJC, sur l’expression d’E6 et E7. Non seulement nos résultats suggèrent que le NMD inhibe l’expression d’E6 et E7, mais nous avons aussi observé que la protéine E6 d’HPV16 réduit l’activité du NMD. Cette inhibition permettrait à HPV16 d’avoir un contrôle sur ses transcrits mais d’affecter aussi des cibles cellulaires du NMD. Etant donné l’implication des gènes régulés par le NMD dans le maintien de l’homéostasie et l’adaptation cellulaires, il serait intéressant d’appréhender le rôle de cette nouvelle activité d’E6 dans la carcinogenèse associée aux HPV-HR. / Human papillomaviruses (HPV) are double strand DNA viruses that infect skin and mucosa. HPV infections, although mostly asymptomatic, cause cell proliferation defects that can sometimes give rise to cancer. According to their carcinogenic potential, we distinguish low-risk HPVs (lr-HPV) causing benign lesions, and high-risk HPV (hr-HPV) responsible for the appearance of numerous anogenital and some head and neck squamous-cell cancers. Among the hr-HPV, HPV16 is the most prevalent. Hr-HPV-induced carcinogenesis is correlated with the expression of the viral oncoproteins, E6 and E7, which deregulate many cellular processes. Viral gene expression, performed by the host cell machine, is finely regulated particularly at the post-transcriptional level. Besides, alternative splicing generates about twenty viral transcripts, leading to the expression of viral proteins. The splicing within the E6 open reading frame that generates an E6*I mRNA only in hr-HPV, but not in the lr-HPV, suggests its involvement in hr-HPV-induced carcinogenesis. However, the biological role of E6*I protein produced by HPV-HR is still controversial.In order to better understand the mechanisms of hr-HPV-induced carcinogenesis, we have interested in: (i) the study of the biological functions of the E6*I isoform, and (ii) the mechanisms involved in the regulation of E6 and E7 expression.To get insight the biological role of HPV16 E6*I, we used RNA sequencing to identify targets deregulated by its ectopic expression. Expression of HPV16 E6 and E6*I isoforms in negative HPV cells deregulate several transcripts involved in biological processes related to viral gene expression, viral carcinogenesis, signal transduction and translation. The expression of E6*I alone, deregulates transcripts involved in the organization of the extracellular matrix, signaling pathways and cell adhesion. Interestingly, it was shown that the genes deregulated by E6*I expression are commonly affected by the intracellular level of ROS (reactive oxygen species). These results support the role of E6*I in increasing ROS production. The ROS-associated oxidative stress could favor viral genome integration with that of the host cell, a characteristic of hr-induced carcinogenesis. In sum, E6*I may have an oncogenic role independent of E6, and intervene in the carcinogenesis associated with hr-HPV.We also studied the role of the exon junction complex (EJC) in the posttranscriptional regulation of E6 and E7 expression. EJC is a multiprotein complex deposited on mRNAs via splicing, thus influencing their fate. We have shown that a factor of EJC, eukaryotic initiation factor 4A3 (eIF4A3), binds to viral mRNAs. Moreover, we have observed that the components of the EJC affected, in different ways, the expression of E6 and E7. Finally, we also studied the effect of nonsense-mediated mRNA decay (NMD), a mechanism linked to the EJC, on the expression of E6 and E7. Our results suggest that not only NMD inhibits the expression of E6 and E7, but we have also observed that HPV16 E6 protein reduces NMD activity. This inhibition would allow HPV16 to have control over its transcripts but also to affect NMD cellular targets. Given the involvement of NMD-regulated genes in the maintenance of cellular homeostasis and adaptation, it would be interesting to understand the role of this new E6 activity in carcinogenesis associated with HPV-HR.

Kaposi's sarcoma-associated herpesvirus (KSHV) expresses numerous intronless mRNAs that are unable to access splicing-dependent cellular mRNA nuclear export pathways. To circumvent this problem, KSHV encodes the open reading frame 57 (ORF57) protein, which orchestrates the formation of an export-competent virus ribonucleoprotein particle comprising the nuclear export complex hTREX, but not the exon-junction complex (EJC). Interestingly, EJCs stimulate mRNA translation, which raises the intriguing question of how intronless KSHV transcripts are efficiently translated. Herein, we show that ORF57 associates with components of the 48S pre-initiation complex and co-sediments with the 40S ribosomal subunits. Strikingly, we observed a direct interaction between ORF57 and PYM, a cellular protein that enhances translation by recruiting the 48S pre-initiation complex to newly exported mRNAs, through an interaction with the EJC. Moreover, detailed biochemical analysis suggests that ORF57 recruits PYM to intronless KSHV mRNA and PYM then facilitates the association of ORF57 and the cellular translation machinery. We, therefore, propose a model whereby ORF57 interacts directly with PYM to enhance translation of intronless KSHV transcripts.

