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Ion channels in excitable cells function in macromolecular complexes in which auxiliary proteins modulate the biophysical properties of the pore-forming subunits. Hyperpolarization-activated, cyclic nucleotide-sensitive HCN4 channels are critical determinants of membrane excitability in cells throughout the body, including thalamocortical neurons and cardiac pacemaker cells. We previously showed that the properties of HCN4 channels differ dramatically in different cell types, possibly due to the endogenous expression of auxiliary proteins. Here, we report the discovery of a family of endoplasmic reticulum transmembrane proteins that interact with and modulate HCN4. Lymphoid-restricted membrane protein (LRMP, Jaw1) and inositol trisphosphate receptor-associated guanylate kinase substrate (IRAG, Mrvi1, Jaw1L) are homologous proteins with small ER luminal domains and large cytoplasmic domains. Despite their homology, LRMP and IRAG have distinct effects on HCN4. LRMP is a loss-of-function modulator that inhibits the canonical depolarizing shift in the voltage-dependence of HCN4 activation in response to binding of cAMP. In contrast, IRAG causes a gain of HCN4 function by depolarizing the basal voltage-dependence of activation in the absence of cAMP. The mechanisms of action of LRMP and IRAG are novel; they are independent of trafficking and cAMP binding, and they are specific to the HCN4 isoform. We also found that IRAG is highly expressed in the mouse sinoatrial node where computer modeling predicts that its presence increases HCN4 availability. Our results suggest important roles for LRMP and IRAG in regulation of cellular excitability and as tools for advancing mechanistic understanding of HCN4 channel function.Significance statementThe pore-forming subunits of ion channels are regulated by auxiliary interacting proteins. Hyperpolarization-activated cyclic nucleotide-sensitive isoform 4 (HCN4) channels are critical determinants of electrical excitability in many types of cells including neurons and cardiac pacemaker cells. Here we report the discovery of two novel HCN4 regulatory proteins. Despite their homology, the two proteins — lymphoid-restricted membrane protein (LRMP) and inositol trisphosphate receptor-associated guanylate kinase substrate (IRAG) — have opposing effects on HCN4, causing loss- and gain-of-function, respectively. LRMP and IRAG are expected to play critical roles in regulation of physiological processes ranging from wakefulness to heart rate through their modulation of HCN4 channel function.

Stac protein (named for its SH3- and cysteine-rich domains) was first identified in brain 20 years ago and is currently known to have three isoforms. Stac2, Stac1, and Stac3 transcripts are found at high, modest, and very low levels, respectively, in the cerebellum and forebrain, but their neuronal functions have been little investigated. Here, we tested the effects of Stac proteins on neuronal, high-voltage-activated Ca(2+) channels. Overexpression of the three Stac isoforms eliminated Ca(2+)-dependent inactivation (CDI) of l-type current in rat neonatal hippocampal neurons (sex unknown), but not CDI of non-l-type current. Using heterologous expression in tsA201 cells (together with β and α(2)-δ(1) auxiliary subunits), we found that CDI for Ca(V)1.2 and Ca(V)1.3 (the predominant, neuronal l-type Ca(2+) channels) was suppressed by all three Stac isoforms, whereas CDI for the P/Q channel, Ca(V)2.1, was not. For Ca(V)1.2, the inhibition of CDI by the Stac proteins appeared to involve their direct interaction with the channel's C terminus. Within the Stac proteins, a weakly conserved segment containing ∼100 residues and linking the structurally conserved PKC C1 and SH3_1 domains was sufficient to fully suppress CDI. The presence of CDI for l-type current in control neonatal neurons raised the possibility that endogenous Stac levels are low in these neurons and Western blotting indicated that the expression of Stac2 was substantially increased in adult forebrain and cerebellum compared with neonate. Together, our results indicate that one likely function of neuronal Stac proteins is to tune Ca(2+) entry via neuronal l-type channels. SIGNIFICANCE STATEMENT Stac protein, first identified 20 years ago in brain, has recently been found to be essential for proper trafficking and function of the skeletal muscle l-type Ca2+ channel and is the site of mutations causing a severe, inherited human myopathy. In neurons, however, functions for Stac protein have remained unexplored. Here, we report that one likely function of neuronal Stac proteins is tuning Ca2+ entry via l-type, but not that via non-l-type, Ca2+ channels. Moreover, there is a large postnatal increase in protein levels of the major neuronal isoform (Stac2) in forebrain and cerebellum, which could provide developmental regulation of l-type channel Ca2+ signaling in these brain regions.

