Age and gender-dependent alternative splicing of P/Q-type calcium channel EF-hand.
Ontology highlight
ABSTRACT:
SUBMITTER: Chang SY
PROVIDER: S-EPMC1978091 | biostudies-literature | 2007 Mar
REPOSITORIES: biostudies-literature
Publications
Age and gender-dependent alternative splicing of P/Q-type calcium channel EF-hand.
Chang S Y SY Yong T F TF Yu C Y CY Liang M C MC Pletnikova O O Troncoso J J Burgunder J-M JM Soong T W TW
Neuroscience 20070208 3
Ca(v)2.1 Ca(2+) channels (P/Q-type), which participate in various key roles in the CNS by mediating calcium influx, are extensively spliced. One of its alternatively-spliced exons is 37, which forms part of the EF hand. The expression of exon 37a (EFa form), but not exon 37b (EFb form), confers the channel an activity-dependent enhancement of channel opening known as Ca(2+)-dependent facilitation (CDF). In this study, we analyzed the trend of EF hand splice variant distributions in mouse, rat an ...[more]
Similar Datasets
Project description:Neurotransmitter release from mammalian sensory neurons is controlled by Ca(V)2.2 N-type calcium channels. N-type channels are a major target of neurotransmitters and drugs that inhibit calcium entry, transmitter release and nociception through their specific G protein-coupled receptors. G protein-coupled receptor inhibition of these channels is typically voltage-dependent and mediated by Gbetagamma, whereas N-type channels in sensory neurons are sensitive to a second G protein-coupled receptor pathway that inhibits the channel independent of voltage. Here we show that preferential inclusion in nociceptors of exon 37a in rat Cacna1b (encoding Ca(V)2.2) creates, de novo, a C-terminal module that mediates voltage-independent inhibition. This inhibitory pathway requires tyrosine kinase activation but not Gbetagamma. A tyrosine encoded within exon 37a constitutes a critical part of a molecular switch controlling N-type current density and G protein-mediated voltage-independent inhibition. Our data define the molecular origins of voltage-independent inhibition of N-type channels in the pain pathway.
| S-EPMC3027493 | biostudies-literature
Project description:The Ca(V)1.4 voltage-gated calcium channel is predominantly expressed in the retina, and mutations to this channel have been associated with human congenital stationary night blindness type-2. The L-type Ca(V)1.4 channel displays distinct properties such as absence of calcium-dependent inactivation (CDI) and slow voltage-dependent inactivation (VDI) due to the presence of an autoinhibitory domain (inhibitor of CDI) in the distal C terminus. We hypothesized that native Ca(V)1.4 is subjected to extensive alternative splicing, much like the other voltage-gated calcium channels, and employed the transcript scanning method to identify alternatively spliced exons within the Ca(V)1.4 transcripts isolated from the human retina. In total, we identified 19 alternative splice variations, of which 16 variations have not been previously reported. Characterization of the C terminus alternatively spliced exons using whole-cell patch clamp electrophysiology revealed a splice variant that exhibits robust CDI. This splice variant arose from the splicing of a novel alternate exon (43*) that can be found in 13.6% of the full-length transcripts screened. Inclusion of exon 43* inserts a stop codon that truncates half the C terminus. The Ca(V)1.4 43* channel exhibited robust CDI, a larger current density, a hyperpolarized shift in activation potential by ?10 mV, and a slower VDI. Through deletional experiments, we showed that the inhibitor of CDI was responsible for modulating channel activation and VDI, in addition to CDI. Calcium currents in the photoreceptors were observed to exhibit CDI and are more negatively activated as compared with currents elicited from heterologously expressed full-length Ca(V)1.4. Naturally occurring alternative splice variants may in part contribute to the properties of the native Ca(V)1.4 channels.
