1,721,020 research outputs found
The Builders of the Junction: Roles of Junctophilin1 and Junctophilin2 in the Assembly of the Sarcoplasmic Reticulum–Plasma Membrane Junctions in Striated Muscle
Full text linkContraction of striated muscle is triggered by a massive release of calcium from the sarcoplasmic reticulum (SR) into the cytoplasm. This intracellular calcium release is initiated by membrane depolarization, which is sensed by voltage-gated calcium channels CaV1.1 (in skeletal muscle) and CaV1.2 (in cardiac muscle) in the plasma membrane (PM), which in turn activate the calcium-releasing channel ryanodine receptor (RyR) embedded in the SR membrane. This cross-communication between channels in the PM and in the SR happens at specialized regions, the SR-PM junctions, where these two compartments come in close proximity. Junctophilin1 and Junctophilin2 are responsible for the formation and stabilization of SR-PM junctions in striated muscle and actively participate in the recruitment of the two essential players in intracellular calcium release, CaV and RyR. This short review focuses on the roles of junctophilins1 and 2 in the formation and organization of SR-PM junctions in skeletal and cardiac muscle and on the functional consequences of the absence or malfunction of these proteins in striated muscle in light of recently published data and recent advancements in protein structure prediction
Interactions of Junctophilins and STIM1 with ER Calcium-Releasing Channels
No full textJunctions between the endoplasmic reticulum (ER) and plasma membrane (PM) are found in many cell types, where they mediate local Ca2+ signaling by recruiting a variety of ER and PM channels to the two sides of these junctions. Stim1 and Junctophilins (JPs) are among the major proteins responsible for forming junctions. Stim1 organizes mostly transient junctions in response to ER Ca2+ depletion and is essential for store-operated Ca2+ entry. Junctophilins 1 and 2 coordinate the organization of the calcium releasing units in striated muscle, but the role of the neuronal junctophilins, JP3 and JP4, is less understood. Because studies on double knock-out mice indicated the importance of JP3/JP4 for brain function, we are using expression in tsA201 cells to compare JP3, JP4 and Stim1. Previously, we showed that JP3 and JP4 were both able to recruit the PM channels CaV1.2, CaV2.1 and CaV2.2 to junctions, and that JP3, but not JP4, could recruit the ER channels RyR1 and RyR2. Here we show that RyR3 is recruited to junctions by JP3, and also by JP4 (less efficiently than JP3). RyR1 and RyR3 constructs lacking ER transmembrane domains co-localized with JP3, indicative of an interaction which likely contributes to the ability of JP3 to recruit the full-length proteins to junctions. By contrast, RyR3 lacking transmembrane domains did not co-localize with JP4. Co-localization also did not occur between IP3R1 and either JPs. On the other hand, constitutively active Stim1D76A displayed moderate co-localization with IP3R1 but none with the RyRs. These results suggest that neuronal ER-PM junctions formed by Stim1, JP3 and JP4 likely harbor different sets of channels and that the JP3/JP4 ratio in junctophilin-formed junctions might modulate the composition of such microdomains with possible functional implications
Role of Neuronal Junctophilins in Recruitment and Modulation of Voltage-Gated Calcium Channels in PM-ER Junctions
No full textSmall conductance calcium activated potassium (SK) channels are predominantly expressed in heart atria and contribute to late repolarization of the action potential. Inhibition of SK current has been proposed as a therapy for atrial fibrillation (AF). We aim to develop an accurate model of SK channel gating and drug interaction that can be used to probe the role of SK channels in atrial health and disease. The model, based on Hirschberg et al's (1998) SK2 model, incorporates 4 closed and 2 open states. To generate a model with utility at physiologic temperature, we performed inside-out macropatch voltage clamp ramps at 23° and 37° C, and observed a pronounced