A pinboard by
this curator

postdoc, University of Colorado


Unveiling the essential proteins involved in calcium signaling in muscle and nerve

Stable junctions between the endoplasmic reticulum (ER) and plasma membrane (PM) were first discovered in skeletal muscle by Porter and Palade in 1957, and later described for other cell types including cardiac muscle and neuronal cells. Junctophilins (JPs) have been identified as responsible for the formation of stable ER-PM junctions in muscle (JP1, JP2) and neurons (JP3, JP4). In both skeletal and cardiac muscle, these junctions are the sites at which L-type calcium channels (CaV’s) in the PM trigger calcium release via ryanodine receptors (RyR’s) located in the ER (or “SR”) and are, therefore, essential for muscle contraction. The role of ER-PM junctions in the nervous system is less known, in part because these junctions are likely to be very heterogeneous, with calcium efflux from the ER occurring via different RyR isoforms or IP3Rs in turn triggered by voltage-gated or ligand-gated ion channels in the PM. We have begun to examine 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. 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 junctophilins 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 physiological roles. In addition to causing channel clustering, the junctophilins appear to have the ability to modify function in that we found CaV2.1 inactivation is slowed by JP3 and, to a greater extent, by JP4. Overall our results show that junctophilins are not just structural proteins necessary for ER-PM formation and stability, but that they actively recruit neuronal channels to spatially restricted PM domains and are potentially able to modulate their activity.


ER-PM junctions: Structure, function and dynamics.

Abstract: ER-PM junctions are contact sites between the endoplasmic reticulum (ER) and the plasma membrane (PM). The distance between the two organelles in the junctions is below 40 nm and the membranes are connected by protein tethers. A number of molecular tools and technical approaches have been recently developed to visualise, modify and characterise properties of ER-PM junctions. The junctions serve as the platforms for lipid exchange between the organelles and for cell signalling, notably Ca(2+) and cAMP signalling. Vice versa, signalling events regulate the development and properties of the junctions. Two Ca(2+) -dependent mechanisms of de novo formation of ER-PM junctions have been recently described and characterised. The junction-forming proteins and lipids are currently the focus of vigorous investigations. Junctions can be relatively short-lived and simple structures, forming and dissolving on the time-scale of a few minutes. However, complex, sophisticated and multifunctional ER-PM junctions, capable of attracting numerous protein residents and other cellular organelles, have been described in some cell types. The road from simplicity to complexity i.e. the transformation from simple 'nascent' ER-PM junctions to advanced stable multiorganellar complexes, is likely to become an attractive research avenue for current and future junctologists. Another area of considerable research interest is the downstream cellular processes that can be activated by specific local signalling events in the ER-PM junctions. Studies of the cell physiology and indeed pathophysiology of ER-PM junctions have already produced some surprising discoveries, likely to expand with advances in our understanding of these remarkable organellar contact sites. This article is protected by copyright. All rights reserved.

Pub.: 05 Mar '16, Pinned: 15 Jun '17

Organization of junctional sarcoplasmic reticulum proteins in skeletal muscle fibers.

Abstract: The sarcoplasmic reticulum (SR) of striated muscles is specialized for releasing Ca(2+) following sarcolemma depolarization in order to activate muscle contraction. To this end, the SR forms a network of longitudinal tubules and cisternae that surrounds the myofibrils and, at the same time, participates to the assembly of the triadic junctional membrane complexes formed by the close apposition of one t-tubule, originated from the sarcolemma, and two SR terminal cisternae. Advancements in understanding the molecular basis of the SR structural organization have identified an interaction between sAnk1, a transmembrane protein located on the longitudinal SR (l-SR) tubules, and obscurin, a myofibrillar protein. The direct interaction between these two proteins results in molecular contacts that have the overall effect to stabilize the l-SR tubules along myofibrils in skeletal muscle fibers. Less known is the structural organization of the sites in the SR that are specialized for Ca(2+) release and are positioned at the junctional SR (j-SR), i.e. the region of the terminal cisternae that faces the t-tubule at triads. At the j-SR, several trans-membrane proteins like triadin, junctin, or intra-luminal SR proteins like calsequestrin, are assembled together with the ryanodine receptor, the SR Ca(2+) release channel, into a macromolecular complex specialized in releasing Ca(2+). At triads, the 12 nm-wide gap between the t-tubule and the j-SR allows the ryanodine receptor on the j-SR to be functionally coupled with the voltage-gated L-type calcium channel on the t-tubule in order to allow the transduction of the voltage-induced signal into Ca(2+) release through the ryanodine receptor channels. The muscle-specific junctophilin isoforms (JPH1 and JPH2) are anchored to the j-SR with a trans-membrane segment present at the C-terminus and are capable to bind the sarcolemma with a series of phospholipid-binding motifs localized at the N-terminus. Accordingly, through this dual interaction, JPH1 and JPH2 are responsible for the assembly of the triadic junctional membrane complexes. Recent data indicate that junctophilins seem also to interact with other proteins of the excitation-contraction machinery, suggesting that they may contribute to hold excitation-contraction coupling proteins to the sites where the j-SR is being organized.

Pub.: 17 Sep '15, Pinned: 15 Jun '17

Stac adaptor proteins regulate trafficking and function of muscle and neuronal L-type Ca2+ channels.

Abstract: Excitation-contraction (EC) coupling in skeletal muscle depends upon trafficking of CaV1.1, the principal subunit of the dihydropyridine receptor (DHPR) (L-type Ca(2+) channel), to plasma membrane regions at which the DHPRs interact with type 1 ryanodine receptors (RyR1) in the sarcoplasmic reticulum. A distinctive feature of this trafficking is that CaV1.1 expresses poorly or not at all in mammalian cells that are not of muscle origin (e.g., tsA201 cells), in which all of the other nine CaV isoforms have been successfully expressed. Here, we tested whether plasma membrane trafficking of CaV1.1 in tsA201 cells is promoted by the adapter protein Stac3, because recent work has shown that genetic deletion of Stac3 in skeletal muscle causes the loss of EC coupling. Using fluorescently tagged constructs, we found that Stac3 and CaV1.1 traffic together to the tsA201 plasma membrane, whereas CaV1.1 is retained intracellularly when Stac3 is absent. Moreover, L-type Ca(2+) channel function in tsA201 cells coexpressing Stac3 and CaV1.1 is quantitatively similar to that in myotubes, despite the absence of RyR1. Although Stac3 is not required for surface expression of CaV1.2, the principle subunit of the cardiac/brain L-type Ca(2+) channel, Stac3 does bind to CaV1.2 and, as a result, greatly slows the rate of current inactivation, with Stac2 acting similarly. Overall, these results indicate that Stac3 is an essential chaperone of CaV1.1 in skeletal muscle and that in the brain, Stac2 and Stac3 may significantly modulate CaV1.2 function.

Pub.: 31 Dec '14, Pinned: 15 Jun '17