| S-EPMC3256920 | biostudies-literature
Project description:Leucine Zipper EF-hand containing transmembrane protein-1 (LETM1) is an inner mitochondrial membrane protein that mediates mitochondrial calcium (Ca2+ )/proton exchange. The matrix residing carboxyl (C)-terminal domain contains a sequence identifiable EF-hand motif (EF1) that is highly conserved among orthologues. Deletion of EF1 abrogates LETM1 mediated mitochondrial Ca2+ flux, highlighting the requirement of EF1 for LETM1 function. To understand the mechanistic role of this EF-hand in LETM1 function, we characterized the biophysical properties of EF1 in isolation. Our data show that EF1 exhibits α-helical secondary structure that is augmented in the presence of Ca2+ . Unexpectedly, EF1 features a weak (~mM), but specific, apparent Ca2+ -binding affinity, consistent with the canonical Ca2+ coordination geometry, suggested by our solution NMR. The low affinity is, at least in part, due to an Asp at position 12 of the binding loop, where mutation to Glu increases the affinity by ~4-fold. Further, the binding affinity is sensitive to pH changes within the physiological range experienced by mitochondria. Remarkably, EF1 unfolds at high and low temperatures. Despite these unique EF-hand properties, Ca2+ binding increases the exposure of hydrophobic regions, typical of EF-hands; however, this Ca2+ -induced conformational change shifts EF1 from a monomer to higher order oligomers. Finally, we showed that a second, putative EF-hand within LETM1 is unreactive to Ca2+ either in isolation or tandem with EF1. Collectively, our data reveal that EF1 is structurally and biophysically responsive to pH, Ca2+ and temperature, suggesting a role as a multipartite environmental sensor within LETM1.
| S-EPMC7980503 | biostudies-literature
Project description:The EF hand, a helix-loop-helix structure, is one of the most common motifs found in animal genomes, and EF-hand Ca(2+)-binding proteins (EFCaBPs) are widely distributed throughout the cell. However, researchers remain confounded by a lack of understanding of how peptide sequences code for specific functions and by uncertainty about the molecular mechanisms that enable EFCaBPs to distinguish among many diverse cellular targets. Such knowledge could define the roles of EFCaBPs in health and disease and ultimately enable control or even design of Ca(2+)-dependent functions in medicine and biotechnology. In this Account, we describe our structural and biochemical research designed to understand the sequence-to-function relationship in EFCaBPs. The first structural goal was to define conformational changes induced by binding Ca(2+), and our group and others established that solution NMR spectroscopy is well suited for this task. We pinpointed residues critical to the differences in Ca(2+) response of calbindin D(9k) and calmodulin (CaM), homologous EFCaBPs from different functional classes, by using direct structure determination with site-directed mutagenesis and protein engineering. Structure combined with biochemistry provided the foundation for identifying the fundamental mechanism of cooperativity in the binding of Ca(2+) ions: this cooperativity provides EFCaBPs with the ability to detect the relatively small changes in concentration that constitute Ca(2+) signals. Using calbindin D(9k) as a model system, studies of the structure and fast time scale dynamics of each of the four ion binding states in a typical EF-hand domain provided direct evidence that site-site communication lowers the free energy cost of reorganization for binding the second ion. Our work has also extended models of how EFCaBPs interact with their cellular targets. We determined the unique dimeric architecture of S100 proteins, a specialized subfamily of EFCaBPs found exclusively in vertebrates. We described the implications for how these proteins transduce signals and went on to characterize interactions with peptide fragments of important cellular targets. Studies of the CaM homolog centrin revealed novel characteristics of its binding of Ca(2+) and its interaction with its cellular target Kar1. These results provided clear examples of how subtle differences in sequence fine-tune EFCaBPs to interact with their specific targets. The structural approach stands at a critical crossroad, shifting in emphasis from descriptive structural biochemistry to integrated biology and medicine. We present our dual-molecular-switch model for Ca(2+) regulation of gating functions of voltage-gated sodium channels in which both CaM and an intrinsic EF-hand domain serve as coupled Ca(2+) sensors. A second example involves novel EFCaBP extracellular function, that is, the role of S100A8/S100A9 heterodimer in the innate immune response to bacterial pathogens. A mechanism for the antimicrobial activity of S100A8/S100A9 was discovered. We describe interactions of S100A8/S100A9 and S100B with the cell surface receptor for advanced glycation end products. Biochemical and structural studies are now uncovering the mechanisms by which EFCaBPs work and are helping to define their biological activities, while simultaneously expanding knowledge of the roles of these proteins in normal cellular physiology and the pathology of disease.