Stac protein (named for its SH3- and cysteine-rich domains) was first identified in brain 20 years ago and is currently known to have three isoforms. Stac2, Stac1, and Stac3 transcripts are found at high, modest, and very low levels, respectively, in the cerebellum and forebrain, but their neuronal functions have been little investigated. Here, we tested the effects of Stac proteins on neuronal, high-voltage-activated Ca2+channels. Overexpression of the three Stac isoforms eliminated Ca2+-dependent inactivation (CDI) ofl-type current in rat neonatal hippocampal neurons (sex unknown), but not CDI of non-l-type current. Using heterologous expression in tsA201 cells (together with β and α2-δ1auxiliary subunits), we found that CDI for CaV1.2 and CaV1.3 (the predominant, neuronall-type Ca2+channels) was suppressed by all three Stac isoforms, whereas CDI for the P/Q channel, CaV2.1, was not. For CaV1.2, the inhibition of CDI by the Stac proteins appeared to involve their direct interaction with the channel's C terminus. Within the Stac proteins, a weakly conserved segment containing ∼100 residues and linking the structurally conserved PKC C1 and SH3_1 domains was sufficient to fully suppress CDI. The presence of CDI forl-type current in control neonatal neurons raised the possibility that endogenous Stac levels are low in these neurons and Western blotting indicated that the expression of Stac2 was substantially increased in adult forebrain and cerebellum compared with neonate. Together, our results indicate that one likely function of neuronal Stac proteins is to tune Ca2+entry via neuronall-type channels.SIGNIFICANCE STATEMENTStac protein, first identified 20 years ago in brain, has recently been found to be essential for proper trafficking and function of the skeletal musclel-type Ca2+ channel and is the site of mutations causing a severe, inherited human myopathy. In neurons, however, functions for Stac protein have remained unexplored. Here, we report that one likely function of neuronal Stac proteins is tuning Ca2+ entry vial-type, but not that via non-l-type, Ca2+ channels. Moreover, there is a large postnatal increase in protein levels of the major neuronal isoform (Stac2) in forebrain and cerebellum, which could provide developmental regulation ofl-type channel Ca2+ signaling in these brain regions.

Type 1 RyRs (RyR1s) of the sarcoplasmic reticulum signal bidirectionally with plasma membrane dihydropyridine receptors in skeletal muscle. Polster et al. show that isolated cytoplasmic domains of RyR1 can oligomerize, localize to membrane junctions, and restore retrograde signaling., The type 1 ryanodine receptor (RyR1) in skeletal muscle is a homotetrameric protein that releases Ca2+ from the sarcoplasmic reticulum (SR) in response to an “orthograde” signal from the dihydropyridine receptor (DHPR) in the plasma membrane (PM). Additionally, a “retrograde” signal from RyR1 increases the amplitude of the Ca2+ current produced by CaV1.1, the principle subunit of the DHPR. This bidirectional signaling is thought to depend on physical links, of unknown identity, between the DHPR and RyR1. Here, we investigate whether the isolated cytoplasmic domain of RyR1 can interact structurally or functionally with CaV1.1 by producing an N-terminal construct (RyR11:4300) that lacks the C-terminal membrane domain. In CaV1.1-null (dysgenic) myotubes, RyR11:4300 is diffusely distributed, but in RyR1-null (dyspedic) myotubes it localizes in puncta at SR–PM junctions containing endogenous CaV1.1. Fluorescence recovery after photobleaching indicates that diffuse RyR11:4300 is mobile, whereas resistance to being washed out with a large-bore micropipette indicates that the punctate RyR11:4300 stably associates with PM–SR junctions. Strikingly, expression of RyR11:4300 in dyspedic myotubes causes an increased amplitude, and slowed activation, of Ca2+ current through CaV1.1, which is almost identical to the effects of full-length RyR1. Fast protein liquid chromatography indicates that ∼25% of RyR11:4300 in diluted cytosolic lysate of transfected tsA201 cells is present in complexes larger in size than the monomer, and intermolecular fluorescence resonance energy transfer implies that RyR11:4300 is significantly oligomerized within intact tsA201 cells and dyspedic myotubes. A large fraction of these oligomers may be homotetramers because freeze-fracture electron micrographs reveal that the frequency of particles arranged like DHPR tetrads is substantially increased by transfecting RyR-null myotubes with RyR11:4300. In summary, the RyR1 cytoplasmic domain, separated from its SR membrane anchor, retains a tendency toward oligomerization/tetramerization, binds to SR–PM junctions in myotubes only if CaV1.1 is also present and is fully functional in retrograde signaling to CaV1.1.

