Voltage-dependent calcium channels (CaV) open in response to changes in membrane potential, but their activity is modulated by Ca 2؉ binding to calmodulin (CaM). Structural studies of this family of channels have focused on CaM bound to the IQ motif; however, the minimal differences between structures cannot adequately describe CaM's role in the regulation of these channels. We report a unique crystal structure of a 77-residue fragment of the Ca V1.2 ␣1 subunit carboxyl terminus, which includes a tandem of the pre-IQ and IQ domains, in complex with Ca 2؉ ⅐CaM in 2 distinct binding modes. The structure of the Ca V1.2 fragment is an unusual dimer of 2 coiled-coiled pre-IQ regions bridged by 2 Ca 2؉ ⅐CaMs interacting with the pre-IQ regions and a canonical Ca V1-IQ-Ca 2؉ ⅐CaM complex. Native Ca V1.2 channels are shown to be a mixture of monomers/dimers and a point mutation in the pre-IQ region predicted to abolish the coiled-coil structure significantly reduces Ca 2؉ -dependent inactivation of heterologously expressed CaV1.2 channels.structure ͉ function ͉ voltage-gated calcium channel E xcitation-contraction coupling and other important cellular processes are controlled by the voltage-gated Ca 2ϩ channels (Ca V ). The ubiquitous Ca 2ϩ sensor and regulator molecule, calmodulin, is an essential component of Ca V regulation by Ca 2ϩ , and several regions of the cytoplasmic carboxyl terminus of the Ca V ␣1 subunit have been identified as critical molecular determinants for CaM's regulation of Ca V . Ca 2ϩ ⅐CaM bound to the IQ motif of the carboxyl terminus of the ␣ 1 subunit of L-type Ca 2ϩ channels is required for both a feed-forward regulation, Ca 2ϩ -dependent facilitation (CDF), and a feed-back regulation, Ca 2ϩ -dependent inactivation (CDI) (1, 2). CaM acts as the Ca 2ϩ sensor for CDI in Ca V 1.2, Ca V 2.1, Ca V 2.2 and Ca V 2.3, and CDF in Ca V 1.2 and Ca V 2.1. This duality of Ca V regulation by CaM suggests that there are either multiple binding sites or alternative interactions exist to regulate the channel based on different functional states of the channel.Regions upstream of the IQ motif, designated the pre-IQ region, have been implicated in Ca 2ϩ ⅐CaM regulation of the channel (3-5). Consistent with this, 2 different segments within the pre-IQ motif (1606-1627 and 1618-1652, identified as the A and C sequences, respectively) have been shown to bind CaM (4). Moreover, both the IQ motif and amino acids within the pre-IQ region (N1630-E1631) have been indicated to be critical for Ca V 1.2 CDI (6).Recent crystal structures of CaM in complex with the IQ motifs from Ca V 1.2 (7, 8), Ca V 2.2 and Ca V 2.3 (5) reveal few structural differences that could account for the differences in regulation of Ca V 1.2 and Ca V 2.2 by CaM. However, very little is known about the structure of the pre-IQ region or the molecular basis for its interactions with CaM.Here, we report the isolation and determination of the crystal structure at 2.1 Å resolution of a 77-residue (1609-1685) fragment of the carboxyl terminus of the ␣ 1 ...

A heterozygous Ile4898 to Thr (I4898T) mutation in the human type 1 ryanodine receptor/Ca 2+ release channel (RyR1) leads to a severe form of central core disease. We created a mouse line in which the corresponding Ryr1 I4895T mutation was introduced by using a “knockin” protocol. The heterozygote does not exhibit an overt disease phenotype, but homozygous (IT/IT) mice are paralyzed and die perinatally, apparently because of asphyxia. Histological analysis shows that IT/IT mice have greatly reduced and amorphous skeletal muscle. Myotubes are small, nuclei remain central, myofibrils are disarranged, and no cross striation is obvious. Many areas indicate probable degeneration, with shortened myotubes containing central stacks of pyknotic nuclei. Other manifestations of a delay in completion of late stages of embryogenesis include growth retardation and marked delay in ossification, dermatogenesis, and cardiovascular development. Electron microscopy of IT/IT muscle demonstrates appropriate targeting and positioning of RyR1 at triad junctions and a normal organization of dihydropyridine receptor (DHPR) complexes into RyR1-associated tetrads. Functional studies carried out in cultured IT/IT myotubes show that ligand-induced and DHPR-activated RyR1 Ca 2+ release is absent, although retrograde enhancement of DHPR Ca 2+ conductance is retained. IT/IT mice, in which RyR1-mediated Ca 2+ release is abolished without altering the formation of the junctional DHPR-RyR1 macromolecular complex, provide a valuable model for elucidation of the role of RyR1-mediated Ca 2+ signaling in mammalian embryogenesis.