| S-EPMC3059389 | biostudies-literature
Project description:Ca2+ is a universal second messenger that plays a pivotal role in diverse signaling mechanisms in almost all life forms. Since the evolution of life from an aquatic to a terrestrial environment, Ca2+ signaling systems have expanded and diversified enormously. Although there are several Ca2+ sensing molecules found in a cell, EF-hand containing proteins play a principal role in calcium signaling event in plants. The major EF-hand containing proteins are calmodulins (CaMs), calmodulin like proteins (CMLs), calcineurin B-like (CBL) and calcium dependent protein kinases (CDPKs/CPKs). CaMs and CPKs contain calcium binding conserved D-x-D motifs in their EF-hands (one motif in each EF-hand) whereas CMLs contain a D-x₃-D motif in the first and second EF-hands that bind the calcium ion. Calcium signaling proteins form a complex interactome network with their target proteins. The CMLs are the most primitive calcium binding proteins. During the course of evolution, CMLs are evolved into CaMs and subsequently the CaMs appear to have merged with protein kinase molecules to give rise to calcium dependent protein kinases with distinct and multiple new functions. Ca2+ signaling molecules have evolved in a lineage specific manner with several of the calcium signaling genes being lost in the monocot lineage.
| S-EPMC6471108 | biostudies-literature
Project description:Troponin is the singular Ca(2+)-sensitive protein in the contraction of vertebrate striated muscles. Troponin C (TnC), the Ca(2+)-binding subunit of the troponin complex, has two distinct domains, C and N, which have different properties despite their extensive structural homology. In this work, we analyzed the thermodynamic stability of the isolated N-domain of TnC using a fluorescent mutant with Phe 29 replaced by Trp (F29W/N-domain, residues 1-90). The complete unfolding of the N-domain of TnC in the absence or presence of Ca(2+) was achieved by combining high hydrostatic pressure and urea, a maneuver that allowed us to calculate the thermodynamic parameters (DeltaV and DeltaG(atm)). In this study, we propose that part of the affinity for Ca(2+) is contributed by the free-energy change of folding of the N- and C-domains that takes place when Ca(2+) binds. The importance of the free-energy change for the structural and regulatory functions of the TnC isolated domains was evaluated. Our results shed light on how the coupling between folding and ion binding contributes to the fine adjustment of the affinity for Ca(2+) in EF-hand proteins, which is crucial to function.
| S-EPMC2576379 | biostudies-literature
Project description:The Ca(V)2 family of voltage-gated calcium channels, present in presynaptic nerve terminals, regulates exocytosis and synaptic transmission. Cumulative inactivation of these channels occurs during trains of action potentials, and this may control short-term dynamics at the synapse. Inactivation during brief, repetitive stimulation is primarily attributed to closed-state inactivation, and several factors modulate the susceptibility of voltage-gated calcium channels to this form of inactivation. We show that alternative splicing of an exon in a cytoplasmic region of the Ca(V)2.2 channel modulates its sensitivity to inactivation during trains of action potential waveforms. The presence of this exon, exon 18a, protects the Ca(V)2.2 channel from entry into closed-state inactivation specifically during short (10 ms to 3 s) and small depolarizations of the membrane potential (-60 mV to -50 mV). The reduced sensitivity to closed-state inactivation within this dynamic range likely underlies the differential responsiveness of Ca(V)2.2 splice isoforms to trains of action potential waveforms. Regulated alternative splicing of Ca(V)2.2 represents a possible mechanism for modulating short-term dynamics of synaptic efficacy in different regions of the nervous system.