Thesis (M.S.)--Miami University, Dept. of Zoology, 2005. / Title from first page of PDF document. Document formatted into pages; contains [1], ix, 75 p. : ill. Includes bibliographical references (p. 69-75).

Calcium ions (Ca2+) are involved in almost all neuronal functions, providing the link between electrical signals and cellular activity. This work examines the mechanisms by which a neuron can regulate the movement and sequestration of Ca2+ through specific channels such that this ubiquitous ion can encode specific functions. My initial focus was using intracellular calcium ([Ca2+]i) imaging techniques to study the influence of the inhibition of specific voltage gated calcium channels (VGCC) by ethanol on a depolarization induced rise in [Ca2+]i in neurohypophysial nerve terminals. This research took an unexpected turn when I observed an elevation of [Ca2+]i during perfusion with ethanol containing solutions. Control experiments showed this to be an artifactual result not directly attributable to ethanol. It was necessary to track down the source of this artifact in order to proceed with future ethanol experiments. The source of the artifact turned out to be a contaminant leaching from I.V. drip chambers. Due to potential health implications stemming from the use of these drip chambers in a clinical setting as well as potential artifactual results in the ethanol field where these chambers are commonly used, I choose to investigate this phenomenon more rigorously. The agent responsible for this effect was shown to be di(2-ethylhexyl)phthalate (DEHP), a widely used plasticizer that has been shown to be carcinogenic in rats and mice. The extraction of this contaminant from the I.V. drip chamber, as measured by spectrophotometry, was time-dependent, and was markedly accelerated by the presence of ethanol in the solution. DEHP added to saline solution caused a rise in [Ca2+]i similar to that elicited by the contaminant containing solution. The rise in calcium required transmembrane flux through membrane channels. Blood levels of DEHP in clinical settings have been shown to exceed the levels which we found to alter [Ca2+]i. This suggests that acute alterations in intracellular calcium should be considered in addition to long-term effects when determining the safety of phthalate-containing plastics. As part of a collaboration between Steven Treistman and Robert Messing's laboratory at UCSF, I participated in a study of how ethanol regulates N-type calcium channels which are known to be inhibited acutely, and upregulated in the chronic presence of ethanol. Specific mRNA splice variants encoding N-type channels were investigated using ribonuclease protection assays and real-time PCR. Three pairs of N-type specific α-subunit Cav2.2 splice variants were examined, with exposure to ethanol observed to increase expression of one alternative splice form in a linker that lacks six bases encoding the amino acids glutamate and threonine (ΔET). Whole cell electrophysiological recordings that I carried out demonstrated a faster rate of channel activation and a shift in the voltage dependence of activation to more negative potentials after chronic alcohol exposure, consistent with increased expression of ΔET variants. These results demonstrate that chronic ethanol exposure not only increases the abundance of N-type calcium channels, but also increases the expression of a Cav2.2 splice variant with kinetics predicted to support a larger and faster rising intracellular calcium signal. This is the first demonstration that ethanol can up-regulate ion channel function through expression of a specific mRNA splice variant, defining a new mechanism underlying the development of drug addiction. Depolarizing a neuron opens voltage gated Ca2+ channels (VGCC), leading to an influx of Ca2+ ions into the cytoplasm, where Ca2+ sensitive signaling cascades are stimulated. How does the ubiquitous calcium ion selectively modulate a large array of neuronal functions? Concurrent electrophysiology and ratiometric calcium imaging were used to measure transmembrane Ca2+ current and the resulting rise and decay of [Ca2+]i, showing that equal amounts of Ca2+ entering through N-type and L-type voltage gated Ca2+ channels result in significantly different [Ca2+]i temporal profiles. When the contribution of N-type channels was reduced, a faster [Ca2+]i decay was observed. Conversely, when the contribution of L-type channels was reduced, [Ca2+]i decay was slower. Potentiating L-type current or inactivating N-type channels both resulted in a more rapid decay of [Ca2+]i. Channel-specific differences in [Ca2+]i decay rates were abolished by depleting intracellular Ca2+ stores suggesting the involvement of Ca2+-induced Ca2+ release (CICR). I was able to conclude that Ca2+ entering through N-type, but not L-type channels, is amplified by ryanodine receptor mediated CICR. Channel-specific activation of CICR generates a unique intracellular Ca2+ signal depending on the route of entry, potentially encoding the selective activation of a subset of Ca2+ -sensitive processes within the neuron.