Inactivation of the sarcoplasmic reticulum calcium channel by protein kinase.

The ryanodine receptor protein of skeletal muscle sarcoplasmic reticulum (SR) membranes is a calcium ion channel which allows movement of calcium from the SR lumen into the cytoplasm during muscle activation. Gating of this channel is modulated by a number of physiologically important substances including calcium. Interestingly, calcium has both activating and inactivating effects which are concentration- and tissue-specific. In skeletal muscle, calcium-dependent inactivation of calcium release occurs at concentrations reached physiologically, suggesting that calcium may modulate the release process by a negative feedback mechanism. To determine the cellular mechanism responsible for calcium-dependent inactivation, we have investigated the ability of protein phosphorylation to affect single channel gating behaviour using the patch clamp technique. Here we demonstrate that the ryanodine receptor protein/calcium release channel of skeletal muscle SR is inactivated under conditions permissive for protein phosphorylation. This inactivation is reversed by the application of phosphatase and prevented by a peptide inhibitor specific for calcium/calmodulin-dependent protein kinase II. The results provide evidence for an endogenous protein kinase which is closely associated with the ryanodine receptor protein and regulates channel gating.     

Crystal structure of the calcium pump of sarcoplasmic reticulum at 2.6 A resolution

Calcium ATPase is a member of the P-type ATPases that transport ions across the membrane against a concentration gradient. Here we have solved the crystal structure of the calcium ATPase of skeletal muscle sarcoplasmic reticulum (SERCA1a) at 2.6 A resolution with two calcium ions bound in the transmembrane domain, which comprises ten alpha-helices. The two calcium ions are located side by side and are surrounded by four transmembrane helices, two of which are unwound for efficient coordination geometry. The cytoplasmic region consists of three well separated domains, with the phosphorylation site in the central catalytic domain and the adenosine-binding site on another domain. The phosphorylation domain has the same fold as haloacid dehalogenase. Comparison with a low-resolution electron density map of the enzyme in the absence of calcium and with biochemical data suggests that large domain movements take place during active transport.     

Inositol 1,4,5-trisphosphate activates a channel from smooth muscle sarcoplasmic reticulum

Inositol 1,4,5-trisphosphate (InsP3) can initiate calcium release into the cytoplasm in a variety of cells. From experiments using permeabilized cells, membrane vesicles, and patch-clamp techniques, it has been suggested that InsP3 acts by directly opening calcium channels. Here, we show that InsP3 induced openings of channels in planar lipid bilayers into which vesicles made from aortic muscle sarcoplasmic reticulum (SR) were incorporated. Activation of channels by InsP3 was not observed when vesicles made from SR of cardiac or skeletal muscle were incorporated into planar lipid bilayers. The present study demonstrates for the first time unique properties of an InsP3-gated calcium channel in sarcoplasmic reticulum vesicles from vascular smooth muscle. This InsP3-activated channel from aortic SR differs strikingly from the calcium-gated calcium channel of striated muscle SR in single-channel conductance and pharmacology.     

Inositol 1,4,5-trisphosphate induces calcium release from sarcoplasmic reticulum of skeletal muscle

The sarcoplasmic reticulum of skeletal muscle is a specialized form of endoplasmic reticulum that controls myoplasmic calcium concentration and, therefore, the contraction-relaxation cycle. Ultrastructural studies have shown that the sarcoplasmic reticulum is a continuous but heterogeneous membranous network composed of longitudinal tubules that surround myofibrils and terminal cisternae. These cisternae are junctionally associated, via bridging structures called 'feet', with sarcolemmal invaginations (the transverse tubules) to form the triadic junction. Following transverse tubule depolarization, a signal, transmitted along the triadic junction, triggers Ca2+ release from terminal cisternae, but the mechanism of this coupling is still unknown. Inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) has recently been shown to mobilize Ca2+ from intracellular stores, referable to endoplasmic reticulum, in a variety of cell types (see ref. 8 for review), including smooth muscle cells of the porcine coronary artery and canine cardiac muscle cells. Here we show that Ins(1,4,5)P3 releases Ca2+ from isolated, purified sarcoplasmic reticulum fractions of rabbit fast-twitch skeletal muscle, the effect being more pronounced on a fraction of terminal cisternae that contains morphologically intact feet structures; and elicits isometric force development in chemically skinned muscle fibres.     

Primary structure and functional expression of the cardiac dihydropyridine-sensitive calcium channel

In cardiac muscle, where Ca2+ influx across the sarcolemma is essential for contraction, the dihydropyridine (DHP)-sensitive L-type calcium channel represents the major entry pathway of extracellular Ca2+. We have previously elucidated the primary structure of the rabbit skeletal muscle DHP receptor by cloning and sequencing the complementary DNA. An expression plasmid carrying this cDNA, microinjected into cultured skeletal muscle cells from mice with muscular dysgenesis, has been shown to restore both excitation-contraction coupling and slow calcium current missing from these cells, so that a dual role for the DHP receptor in skeletal muscle transverse tubules is suggested. We report here the complete amino-acid sequence of the rabbit cardiac DHP receptor, deduced from the cDNA sequence. We also show that messenger RNA derived from the cardiac DHP receptor cDNA is sufficient to direct the formation of a functional DHP-sensitive calcium channel in Xenopus oocytes. Furthermore, higher calcium-channel activity is observed when mRNA specific for the polypeptide of relative molecular mass approximately 140,000 (alpha 2-subunit) associated with skeletal muscle DHP receptor is co-injected.     

