II-III Loop Research Articles

The neuronal junctophilins interact with the II-III loop of Cav2.1

The neuronal junctophilins interact with the II-III loop of Cav2.1

CaV 2.1 α1 subunit motifs that control presynaptic CaV 2.1subtype abundance are distinct from CaV 2.1 preference.

Presynaptic voltage-gated Ca2+ channel (CaV ) subtype abundance at mammalian synapses regulates synaptic transmission in health and disease. In the mammalian central nervous system (CNS), most presynaptic terminals are CaV 2.1 dominant with a developmental reduction in CaV 2.2 and CaV 2.3 levels, and CaV 2subtype levels are altered in various diseases. However, the molecular mechanisms controlling presynaptic CaV 2subtype levels are largely unsolved. Because the CaV 2 α1 subunit cytoplasmic regions contain varying levels of sequence conservation, these regions are proposed to control presynaptic CaV 2subtype preference and abundance. To investigate the potential role of these regions, we expressed chimeric CaV 2.1 α1 subunits containing swapped motifs with the CaV 2.2 and CaV 2.3 α1 subunit on a CaV 2.1/CaV 2.2 null background at the calyx of Held presynaptic terminals. We found that expression of CaV 2.1 α1 subunit chimeras containing the CaV 2.3 loop II-III region or cytoplasmic C-terminus (CT) resulted in a large reduction of presynaptic Ca2+ currents compared to the CaV 2.1 α1 subunit. However, the Ca2+ current sensitivity to the CaV 2.1 blocker agatoxin-IVA was the same between the chimeras and the CaV 2.1 α1 subunit. Additionally, we found no reduction in presynaptic Ca2+ currents with CaV 2.1/2.2 cytoplasmic CT chimeras. We conclude that the motifs in the CaV 2.1 loop II-III and CT do not individually regulate CaV 2.1 preference, although these motifs control CaV 2.1 levels and the CaV 2.3 CT contains motifs that negatively regulate presynaptic CaV 2.3 levels. We propose that the motifs controlling presynaptic CaV 2.1 preference are distinct from those regulating CaV 2.1 levels and may act synergistically to impact pathways regulating CaV 2.1 preference and abundance. KEY POINTS: Presynaptic CaV 2subtype abundance regulates neuronal circuit properties, although the mechanisms regulating presynaptic CaV 2subtype abundance and preference remain enigmatic. The CaV α1 subunit determines subtype and contains multiple motifs implicated in regulating presynaptic subtype abundance and preference. The CaV 2.1 α1 subunit domain II-III loop and cytoplasmic C-terminus are positive regulators of presynaptic CaV 2.1 abundance but do not regulate preference. The CaV 2.3 α1 subunit cytoplasmic C-terminus negatively regulates presynaptic CaV 2subtype abundance but not preference, whereas the CaV 2.2 α1 subunit cytoplasmic C-terminus is not a key regulator of presynaptic CaV 2subtype abundance or preference. The CaV 2 α1 subunit motifs determining the presynaptic CaV 2 preference are distinct from abundance.

STAC proteins inhibit the voltage dependent inactivation of l-type calcium channels.

The inactivation of voltage-gated calcium channels is governed by two distinct processes: voltage dependent inactivation (VDI) and calcium-dependent inactivation (CDI). The STAC family of adaptor proteins inhibits the CDI of L-type calcium channels (LTCC). In fact, all LTCC (CaV1.2, CaV1.3 and CaV1.4) showed reduced CDI in the presence of any STAC isoform. However, whether STAC proteins also modulate the CDI of the skeletal muscle channel CaV1.1 was initially not investigated, as CaV1.1 requires STAC3 for its functional expression. Interestingly, we could recently demonstrate that STAC3 does not inhibit the CDI, but the VDI of CaV1.1. Therefore, we hypothesized that STAC proteins inhibit also the VDI of LTCC. Indeed, when co-expressed in HEK cells, all STAC isoforms reduced the VDI of CaV1.2 and CaV1.3 currents. As for CDI, also the VDI inhibition is specific for LTCC, as the VDI of CaV2.1 and CaV2.3 is not affected by STAC proteins. As the predominant contribution to VDI is known to arise from the I-II loop interaction with the CaVβ subunit, and that STAC proteins were shown to interact with distinct cytoplasmic domains (II-III loop and C-terminus), we hypothesized that STAC proteins and membrane tethered β subunits utilize different mechanism to inhibit VDI. Indeed, co-expression of STAC3 with CaV1.3/β2a revealed additional deceleration of VDI in HEK cells. Ongoing experiments, involving STAC and channels mutants and chimeras, will reveal the molecular determinants of the VDI inhibition by STAC proteins.