| S-EPMC397472 | biostudies-literature
Project description:CaV1.2 voltage-gated calcium channels play critical roles in the control of membrane excitability, gene expression, and muscle contraction. These channels show diverse functional properties generated by alternative splicing at multiple sites within the CaV1.2 pre-mRNA. The molecular mechanisms controlling this splicing are not understood. We find that two exons in the CaV1.2 channel are controlled in part by members of the Fox family of splicing regulators. Exons 9* and 33 confer distinct electrophysiological properties on the channel and show opposite patterns of regulation during cortical development, with exon 9* progressively decreasing its inclusion in the CaV1.2 mRNA over time and exon 33 progressively increasing. Both exons contain Fox protein binding elements within their adjacent introns, and Fox protein expression is induced in cortical neurons in parallel with the changes in CaV1.2 splicing. We show that knocking down expression of Fox proteins in tissue culture cells has opposite effects on exons 9* and 33. The loss of Fox protein increases exon 9* splicing and decreases exon 33, as predicted by the positions of the Fox binding elements and by the pattern of splicing in development. Conversely, overexpression of Fox1 and Fox2 proteins represses exon 9* and enhances exon 33 splicing in the endogenous CaV1.2 mRNA. These effects of Fox proteins on exons 9* and 33 can be recapitulated in transfected minigene reporters. Both the repressive and the enhancing effects of Fox proteins are dependent on the Fox binding elements within and adjacent to the target exons, indicating that the Fox proteins are directly regulating both exons. These results demonstrate that the Fox protein family is playing a key role in tuning the properties of CaV1.2 calcium channels during neuronal development.
| S-EPMC2725710 | biostudies-literature
Project description:Many essential physiological processes are regulated by the modulation of calcium concentration in the cell. The EF-hand proteins represent a superfamily of calcium-binding proteins involved in calcium signaling and homeostasis. Secretagogin is a hexa-EF-hand protein that is highly expressed in pancreatic islet of Langerhans and neuroendocrine cells and may play a role in the trafficking of secretory granules. We present the X-ray structure of Danio rerio secretagogin, which is 73% identical to human secretagogin, in calcium-free form at 2.1-A resolution. Secretagogin consists of the three globular domains each of which contains a pair of EF-hand motifs. The domains are arranged into a V-shaped molecule with a distinct groove formed at the interface of the domains. Comparison of the secretagogin structure with the solution structure of calcium-loaded calbindin D(28K) revealed a striking difference in the spatial arrangement of their domains, which involves approximately 180 degrees rotation of the first globular domain with respect to the module formed by the remaining domains.
| S-EPMC2742686 | biostudies-literature
Project description:The juvenile phase is characterized by continuously progressing physiological processes such as growth and maturation, which are accompanied by transitions in gene expression. The contribution of transcriptome dynamics to the establishment of juvenile properties remains unclear. Here, we investigated alternative splicing (AS) events in postnatal growth and elucidated the landscape of age-dependent alternative splicing (ADAS) in C57BL/6 mice. Our analysis of ADAS in the cerebral cortex, cardiomyocytes, and hepatocytes revealed numerous juvenile-specific splicing isoforms that shape the juvenile transcriptome, which in turn functions as a basis for the highly anabolic status of juvenile cells. Mechanistically, the juvenile-expressed splicing factor Srsf7 mediates ADAS, as exemplified by switching from juvenile to adult forms of anabolism-associated genes Eif4a2 and Rbm7. Suppression of Srsf7 results in "fast-forwarding" of this transcriptome transition, causing impaired anabolism and growth in mice. Thus, juvenile-specific AS is indispensable for the anabolic state of juveniles and differentiates juveniles from adults.
| S-EPMC7063262 | biostudies-literature