A number of G-protein-coupled receptors are expressed in skeletal muscle but their roles in muscle physiology and downstream effector systems remain poorly investigated. Here we explored the functional importance of the G-protein betagamma (Gbetagamma) signalling pathway on voltage-controlled Ca(2+) homeostasis in single isolated adult skeletal muscle fibres. A GFP-tagged Gbeta(1)gamma(2) dimer was expressed in vivo in mice muscle fibres. The GFP fluorescence pattern was consistent with a Gbeta(1)gamma(2) dimer localization in the transverse-tubule membrane. Membrane current and indo-1 fluorescence measurements performed under voltage-clamp conditions reveal a drastic reduction of both L-type Ca(2+) current density and of peak amplitude of the voltage-activated Ca(2+) transient in Gbeta(1)gamma(2)-expressing fibres. These effects were not observed upon expression of Gbeta(2)gamma(2), Gbeta(3)gamma(2) or Gbeta(4)gamma(2). Our data suggest that the G-protein beta(1)gamma(2) dimer may play an important regulatory role in skeletal muscle excitation-contraction coupling.

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.

International audience; The scorpion toxin maurocalcine acts as a high affinity agonist of the type-1 ryanodine receptor expressed in skeletal muscle. Here, we investigated the effects of the reducing agent dithiothreitol or the oxidizing reagent thimerosal on type-1 ryanodine receptor stimulation by maurocalcine. Maurocalcine addition to sarcoplasmic reticulum vesicles actively loaded with calcium elicited Ca(2+) release from native vesicles and from vesicles pre-incubated with dithiothreitol; thimerosal addition to native vesicles after Ca(2+) uptake completion prevented this response. Maurocalcine enhanced equilibrium [(3)H]-ryanodine binding to native and to dithiothreitol-treated reticulum vesicles, and increased 5-fold the apparent Ki for Mg(2+) inhibition of [(3)H]-ryanodine binding to native vesicles. Single calcium release channels incorporated in planar lipid bilayers displayed a long-lived open sub-conductance state after maurocalcine addition. The fractional time spent in this sub-conductance state decreased when lowering cytoplasmic [Ca(2+)] from 10μM to 0.1μM or at cytoplasmic [Mg(2+)]≥30μM. At 0.1μM [Ca(2+)], only channels that displayed poor activation by Ca(2+) were readily activated by 5nM maurocalcine; subsequent incubation with thimerosal abolished the sub-conductance state induced by maurocalcine. We interpret these results as an indication that maurocalcine acts as a more effective type-1 ryanodine receptor channel agonist under reducing conditions.

International audience; Maurocalcine is the first demonstrated example of an animal toxin peptide with efficient cell penetration properties. Although it is a highly competitive cell-penetrating peptide (CPP), its relatively large size of 33 amino acids and the presence of three internal disulfide bridges may hamper its development for in vitro and in vivo applications. Here, we demonstrate that several efficient CPPs can be derived from maurocalcine by replacing Cys residues by isosteric 2-aminobutyric acid residues and sequence truncation down to peptides of up to 9 residues in length. A surprising finding is that all of the truncated maurocalcine analogues possessed cell penetration properties, indicating that the maurocalcine is a highly specialized CPP. Careful examination of the cell penetration properties of the truncated analogues indicates that several maurocalcine-derived peptides should be of great interest for cell delivery applications where peptide size matters.