Identifying the STAC3/CaV1.1 interactions responsible for CaV1.1 expression in skeletal muscle.

The adaptor protein STAC3 was shown to be crucial for membrane and functional expression of the voltage sensor of EC coupling CaV1.1. In fact, CaV1.1 membrane expression is reduced and calcium currents are abolished in STAC3 KO animals. Two distinct STAC3/CaV1.1 interactions have been identified: one between the II-III loop of CaV1.1 and the SH3-1 domain of STAC3 and a second between the proximal C-terminus of CaV1.1 channels and the linker region of STAC3. Here, we investigated whether a single interaction is responsible for the CaV1.1 membrane and functional expression or if both synergistically contribute to CaV1.1 expression. To this end, we generated two STAC3 fragments, STAC3-NT, consisting of the N-terminal part, including the C1 domain and the linker region, expected to interact with the proximal C-terminus of CaV1.1, and STAC3-CT, consisting of only the SH3 domains, expected to interact with the II-III loop of CaV1.1. Expression of either one of these fragments, in a novel C2C12 STAC3 KO cell line still promoted CaV1.1 membrane expression, but at a reduced level compared to full length STAC3, suggesting that both interactions are contributing to CaV1.1 membrane expression. However, only expression of STAC3-NT supported CaV1.1 functional expression in CaV1.1 reconstituted double CaV1.1/STAC3 KO cells, indicating that only the STAC3 interaction with the proximal C-terminus is essential for CaV1.1 function in muscle cells. In addition, we analysed the ability of each STAC3 fragment to colocalize with CaV1.1 in the triads of myotubes. While the STAC3-NT fragment is still able to incorporate in CaV1.1 clusters, albeit with a 50% reduction in the Pearson's coefficient compared to full length STAC3, the STAC3-CT fragment remained diffusely localized in the cytoplasm. Experiments are planned to establish the importance of each fragment for EC coupling.

The Cav2.1 II-III loop binds to both junctophilin 3 and junctophilin 4

Junctophilin 3 (JPH3) and junctophilin 4 (JPH4) are both expressed in neurons, where they form ER-PM junctions, regions in which the endoplasmic reticulum comes into close proximity with the plasma membrane. By allowing cross-communication between voltage- and ligand-gated channels in the PM and Ca2+-releasing channels in the ER, junctophilin-induced ER-PM junctions are critical for neuronal function. We previously showed that JPH3 and JPH4 recruited high-voltage activated Ca2+ channels into ER-PM junctions when transfected in tsA201 cells.

Regulation of CaV3.2 channels by the receptor for activated C kinase 1 (Rack-1).

This study describes the interaction between CaV3.2 calcium channels and the receptor for activated C kinase 1 (Rack-1), a scaffold protein which has recently been implicated in neuropathic pain. The coexpression of CaV3.2 and Rack-1 in tsA-201 cells led to a reduction in the magnitude of whole-cell CaV3.2 currents and CaV3.2 channel expression at the plasma membrane. Co-immunoprecipitations from transfected cells show the formation of a molecular protein complex between Cav3.2 channels and Rack-1. We determined that the interaction of Rack-1 occurs at the intracellular II-III loop and the C-terminus of the channel. Finally, the coexpression of PKCβII abolished the effect of Rack-1 on current densities. Altogether, our findings show that Rack-1 regulates CaV3.2-mediated calcium entry in a PKC-dependent manner.