leftward EC50 shift at 37° C (0.53 ± 0.07 μM and 0.38 ± 0.02 μM to 0.23 ± 0.02 μM and 0.28 ± 0.01 μM for hSK3 and hSK2, respectively). Thus, taking calcium activation of the major hSK isoforms into the diastolic calcium range. To provide a first-principles basis for constraining the kinetics of multi-step calcium-dependent activation, and binding of known and novel pharmacologic inhibitors, we performed single-channel recordings of SK2 at 23° C. Opening events without inhibitors were best fit with two major open-state conductances (low: 10.9 ± 1.6 pS, 60 ± 41% of events; high: 14.9 ± 1.4 pS, 40 ± 41% of events), justifying the multiple open states included in the model and to be further analysed regarding state specific kinetics. We will also present single channel data on known inhibitor apamin and novel modulator AP14145 constraining a pharmacological model. In conclusion, these experiments provided novel findings on temperature sensitivity of SK channels. These will be incorporated into an improved Markov model of SK channel dynamics to elucidate the roles of SK channels in health and disease
Neuronal Junctophilin 3 Can Replace Muscle Junctophilin 2 in Voltage-induced Calcium Release
No full textNovel Details of Calsequestrin Gel Conformation in Situ
Full text linkCalsequestrin (CASQ) is the major component of the sarcoplasmic reticulum (SR) lumen in skeletal and cardiac muscles. This calcium-binding protein localizes to the junctional SR (jSR) cisternae, where it is responsible for the storage of large amounts of Ca2+, whereas it is usually absent, at least in its polymerized form, in the free SR. The retention of CASQ inside the jSR is due partly to its association with other jSR proteins, such as junctin and triadin, and partly to its ability to polymerize, in a high Ca2+ environment, into an intricate gel that holds the protein in place. In this work, we shed some light on the still poorly described in situ structure of polymerized CASQ using detailed EM images from thin sections, with and without tilting, and from deep-etched rotary-shadowed replicas. The latter directly illustrate the fundamental network nature of polymerized CASQ, revealing repeated nodal points connecting short segments of the linear polymer. © 2013 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A
De Novo Reconstitution of Skeletal Muscle Voltage-Induced Calcium Release
No full textDepolarization of vertebrate skeletal muscle causes intracellular calcium
release, an event depending on specialized junctions between the plasma
membrane (PM) and sarcoplasmic reticulum (SR). It is known that depolarization is ‘‘sensed’’ by a voltage-gated calcium channel located in the PM
and containing CaV1.1 as its principal subunit, that calcium is released
from the SR via RyR1, and that this does not require external Ca2þ entry.
Thus, it is thought that activation of RyR1 is driven by depolarizationinduced conformational changes in CaV1.1 which are mechanically transmitted to RyR1, either directly or via intervening proteins. Based on muscle
gene knockouts, at least two additional proteins are required for this conformational coupling, but knockouts cannot reveal whether yet-to-be identified
proteins, or sets of proteins with overlapping functions, are also critical.
Thus, we used reconstitution in tsA201 cells to identify a minimal set of proteins required for conformational coupling. We found that expression of junctophilin2 was effective at promoting ER-PM junctions, and that CaV1.1 and
RyR1 both targeted to these junctions in the presence of the CaV1.1 auxiliary
subunit b1a. Moreover, tsA201 cells expressing these four proteins, and the
adapter protein Stac3, produced depolarization-triggered Ca2þ transients
which were independent of extracellular Ca2þ entry and which increased in
amplitude as a saturating function of voltage, as expected for conformational
coupling. Additionally, freeze-fracture electron microscopy indicated that
CaV1.1 and RyR1 were physically linked in these cells, thus establishing
that the five expressed proteins are sufficient for conformational coupling.
