Abstract
Junctate is a 33 kDa integral protein of sarco(endo)plasmic reticulum membranes that forms a macromolecular complex with inositol 1,4,5-trisphosphate [Ins(1,4,5)P(3)] receptors and TRPC3 channels. TIRF microscopy shows that junctate enhances the number of fluorescent puncta on the plasma membrane. The size and distribution of these puncta are not affected by the addition of agonists that mobilize Ca(2+) from Ins(1,4,5)P(3)-sensitive stores. Puncta are associated with a significantly larger number of peripheral junctions between endoplasmic reticulum and plasma membrane, which are further enhanced upon stable co-expression of junctate and TRPC3. The gap between the membranes of peripheral junctions is bridged by regularly spaced electron-dense structures of 10 nm. Ins(1,4,5)P(3) inhibits the interaction of the cytoplasmic N-terminus of junctate with the ligand-binding domain of the Ins(1,4,5)P(3) receptor. Furthermore, Ca(2+) influx evoked by activation of Ins(1,4,5)P(3) receptors is increased where puncta are located. We conclude that stable peripheral junctions between the plasma membrane and endoplasmic reticulum are the anatomical sites of agonist-activated Ca(2+) entry.
Publication types
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Research Support, N.I.H., Extramural
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Research Support, Non-U.S. Gov't
MeSH terms
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Biological Transport
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Calcium / metabolism*
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Calcium-Binding Proteins / agonists
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Calcium-Binding Proteins / genetics
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Calcium-Binding Proteins / metabolism
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Cell Membrane / genetics
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Cell Membrane / metabolism*
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Endoplasmic Reticulum / genetics
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Endoplasmic Reticulum / metabolism*
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HEK293 Cells
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Humans
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Inositol 1,4,5-Trisphosphate / metabolism
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Inositol 1,4,5-Trisphosphate Receptors / agonists
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Inositol 1,4,5-Trisphosphate Receptors / genetics
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Inositol 1,4,5-Trisphosphate Receptors / metabolism
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Membrane Proteins / agonists
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Membrane Proteins / genetics
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Membrane Proteins / metabolism
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Mixed Function Oxygenases / genetics
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Mixed Function Oxygenases / metabolism
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Muscle Proteins / agonists
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Muscle Proteins / genetics
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Muscle Proteins / metabolism
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Protein Binding
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TRPC Cation Channels / agonists
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TRPC Cation Channels / genetics
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TRPC Cation Channels / metabolism
Substances
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Calcium-Binding Proteins
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Inositol 1,4,5-Trisphosphate Receptors
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Membrane Proteins
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Muscle Proteins
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TRPC Cation Channels
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TRPC3 cation channel
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Inositol 1,4,5-Trisphosphate
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Mixed Function Oxygenases
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ASPH protein, human
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Calcium