Multiple Sequence Variants in STAC3 Affect Interactions with CaV1.1 and Excitation-Contraction Coupling

STAC3 is a soluble protein essential for skeletal muscle excitation-contraction (EC) coupling. Through its tandem SH3 domains, it interacts with the cytosolic II-III loop of the skeletal muscle voltage-gated calcium channel. STAC3 is the target for a mutation (W284S) that causes Native American myopathy, but multiple other sequence variants have been reported. Here, we report a crystal structure of the human STAC3 tandem SH3 domains. We analyzed the effect of five disease-associated variants, spread over both SH3 domains, on their ability to bind to the CaV1.1 II-III loop and on muscle EC coupling. In addition to W284S, we find the F295L and K329N variants to affect both binding and EC coupling. The ability of the K329N variant, located in the second SH3 domain, to affect the interaction highlights the importance of both SH3 domains in association with CaV1.1. Our results suggest that multiple STAC3 variants may cause myopathy.

Which CaV1.1 Voltage Sensor(s) Activate RYR1?

The skeletal muscle L-type Ca­V channel is composed by the pore-forming α1s (CaV1.1) in association with α2δ-1, β1a and γ1 subunits in 1:1:1:1 stoichiometry. This large protein complex makes physical contact with Ryanodine (RyR1) channel located in the sarcoplasmic reticulum (SR), conferring voltage-dependence to the Ca2+ release channel, thus coupling electrical excitation with contraction. CaV1.1 channels possess four homologous but non-identical voltage sensing domains (VSDs), which undergo voltage-dependent rearrangements: we propose that one VSD is a stronger candidate than all others to be the sensor responsible for RyR1 voltage-dependent activation. We reconstituted human CaV1.1 complexes (α1s, α2δ-1, β1a, γ1 and stac3) in Xenopus oocytes. We used the voltage clamp fluorometry technique to simultaneously record Ca2+ currents (2mM external Ca2+) and the structural rearrangements of the four individual VSDs. We found that the voltage-dependent activation curves of the four VSDs are spread from −150 to +80mV, all preceding pore opening, with VSD-I and VSD-IV operating at more hyperpolarized potentials (VSD-I Vhalf=-35.6±2.2mV, z=1.0±0.01e0, VSD-IV Vhalf=-64.9±2.7mV, z=0.74±0.02e0) than VSD-II and VSD-III (VSD-II Vhalf=7.2±2.8mV, z=1.6±0.1e0, VSD-III Vhalf=-23.9±3mV, z=1.6±0.04e0). Each VSD exhibited distinct time-dependent properties, with VSD-I and VSD-II displaying slow activation kinetics (VSD-I τ=39.8±2ms, VSD-II τ=132±16ms, at 20mV), while VSD-III and VSD-IV activated rapidly (VSD-III τ=1.8±0.2ms, VSD-IV τ=2±1ms, at 20mV). The operation of the four voltage sensors was then investigated under a physiologically-relevant action potential (AP) clamp (skeletal muscle AP). Within the short AP clamp (∼5ms), no clear structural rearrangements of the slowest sensors (VSD-I and VSD-II) could be observed, while VSD-III and VSD-IV were readily activated. Based on i) kinetics, ii) voltage-dependence, iii) proximity with intracellular II-III loop (known to be a critical element in EC coupling), VSD-III is a strong candidate as sensor conferring voltage-dependent properties to RyR1 and the SR Ca2+ release.

Stac Proteins Associate with the Domain of the CaV1.1 II-III Loop Critical for EC Coupling

Stac Proteins Associate with the Domain of the CaV1.1 II-III Loop Critical for EC Coupling

Structural insights into binding of STAC proteins to voltage-gated calcium channels

Excitation-contraction (EC) coupling in skeletal muscle requires functional and mechanical coupling between L-type voltage-gated calcium channels (CaV1.1) and the ryanodine receptor (RyR1). Recently, STAC3 was identified as an essential protein for EC coupling and is part of a group of three proteins that can bind and modulate L-type voltage-gated calcium channels. Here, we report crystal structures of tandem-SH3 domains of different STAC isoforms up to 1.2-Å resolution. These form a rigid interaction through a conserved interdomain interface. We identify the linker connecting transmembrane repeats II and III in two different CaV isoforms as a binding site for the SH3 domains and report a crystal structure of the complex with the STAC2 isoform. The interaction site includes the location for a disease variant in STAC3 that has been linked to Native American myopathy (NAM). Introducing the mutation does not cause misfolding of the SH3 domains, but abolishes the interaction. Disruption of the interaction via mutations in the II-III loop perturbs skeletal muscle EC coupling, but preserves the ability of STAC3 to slow down inactivation of CaV1.2.