International audience; Caveolin-3 is the striated muscle specific isoform of the scaffolding protein family of caveolins and has been shown to interact with a variety of proteins, including ion channels. Mutations in the human CAV3 gene have been associated with several muscle disorders called caveolinopathies and among these, the P104L mutation (Cav-3(P104L)) leads to limb girdle muscular dystrophy of type 1C characterized by the loss of sarcolemmal caveolin. There is still no clear-cut explanation as to specifically how caveolin-3 mutations lead to skeletal muscle wasting. Previous results argued in favor of a role for caveolin-3 in dihydropyridine receptor (DHPR) functional regulation and/or T-tubular membrane localization. It appeared worth closely examining such a functional link and investigating if it could result from the direct physical interaction of the two proteins. Transient expression of Cav-3(P104L) or caveolin-3 specific siRNAs in C2C12 myotubes both led to a significant decrease of the L-type Ca(2+) channel maximal conductance. Immunolabeling analysis of adult skeletal muscle fibers revealed the colocalization of a pool of caveolin-3 with the DHPR within the T-tubular membrane. Caveolin-3 was also shown to be present in DHPR-containing triadic membrane preparations from which both proteins co-immunoprecipitated. Using GST-fusion proteins, the I-II loop of Ca(v)1.1 was identified as the domain interacting with caveolin-3, with an apparent affinity of 60nM. The present study thus revealed a direct molecular interaction between caveolin-3 and the DHPR which is likely to underlie their functional link and whose loss might therefore be involved in pathophysiological mechanisms associated to muscle caveolinopathies.

In skeletal muscle, RyR1 (5,037 residues) releases calcium from the sarcoplasmic reticulum (SR) in response to an orthograde signal from the DHPR in the plasma membrane (PM), and transmits a retrograde signal which increases the L-type calcium current via the DHPR (which contains CaV1.1 as its principal subunit). Previously, we tested the behavior of a RyR1 construct (YFP-RyR11:4300), which encodes the cytoplasmic domain of RyR1 ("foot") but lacks the C-terminal domains which form the ion pore for SR calcium release and which anchor RyR1 in the SR membrane. We found that YFP-RyR11:4300 targets to PM-SR junctions in dyspedic myotubes (null for endogenous RyR1) and transmits the retrograde signal to the DHPRs present in those myotubes. We have now begun to examine whether these interactions between the RyR1 foot and DHPR can occur in the absence of other proteins which are present in PM-SR junctions of muscle cells. Toward this end, we used tsA201 cells to co-express CFP-RyR11:4300, YFP-CaV1.1 (or CaV1.1/CaV1.2 chimeras) and the DHPR auxiliary subunit β1a. Most of the CaV constructs appeared to be retained in a peri-nuculear compartment (CaV1.1, and chimeras in which three CaV1.1 repeats were replaced by the corresponding CaV1.2 repeats). PM-targeting was observed for a construct composed of CaV1.2 with the cytoplasmic II-III loop replaced by that of CaV1.1. However, we have not yet observed co-localization of CFP-RyR11:4300 with any of the CaV constructs. We are currently testing whether the presence of additional, junctional proteins will result in co-localization of RyR11:4300 and CaV. Supported by grants NIH AR055104 and MDA 277475 to K.G.B.

International audience; Maurocalcine has been the first demonstrated animal toxin acting as a cell-penetrating peptide. Although it possesses competitive advantages, its use as a cell-penetrating peptide (CPP) requires that analogues be developed that lack its characteristic pharmacological activity on ryanodine-sensitive calcium channels without affecting its cell-penetrating and vector efficiencies. Here, we present the synthesis, three-dimensional (1)H NMR structure, and activity of D-maurocalcine. We demonstrate that it possesses all of the desired features for an excellent CPP: preserved structure, lack of pharmacological action, conserved vector properties, and absence of cell toxicity. This is the first report of a folded/oxidized animal toxin in its D-diastereomer conformation for use as a CPP. The protease resistance of this new peptide analogue, combined with its efficient cell penetration at concentrations devoid of cell toxicity, suggests that D-maurocalcine should be an excellent vector for in vivo applications.