Primary structure of the receptor for calcium channel blockers from skeletal muscle

The complete amino-acid sequence of the receptor for dihydropyridine calcium channel blockers from rabbit skeletal muscle is predicted by cloning and sequence analysis of DNA complementary to its messenger RNA. Structural and sequence similarities to the voltage-dependent sodium channel suggest that in the transverse tubule membrane of skeletal muscle the dihydropyridine receptor may act both as voltage sensor in excitation-contraction coupling and as a calcium channel.     

Three-dimensional architecture of a polytene nucleus

The three-dimensional chromosome topography in an intact nucleus has been determined using fluorescently stained Drosophila polytene chromosomes, optical fluorescence microscopy and newly developed, generally applicable, cellular image reconstruction techniques. The folding pattern is a complex mixture of parallel chromosomal segments and intertwined coils and shows extensive interaction of the chromosomes with the nuclear envelope.     

Decamethonium and hexamethonium block K+ channels of sarcoplasmic reticulum

The sarcoplasmic reticulum membrane (SR) of skeletal muscle contains cation-selective channels which have been detected by isotope fluxes in fragmented SR vesicles, fluorimetric dyes and direct incorporation of SR vesicles to planar phospholipid bilayers. SR channels incorporated in bilayers have a single open-state conductance of 140 pS in 0.1 MK+ (refs 4,5). We have previously reported blockade of the SR channel by Cs+, a low-affinity blocker with a zero-voltage dissociation constant of 40 mM (ref. 6). We showed that increasing Cs+ concentrations reduced the open-channel conductance, increased the mean open time and conferred voltage dependence on the open-state conductance. Here we report on the blockade induced by the cholinergic drugs decamethonium and hexamethonium on the SR channel. Although blockade by hexamethonium is similar to that of Cs+, decamethonium blocks with a much higher affinity and induces flickering events which are probably due to the interaction of single drug molecules with the open state.     

Occluded bound calcium on the phosphorylated sarcoplasmic transport ATPase

The Ca2+ + Mg2+-activated ATPase of the sarcoplasmic reticulum is responsible for the active Ca2+ transport of this membrane system, the key feature of which is the formation of an energy-rich phosphorylated transport enzyme (EP) and its conversion. To understand the Ca2+-transport mechanism, it is essential to clarify the behaviour of this intermediate in relation to such ligands as ATP, ADP, Mg2+ and, particularly, Ca2+. Recent kinetic studies on the phosphate turnover of this system suggested a relatively slow rate of Ca2+ dissociation from the phosphorylated enzyme, which possibly indicated Ca2+ binding in some occluded form with the intermediate. Here we report direct measurements of the binding and release of Ca2+ during phosphorylation of the sarcoplasmic transport enzyme. The results indicate an occlusion of the Ca2+ binding, accompanying an initial configurational change of the enzyme induced by the energy-rich phosphoryl transfer.     

Three-dimensional structure of the wild-type RHDV

The three-dimensional (3D) structure of the wild-type rabbit hemorrhagic disease virus (RHDV) has been determined to a resolution of 3.2 nm by electron cryomicroscopy and computer image reconstruction techniques. The 3D density map exhibits characteristic structural features of a calicivirus: a T=3 icosahedral capsid with 90 arch-like capsomeres at the icosahedral and local 2-fold axes and 32 large surface hollows at the icosahedral 5- and 3-fold axes. This result confirms that the RHDV isolated in China is a member of the Caliciviridae family. A rather continuous capsid shell was found without channels. However, our RHDV structure also reveals some distinct structural characteristics not observed in other caliciviruses, including interconnected capsomeres and the lack of protuberance on the base of each of the surface hollows. Two types of particles were identified with similar outer capsid structure but different density distributions inside the capsid shells, which could not be distinguished by conventional negative staining electron microscopy. As the genomic and subgenomic RNAs are both packaged into particles for RHDV, we suggest that the two types of particles identified correspond to those containing either the genomic or subgenomic RNAs, respectively.     

Three-dimensional structure of calmodulin

The three-dimensional structure of calmodulin has been determined crystallographically at 3.0 A resolution. The molecule consists of two globular lobes connected by a long exposed alpha-helix. Each lobe binds two calcium ions through helix-loop-helix domains similar to those of other calcium-binding proteins. The long helix between the lobes may be involved in interactions of calmodulin with drugs and various proteins.