Cell-Specific RNA Binding Protein Rbfox2 Regulates CaV2.2 mRNA Exon Composition and CaV2.2 Current Size.

The majority of multiexon mammalian genes contain alternatively spliced exons that have unique expression patterns in different cell populations and that have important cell functions. The expression profiles of alternative exons are controlled by cell-specific splicing factors that can promote exon inclusion or exon skipping but with few exceptions we do not know which specific splicing factors control the expression of alternatively spliced exons of known biological function. Many ion channel genes undergo extensive alternative splicing including Cacna1b that encodes the voltage-gated CaV2.2 α1 subunit. Alternatively spliced exon 18a in Cacna1b RNA encodes 21 amino acids in the II-III loop of CaV2.2, and its expression differs across the nervous system and over development. Genome-wide, protein-RNA binding analyses coupled to high-throughput RNA sequencing show that RNA binding Fox (Rbfox) proteins associate with CaV2.2 (Cacna1b) pre-mRNAs. Here, we link Rbfox2 to suppression of e18a. We show increased e18a inclusion in CaV2.2 mRNAs: (1) after siRNA knockdown of Rbfox2 in a neuronal cell line and (2) in RNA from sympathetic neurons of adult compared to early postnatal mice. By immunoprecipitation of Rbfox2-RNA complexes followed by qPCR, we demonstrate reduced Rbfox2 binding upstream of e18a in RNA from sympathetic neurons of adult compared to early postnatal mice. CaV2.2 currents in cell lines and in sympathetic neurons expressing only e18a-CaV2.2 are larger compared to currents from those expressing only Δ18a-CaV2.2. We conclude that Rbfox2 represses e18a inclusion during pre-mRNA splicing of CaV2.2, limiting the size of CaV2.2 currents early in development in certain neuronal populations.

Ca2+ channel clustering with insulin-containing granules is disturbed in type 2 diabetes.

Loss of first-phase insulin secretion is an early sign of developing type 2 diabetes (T2D). Ca2+ entry through voltage-gated L-type Ca2+ channels triggers exocytosis of insulin-containing granules in pancreatic β cells and is required for the postprandial spike in insulin secretion. Using high-resolution microscopy, we have identified a subset of docked insulin granules in human β cells and rat-derived clonal insulin 1 (INS1) cells for which localized Ca2+ influx triggers exocytosis with high probability and minimal latency. This immediately releasable pool (IRP) of granules, identified both structurally and functionally, was absent in β cells from human T2D donors and in INS1 cells cultured in fatty acids that mimic the diabetic state. Upon arrival at the plasma membrane, IRP granules slowly associated with 15 to 20 L-type channels. We determined that recruitment depended on a direct interaction with the synaptic protein Munc13, because expression of the II–III loop of the channel, the C2 domain of Munc13-1, or of Munc13-1 with a mutated C2 domain all disrupted L-type channel clustering at granules and ablated fast exocytosis. Thus, rapid insulin secretion requires Munc13-mediated recruitment of L-type Ca2+ channels in close proximity to insulin granules. Loss of this organization underlies disturbed insulin secretion kinetics in T2D.

Fluorescence Resonance Energy Transfer-based Structural Analysis of the Dihydropyridine Receptor α1S Subunit Reveals Conformational Differences Induced by Binding of the β1a Subunit