The type 1 isoform of the ryanodine receptor (RYR1) is the Ca2+ release channel of the sarcoplasmic reticulum (SR) that is activated during skeletal muscle excitation–contraction (EC) coupling. Mutations in the RYR1 gene cause several rare inherited skeletal muscle disorders, including malignant hyperthermia and central core disease (CCD). The human RYR1I4898T mutation is one of the most common CCD mutations. To elucidate the mechanism by which RYR1 function is altered by this mutation, we characterized in vivo muscle strength, EC coupling, SR Ca2+ content, and RYR1 Ca2+ release channel function using adult heterozygous Ryr1I4895T/+ knock-in mice (IT/+). Compared with age-matched wild-type (WT) mice, IT/+ mice exhibited significantly reduced upper body and grip strength. In spite of normal total SR Ca2+ content, both electrically evoked and 4-chloro-m-cresol–induced Ca2+ release were significantly reduced and slowed in single intact flexor digitorum brevis fibers isolated from 4–6-mo-old IT/+ mice. The sensitivity of the SR Ca2+ release mechanism to activation was not enhanced in fibers of IT/+ mice. Single-channel measurements of purified recombinant channels incorporated in planar lipid bilayers revealed that Ca2+ permeation was abolished for homotetrameric IT channels and significantly reduced for heterotetrameric WT:IT channels. Collectively, these findings indicate that in vivo muscle weakness observed in IT/+ knock-in mice arises from a reduction in the magnitude and rate of RYR1 Ca2+ release during EC coupling that results from the mutation producing a dominant-negative suppression of RYR1 channel Ca2+ ion permeation.

The skeletal muscle ryanodine receptor plays a crucial role in excitation–contraction (EC) coupling and is implicated in various congenital myopathies. The periodic paralyses are a heterogeneous, dominantly inherited group of conditions mainly associated with mutations in the SCN4A and the CACNA1S genes. The interaction between RyR1 and DHPR proteins underlies depolarization-induced Ca2+ release during EC coupling in skeletal muscle. We report a 35-year-old woman presenting with signs and symptoms of a congenital myopathy at birth and repeated episodes of generalized, atypical normokalaemic paralysis in her late teens. Genetic studies of this patient revealed three heterozygous RYR1 substitutions (p.Arg2241X, p.Asp708Asn and p.Arg2939Lys) associated with marked reduction of the RyR1 protein and abnormal DHPR distribution. We conclude that RYR1 mutations may give rise to both myopathies and atypical periodic paralysis, and RYR1 mutations may underlie other unresolved cases of periodic paralysis with unusual features.

SummaryThe I4898T (IT) mutation in type 1 ryanodine receptor (RyR1), the Ca 2+ release channel of the sarcoplasmic reticulum (SR) is linked to a form of central core disease (CCD) in humans and results in a nonleaky channel and excitation-contraction uncoupling. We characterized agedependent and fiber-type-dependent alterations in muscle ultrastructure, as well as the magnitude and spatiotemporal properties of evoked Ca 2+ release in heterozygous Ryr1I4895T ⁄ WT (IT ⁄ +) knock-in mice on a mixed genetic background. The results indicate a classical but mild CCD phenotype that includes muscle weakness and the presence of mitochondrial-deficient areas in type I fibers. Electrically evoked Ca 2+ release is significantly reduced in single flexor digitorum brevis (FDB) fibers from young and old IT ⁄ + mice. Structural changes are strongly fiber-type specific, affecting type I and IIB ⁄ IIX fibers in very distinct ways, and sparing type IIA fibers. Ultrastructural alterations in our IT ⁄ + mice are also present in wild type, but at a lower frequency and older ages, suggesting that the disease mutation on the mixed background promotes an acceleration of normal age-dependent changes. The observed functional and structural alterations and their similarity to age-associated changes are entirely consistent with the known properties of the mutated channel, which result in reduced calcium release as is also observed in normal aging muscle. In strong contrast to these observations, a subset of patients with the analogous human heterozygous mutation and IT ⁄ + mice on an inbred 129S2 ⁄ SvPasCrl background exhibit a more severe disease phenotype, which is not directly consistent with the mutated channel properties.