A number of G-protein-coupled receptors are expressed in skeletal muscle but their roles in muscle physiology and downstream effector systems remain poorly investigated. Here we explored the functional importance of the G-protein betagamma (Gbetagamma) signalling pathway on voltage-controlled Ca(2+) homeostasis in single isolated adult skeletal muscle fibres. A GFP-tagged Gbeta(1)gamma(2) dimer was expressed in vivo in mice muscle fibres. The GFP fluorescence pattern was consistent with a Gbeta(1)gamma(2) dimer localization in the transverse-tubule membrane. Membrane current and indo-1 fluorescence measurements performed under voltage-clamp conditions reveal a drastic reduction of both L-type Ca(2+) current density and of peak amplitude of the voltage-activated Ca(2+) transient in Gbeta(1)gamma(2)-expressing fibres. These effects were not observed upon expression of Gbeta(2)gamma(2), Gbeta(3)gamma(2) or Gbeta(4)gamma(2). Our data suggest that the G-protein beta(1)gamma(2) dimer may play an important regulatory role in skeletal muscle excitation-contraction coupling.

Maurocalcine (MCa), initially identified from a tunisian scorpion venom, defines a new member of the family of cell penetrating peptides by its ability to efficiently cross the plasma membrane. The initiating mechanistic step required for the cell translocation of a cell penetrating peptide implicates its binding onto cell surface components such as membrane lipids and/or heparan sulfate proteoglycans. Here we characterized the interaction of wild-type MCa and MCa K20A, a mutant analogue with reduced cell-penetration efficiency, with heparin (HP) and heparan sulfates (HS) through surface plasma resonance. HP and HS bind both to MCa, indicating that heparan sulfate proteoglycans may represent an important entry route of the peptide. This is confirmed by the fact that (i) both compounds bind with reduced affinity to MCa K20A and (ii) the cell penetration of wild-type or mutant MCa coupled to fluorescent streptavidin is reduced by about 50% in mutant Chinese hamster ovary cell lines lacking either all glycosaminoglycans (GAGs) or just HS. Incubating MCa with soluble HS, HP, or chondroitin sulfates also inhibits the cell penetration of MCa-streptavidin complexes. Analyses of the cell distributions of MCa/streptavidin in several Chinese hamster ovary cell lines show that the distribution of the complex coincides with the endosomal marker Lyso-Tracker red and is not affected by the absence of GAGs. The distribution of MCa/streptavidin is not coincident with that of transferrin receptors nor affected by a dominant-negative dynamin 2 K44A mutant, an inhibitor of clathrin-mediated endocytosis. However, entry of the complex is greatly diminished by amiloride, indicating the importance of macropinocytosis in MCa/streptavidin entry. It is concluded that (i) interaction of MCa with GAGs quantitatively improves the cell penetration of MCa, and (ii) GAG-dependent and -independent MCa penetration rely similarly on the macropinocytosis pathway.

In skeletal muscle, the dihydropyridine receptor (DHPR) in the plasma membrane engages in bi-directional interactions with the type 1 ryanodine receptor (RyR1) in the sarcoplasmic reticulum (SR) such that an “orthograde” signal from the DHPR triggers SR Ca2+ release via RyR1, and a retrograde signal from RyR1 enhances the L-type Ca2+ current via the DHPR. RyR1 (5,037 residues) assembles as a homotetramer in which the C-termini form the ion conducting pore across the SR membrane, with the remainder of the protein forming a “foot” structure which spans between the SR and plasma membranes. In an accompanying abstract (Polster et al., this meeting) we showed that a construct (YFP-RyR11:4300) which lacks the C-terminal residues was diffusely distributed in dysgenic myotubes (DHPR null), but that in dyspedic myotubes (RyR1-null but containing DHPRs) it targeted junctionally and retrogradely enhanced L-type Ca2+ current. Here we have examined the oligormerization of YFP-RyR11:4300. Measurement of FRAP for YFP-RyR11:4300 in dysgenic myotubes yielded a diffusion coefficient of 2.17x10−8 cm2/sec, which, based on previous measurements of protein diffusion in muscle, is compatible with YFP-RyR11:4300 existing as a tetramer. We also used SDS PAGE (4-15% gradient) and immunoblotting to compare YFP-RyR11:4300 (expressed in tsA-201 cells) and native RyR1. Without prior cross-linking, YFP-RyR11:4300 displayed a smaller apparent MW than wt RyR1; after glutaraldehyde cross-linking, both proteins migrated as single bands of much higher apparent MW, with YFP-RyR11:4300 again displaying a slightly greater mobility, consistent with it's being a tetramer.