The skeletal muscle dihydropyridine receptor α1S subunit plays a key role in skeletal muscle excitation-contraction coupling by sensing membrane voltage changes and then triggering intracellular calcium release. The cytoplasmic loops connecting four homologous α1S structural domains have diverse functions, but their structural arrangement is poorly understood. Here, we used a novel FRET-based method to characterize the relative proximity of these intracellular loops in α1S subunits expressed in intact cells. In dysgenic myotubes, energy transfer was observed from an N-terminal-fused YFP to a FRET acceptor, ReAsH (resorufin arsenical hairpin binder), targeted to each α1S intracellular loop, with the highest FRET efficiencies measured to the α1S II-III loop and C-terminal tail. However, in HEK-293T cells, FRET efficiencies from the α1S N terminus to the II-III and III-IV loops and the C-terminal tail were significantly lower, thus suggesting that these loop structures are influenced by the cellular microenvironment. The addition of the β1a dihydropyridine receptor subunit enhanced FRET to the II-III loop, thus indicating that β1a binding directly affects II-III loop conformation. This specific structural change required the C-terminal 36 amino acids of β1a, which are essential to support EC coupling. Direct FRET measurements between α1S and β1a confirmed that both wild type and truncated β1a bind similarly to α1S These results provide new insights into the role of muscle-specific proteins on the structural arrangement of α1S intracellular loops and point to a new conformational effect of the β1a subunit in supporting skeletal muscle excitation-contraction coupling.

Characterization of the STAC3-CaV1.1 Interaction in Skeletal Muscle Excitation-Contraction Coupling

Skeletal muscle EC coupling critically depends on the physical interaction between the voltage-gated calcium channel CaV1.1 (DHPR), acting as voltage sensor, and the SR calcium release channel (RyR1). The skeletal muscle-specific DHPR components essential for EC coupling are the CaV1.1 II-III loop and the auxiliary β1a subunit. Recently, STAC3 (src homology 3 and cysteine rich domain 3) has been identified as an additional essential EC coupling protein. Further investigations indicated that STAC3 functions in membrane trafficking of CaV1.1 and linked STAC3 to a rare muscle disease Native American Myopathy (NAM). In contrast STAC3 is not expressed in the heart and thus appears not to participate in cardiac muscle EC coupling. These findings raise the question as to whether STAC3 binds exclusively to the skeletal muscle DHPR, and whether its interaction with the EC coupling complex is stable or dynamic. Here, we applied fluorescence recovery after photobleaching (FRAP) in dysgenic (CaV1.1-null) myotubes reconstituted with CaV1.1 and GFP-tagged STAC3 to demonstrate that STAC3 forms stable complexes with the skeletal muscle DHPR. Furthermore, we examined the subcellular localization of STAC3 and of the other two members of the STAC family of adaptor proteins, STAC1 and STAC2, in dysgenic myotubes reconstituted with either CaV1.1 or CaV1.2. Together these experiments demonstrate that the three members of the STAC family display differential interactions with the skeletal muscle and cardiac DHPRs. These functional differences allow the use of chimeric proteins to identify the molecular domains involved in the association of STAC3 with the skeletal muscle EC coupling apparatus. This work is supported by Austrian Research Fund FWF (P23479, P27031).

Ca2+ influx through L-type Ca2+ channels and Ca2+-induced Ca2+ release regulate cAMP accumulation and Epac1-dependent ERK 1/2 activation in INS-1 cells

We previously reported that INS-1 cells expressing the intracellular II-III loop of the L-type Ca2+ channel Cav1.2 (Cav1.2/II-III cells) are deficient in Ca2+-induced Ca2+ release (CICR). Here we show that glucose-stimulated ERK 1/2 phosphorylation (GSEP) is slowed and reduced in Cav1.2/II-III cells compared to INS-1 cells. This parallels a decrease in glucose-stimulated cAMP accumulation (GS-cAMP) in Cav1.2/II-III cells. Influx of Ca2+ via L-type Ca2+ channels and CICR play roles in both GSEP and GS-cAMP in INS-1 cells since both are inhibited by nicardipine or ryanodine. Further, the Epac1-selective inhibitor CE3F4 abolishes glucose-stimulated ERK activation in INS-1 cells, as measured using the FRET-based sensor EKAR. The non-selective Epac antagonist ESI-09 but not the Epac2-selective antagonist ESI-05 nor the PKA antagonist Rp-cAMPs inhibits GSEP in both INS-1 and Cav1.2/II-III cells. We conclude that L-type Ca2+ channel-dependent cAMP accumulation, that's amplified by CICR, activates Epac1 and drives GSEP in INS-1 cells.