The ability to reconstitute conformational coupling with a minimal set of proteins may provide a system for obtaining high resolutions structures of
CaV1.1 and RyR1 as part of a functioning complex, which will be necessary
for understanding the molecular mechanism of depolarization-evoked calcium release in skeletal muscle
Relationship between natural antioxidants and prevention of oxidative stress related diseases
No full textNeuronal junctophilins recruit specific CaV and RyR isoforms to ER-PM junctions and functionally alter CaV2.1 and CaV2.2
Full text linkJunctions between the endoplasmic reticulum and plasma membrane that are induced by the neuronal junctophilins are of demonstrated importance, but their molecular architecture is still poorly understood and challenging to address in neurons. This is due to the small size of the junctions and the multiple isoforms of candidate junctional proteins in different brain areas. Using colocalization of tagged proteins expressed in tsA201 cells, and electrophysiology, we compared the interactions of JPH3 and JPH4 with different calcium channels. We found that JPH3 and JPH4 caused junctional accumulation of all the tested high-voltage-activated CaV isoforms, but not a low-voltage-activated CaV. Also, JPH3 and JPH4 noticeably modify CaV2.1 and CaV2.2 inactivation rate. RyR3 moderately colocalized at junctions with JPH4, whereas RyR1 and RyR2 did not. By contrast, RyR1 and RyR3 strongly colocalized with JPH3, and RyR2 moderately. Likely contributing to this difference, JPH3 binds to cytoplasmic domain constructs of RyR1 and RyR3, but not of RyR2
Junctophilins 1, 2, and 3 all support voltage-induced Ca2+ release despite considerable divergence
Full text linkIn skeletal muscle, depolarization of the plasma membrane (PM) causes conformational changes of the calcium channel CaV1.1 that then activate RYR1 to release calcium from the SR. Being independent of extracellular calcium entry, this process is termed voltage-induced calcium release. In skeletal muscle, junctophilins (JPHs) 1 and 2 form the SR–PM junctions at which voltageinduced calcium release occurs. Previous work demonstrated that JPH2 is able to recapitulate voltage-induced calcium release when expressed in HEK293 cells together with CaV1.1, β1a, Stac3, and RYR1. However, it is unknown whether JPH1 and the more distantly related neuronal JPH3 and JPH4 might also function in this manner, a question of interest because different JPH isoforms diverge in their interactions with RYR1. Here, we show that, like JPH2, JPH1 and JPH3, coexpressed with CaV1.1, β1a, Stac3, and RYR1 in HEK293 cells, cause colocalization of CaV1.1 and RYR1 at ER–PM junctions. Furthermore, potassium depolarization elicited cytoplasmic calcium transients in cells in which WT CaV1.1 was replaced with the calcium impermeant mutant CaV1.1(N617D), indicating that JPH1, JPH2, and JPH3 can all support voltage-induced calcium release, despite sequence divergence and differences in interaction with RYR1. Conversely, JPH4-induced ER–PM junctions contain CaV1.1 but not RYR1, and cells expressing JPH4 are unable to produce depolarization-induced calcium transients. Thus, JPHs seem to act primarily to form ER–PM junctions and to recruit the necessary signaling proteins to these junctions but appear not to be directly involved in the functional interactions between these proteins
Interactions between Neuronal Junctophilins and Voltage Gated Ion Channels
No full textJunctions between the endoplasmic reticulum (ER) and plasma membrane (PM) occur in diverse cell types. In many of these junctions, calcium efflux from the ER occurs via ryanodine receptors or IP3 receptors, triggered by voltage-gated ion channels or ligand receptors in the PM. Junctophilins (JPs) have been identified as proteins that cause formation of ER-PM junctions in muscle (JP1, JP2) and neurons (JP3, JP4). Knockout of JP1 or JP2 is perinatal or embryonic lethal. Single knockout of neuronal JP produces only a mild (JP3) or no (JP4) phenotype; knockout of both causes hippocampal abnormalities, irregular hindlimb reflexes and impaired memory. Here, we have examined the ability of neuronal junctophilins to cause voltage-gated channels to localize within junctional domains of the PM by co-expressing, in tsA201 cells, voltage-gated channels and JP3 or JP4 tagged with different fluorescent proteins. In the absence of JPs, the L-type channel CaV1.2 had a relatively uniform distribution, while P/Q-type (CaV2.1), N-type (CaV2.2) and T-type (CaV3.1) channels were present in the surface at a low density making them difficult to visualize. Both JP3 and JP4 caused CaV1.2, CaV2.1 and CaV2.2 to cluster at sites co-localized with the junctophilin. Such clusters were not observed for CaV3.1. KV2.1 clustered without junctophilin at sites previously shown to represent PM junctions with the ER. When co-expressed, JP4 co-localized with the KV2.1 clusters, whereas JP3 did not, raising the possibility that the two junctophilins have distinct roles. Overall our results show that junctophilins are not just structural proteins necessary for ER-PM formation, but that they actively recruit neuronal channels to spatially restricted PM domains. Supported by: NIH grants AR070298 to KGB and GM109888 to MM Tamkun (who provided GFP-KV2.1). HM Colecraft provided CaV3.1-YFP
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