Abstract
Ca(2+) influx via voltage-dependent CaV1/CaV2 channels couples electrical signals to biological responses in excitable cells. CaV1/CaV2 channel blockers have broad biotechnological and therapeutic applications. Here we report a general method for developing novel genetically encoded calcium channel blockers inspired by Rem, a small G-protein that constitutively inhibits CaV1/CaV2 channels. We show that diverse cytosolic proteins (CaVβ, 14-3-3, calmodulin and CaMKII) that bind pore-forming α1-subunits can be converted into calcium channel blockers with tunable selectivity, kinetics and potency, simply by anchoring them to the plasma membrane. We term this method 'channel inactivation induced by membrane-tethering of an associated protein' (ChIMP). ChIMP is potentially extendable to small-molecule drug discovery, as engineering FK506-binding protein into intracellular sites within CaV1.2-α1C permits heterodimerization-initiated channel inhibition with rapamycin. The results reveal a universal method for developing novel calcium channel blockers that may be extended to develop probes for a broad cohort of unrelated ion channels.
Publication types
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Research Support, N.I.H., Extramural
MeSH terms
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14-3-3 Proteins / chemistry
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14-3-3 Proteins / genetics
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14-3-3 Proteins / metabolism
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Action Potentials
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Animals
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Calcium Channel Blockers / chemistry
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Calcium Channel Blockers / pharmacology*
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Calcium-Calmodulin-Dependent Protein Kinase Type 2 / chemistry
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Calcium-Calmodulin-Dependent Protein Kinase Type 2 / genetics
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Calcium-Calmodulin-Dependent Protein Kinase Type 2 / metabolism
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Caveolin 1 / antagonists & inhibitors*
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Caveolin 1 / chemistry
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Caveolin 1 / metabolism
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Caveolin 2 / antagonists & inhibitors*
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Caveolin 2 / chemistry
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Caveolin 2 / metabolism
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Cell Membrane / drug effects
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Cell Membrane / metabolism
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Drug Discovery
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Ganglia, Spinal / cytology
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Ganglia, Spinal / drug effects
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Ganglia, Spinal / metabolism
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HEK293 Cells
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Humans
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Ion Channel Gating / drug effects
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Ion Transport / drug effects
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Mice
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Molecular Mimicry
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Neurons / cytology
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Neurons / drug effects
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Neurons / metabolism
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PC12 Cells
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Patch-Clamp Techniques
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Protein Binding
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Rats
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Sirolimus / pharmacology
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Tacrolimus Binding Proteins / chemistry
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Tacrolimus Binding Proteins / genetics*
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Tacrolimus Binding Proteins / metabolism
Substances
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14-3-3 Proteins
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CAV1 protein, human
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CAV2 protein, human
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Calcium Channel Blockers
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Caveolin 1
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Caveolin 2
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Calcium-Calmodulin-Dependent Protein Kinase Type 2
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Tacrolimus Binding Proteins
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Sirolimus