Physiological and Pharmacological Modulation of the Embryonic Skeletal Muscle Calcium Channel Splice Variant CaV1.1e

CaV1.1e is the voltage-gated calcium channel splice variant of embryonic skeletal muscle. It differs from the adult CaV1.1a splice variant by the exclusion of exon 29 coding for 19 amino acids in the extracellular loop connecting transmembrane domains IVS3 and IVS4. Like the adult splice variant CaV1.1a, the embryonic CaV1.1e variant functions as voltage sensor in excitation-contraction coupling, but unlike CaV1.1a it also conducts sizable calcium currents. Consequently, physiological or pharmacological modulation of calcium currents may have a greater impact in CaV1.1e expressing muscle cells. Here, we analyzed the effects of L-type current modulators on whole-cell current properties in dysgenic (CaV1.1-null) myotubes reconstituted with either CaV1.1a or CaV1.1e. Furthermore, we examined the physiological current modulation by interactions with the ryanodine receptor using a chimeric CaV1.1e construct in which the cytoplasmic II-III loop, essential for skeletal muscle excitation-contraction coupling, has been replaced with the corresponding but nonfunctional loop from the Musca channel. Whereas the equivalent substitution in CaV1.1a had abolished the calcium currents, substitution of the II-III loop in CaV1.1e did not significantly reduce current amplitudes. This indicates that CaV1.1e is not subject to retrograde coupling with the ryanodine receptor and that the retrograde coupling mechanism in CaV1.1a operates by counteracting the limiting effects of exon 29 inclusion on the current amplitude. Pharmacologically, CaV1.1e behaves like other L-type calcium channels. Its currents are substantially increased by the calcium channel agonist Bay K 8644 and inhibited by the calcium channel blocker nifedipine in a dose-dependent manner. With an IC50 of 0.37 μM for current inhibition by nifedipine, CaV1.1e is a potential drug target for the treatment of myotonic dystrophy. It might block the excessive calcium influx resulting from the aberrant expression of the embryonic splice variant CaV1.1e in the skeletal muscles of myotonic dystrophy patients.

3D Structure of the Dihydropyridine Receptor of Skeletal Muscle.

Excitation contraction coupling, the rapid and massive Ca2+ release under control of an action potential that triggers muscle contraction, takes places at specialized regions of the cell called triad junctions. There, a highly ordered supramolecular complex between the dihydropyridine receptor (DHPR) and the ryanodine receptor (RyR1) mediates the quasi-instantaneous conversion from T-tubule depolarization into Ca2+ release from the sarcoplasmic reticulum (SR). The DHPR has several key modules required for EC coupling: the voltage sensors and II-III loop in the alpha1s subunit, and the beta subunit. To gain insight into their molecular organization, this review examines the most updated 3D structure of the DHPR as obtained by transmission electron microscopy and image reconstruction. Although structure determination of a heteromeric membrane protein such as the DHPR is challenging, novel technical advances in protein expression and 3D labeling facilitated this task. The 3D structure of the DHPR complex consists of a main body with five irregular corners around its perimeter encompassing the transmembrane alpha 1s subunit besides the intracellular beta subunit, an extended extracellular alpha 2 subunit, and a bulky intracellular II-III loop. The structural definition attained at 19 Å resolution enabled docking of the atomic coordinates of structural homologs of the alpha1s and beta subunits. These structural features, together with their relative location with respect to the RyR1, are discussed in the context of the functional data.

FRET-Based Structural Analysis of the Skeletal Muscle DHPR using Biarsenical Labeling

The α1s skeletal muscle dihydropyridine receptor (DHPR) subunit triggers muscle contraction during excitation contraction (EC) coupling. The intracellular loops of this voltage-gated calcium channel have diverse functions, though the spatial organization of these loops relative to each other and to other DHPR subunits, is poorly understood. We employed a FRET-based assay to determine the proximity of these intracellular loops relative to the N-terminus of the α1s DHPR subunit and then determined how these proximities changed upon binding of the β1a DHPR subunit. Recombinant α1s DHPRs were expressed in HEK293T cells and then FRET was measured from YFP fused to the DHPR N-terminus to the biarsenical reagent ReAsH targeted to a tetracysteine (Tc) tag inserted into each of the DHPR intracellular loops. Energy transfer was highest to ReAsH bound to a Tc tag placed adjacent to the N-terminally fused YFP (E∼40-50%) whereas low FRET (E∼10%) was observed for a negative control construct lacking a Tc tag. Placement of Tc tags in each of the DHPR intracellular loops resulted in intermediate FRET efficiencies whereas insignificant energy transfer was observed between the DHPR N- and C-termini. Addition of purified recombinant DHPR β1a subunit resulted in significant enhancement of energy transfer between the N-terminal YFP and ReAsH targeted to the DHPR α1s II-III loop, a major EC coupling determinant. Our data demonstrate that FRET measurements using biarsenical labeling reagents offer an effective approach to study DHPR structure since disruption of native conformation is minimal. In addition, these studies should allow for subsequent measurements of changes in DHPR structure that occur during EC coupling. Supported by NIH grants R01AR059126 (to JDF) and R03AR066359 (to CFP).

Crystal Structures of the Ryanodine Receptor SPRY2 Domain

The SPRY2 domain is one of three repeats of the same fold that are present within the RyR. It has been suggested as a key protein interaction site with dihydropyridine receptors to mediate excitation-contraction coupling in skeletal muscle tissue. RyR1 and RyR2 SPRY2 domains were crystallized and reveal differences with what was thought to be SPRY2 and with several other known SPRY domain structures. Our RyR1 SPRY2 construct (rabbit, 1070-1246) is 43% larger than a previously reported construct (1085-1208), consists of multiple modules, is highly soluble, and is monomeric in nature. Docking of the RyR1 SPRY2 structure places it in between the central rim and the clamp region. The structure of RyR2 SPRY2 (mouse, 1080-1253) and a loss-of-function disease mutant (human, T1107M) causing cardiomyopathy were also determined. The T1107M mutation is buried and causes a large ∼10°C decrease in thermal stability as compared to wildtype and also causes local misfolding of the domain. Finally, RyR1 SPRY2 binding to the DHPR II-III loop is undetectable by isothermal titration calorimetry.

Uncoupling of Cav1.2 From Ca2+-Induced Ca2+Release and SK Channel Regulation in Pancreatic β-Cells

We investigated the role of Cav1.2 in pancreatic β-cell function by expressing a Cav1.2 II-III loop/green fluorescent protein fusion in INS-1 cells (Cav1.2/II-III cells) to disrupt channel-protein interactions. Neither block of KATP channels nor stimulation of membrane depolarization by tolbutamide was different in INS-1 cells compared with Cav1.2/II-III cells, but whole-cell Cav current density was significantly increased in Cav1.2/II-III cells. Tolbutamide (200 μM) stimulated insulin secretion and Ca(2+) transients in INS-1 cells, and Cav1.2/II-III cells were completely blocked by nicardipine (2 μM), but thapsigargin (1 μM) blocked tolbutamide-stimulated secretion and Ca(2+) transients only in INS-1 cells. Tolbutamide-stimulated endoplasmic reticulum [Ca(2+)] decrease was reduced in Cav1.2/II-III cells compared with INS-1 cells. However, Ca(2+) transients in both INS-1 cells and Cav1.2/II-III cells were significantly potentiated by 8-pCPT-2'-O-Me-cAMP (5 μM), FPL-64176 (0.5 μM), or replacement of extracellular Ca(2+) with Sr(2+). Glucose (10 mM) + glucagon-like peptide-1 (10 nM) stimulated discrete spikes in [Ca(2+)]i in the presence of verapamil at a higher frequency in INS-1 cells than in Cav1.2/II-II cells. Glucose (18 mM) stimulated more frequent action potentials in Cav1.2/II-III cells and primary rat β-cells expressing the Cav1.2/II-II loop than in control cells. Further, apamin (1 μM) increased glucose-stimulated action potential frequency in INS-1 cells, but not Cav1.2/II-III cells, suggesting that SK channels were not activated under these conditions in Cav1.2/II-III loop-expressing cells. We propose the II-III loop of Cav1.2 as a key molecular determinant that couples the channel to Ca(2+)-induced Ca(2+) release and activation of SK channels in pancreatic β-cells.