Ryanodine receptors, voltage-gated calcium channels and their relationship with protein kinase A in the myocardium

We present a review about the relationship between ryanodine receptors and voltage-gated calcium channels in myocardium, and also how both of them are related to protein kinase A. Ryanodine receptors, which have three subtypes (RyR1-3), are located on the membrane of sarcoplasmic reticulum. Different subtypes of voltage-gated calcium channels interact with ryanodine receptors in skeletal and cardiac muscle tissue. The mechanism of excitation-contraction coupling is therefore different in the skeletal and cardiac muscle. However, in both tissues ryanodine receptors and voltage-gated calcium channels seem to be physically connected. FK-506 binding proteins (FKBPs) are bound to ryanodine receptors, thus allowing their concerted activity, called coupled gating. The activity of both ryanodine receptors and voltage-gated calcium channels is positively regulated by protein kinase A. These effects are, therefore, components of the mechanism of sympathetic stimulation of myocytes. The specificity of this enzyme's targeting is achieved by using different A kinase adapting proteins. Different diseases are related to inborn or acquired changes in ryanodine receptor activity in cardiac myocytes. Mutations in the cardiac ryanodine receptor gene can cause catecholamine-provoked ventricular tachycardia. Changes in phosphorylation state of ryanodine receptors can provide a credible explanation for the development of heart failure. The restoration of their normal level of phosphorylation could explain the positive effect of beta-blockers in the treatment of this disease. In conclusion, molecular interactions of ryanodine receptors and voltage-gated calcium channels with PKA have a significant physiological role. However, their defects and alterations can result in serious disturbances.     

Wall-associated kinases are expressed throughout plant development and are required for cell expansion

The mechanism by which events in the angiosperm cell wall are communicated to the cytoplasm is not well characterized. A family of five Arabidopsis wall-associated kinases (WAKs) have the potential to provide a physical and signaling continuum between the cell wall and the cytoplasm. The WAKs have an active cytoplasmic protein kinase domain, span the plasma membrane, and contain an N terminus that binds the cell wall. We show here that WAKs are expressed at organ junctions, in shoot and root apical meristems, in expanding leaves, and in response to wall disturbances. Leaves expressing an antisense WAK gene have reduced WAK protein levels and exhibit a loss of cell expansion. WAKs are covalently bound to pectin in the cell wall, providing evidence that the binding of a structural carbohydrate by a receptor-like kinase may have significance in the control of cell expansion.     

miR-17 family miRNAs are expressed during early mammalian development and regulate stem cell differentiation

MicroRNAs are small non-coding RNAs that regulate protein expression by binding 3′UTRs of target mRNAs, thereby inhibiting translation. Similar to siRNAs, miRNAs are cleaved by Dicer. Mouse and ES cell Dicer mutants demonstrate that microRNAs are necessary for embryonic development and cellular differentiation. However, technical obstacles and the relative infancy of this field have resulted in few data on the functional significance of individual microRNAs. We present evidence that miR-17 family members, miR-17-5p, miR-20a, miR-93, and miR-106a, are differentially expressed in developing mouse embryos and function to control differentiation of stem cells. Specifically, miR-93 localizes to differentiating primitive endoderm and trophectoderm of the blastocyst. We also observe high miR-93 and miR-17-5p expression within the mesoderm of gastrulating embryos. Using an ES cell model system, we demonstrate that modulation of these miRNAs delays or enhances differentiation into the germ layers. Additionally, we demonstrate that these miRNAs regulate STAT3 mRNA in vitro. We suggest that STAT3, a known ES cell regulator, is one target mRNA responsible for the effects of these miRNAs on cellular differentiation.     

Transient receptor potential, TRP channels: a new family of channels broadly expressed

Transient receptor potential, TRP channels are a new superfamily of functionally versatile non-selective cation channels present from yeast to mammals. On the basis of their structural homology, TRP channels are subdivided in 7 groups : TRPC 1-7 Canonical, TRPV 1-6 Vanilloid, TRPM 1-8 Melastatin, TRPP 1-3 Polycystin, TRPML Mucolipin, TRPA Ankyrin and TRPN (NO mechanotransducer potential C), the latter not expressed in mammals. Their cloning and heterologous expression allowed to demonstrating that these channels are generally weakly voltage-dependent. They are activated by various ligands involving a signal transduction cascade as well as directly by multiple compounds, heat and pH. TRP channels are found in a broad range of cell types. TRP channels are essential in allowing animals to sense the outside world and cells to sense their local environment. Following mutations or anomalous behaviour, these channels have a major role in several human diseases.     

DBHR, a gene with homology to dopamine beta-hydroxylase, is expressed in the neural crest throughout early development

In a screen for genes involved in neural crest development, we identified DBHR (DBH-Related), a putative monooxygenase with low homology to dopamine beta-hydroxylase (DBH). Here, we describe novel expression patterns for DBHR in the developing embryo and particularly the neural crest. DBHR is an early marker for prospective neural crest, with earliest expression at the neural plate border where neural crest is induced. Furthermore, DBHR expression persists in migrating neural crest and in many, though not all, crest derivatives. DBHR is also expressed in the myotome, from the earliest stages of its formation, and in distinct regions of the neural tube, including even-numbered rhombomeres of the hindbrain. In order to investigate the signals that regulate its segmented pattern in the hindbrain, we microsurgically rotated the rostrocaudal positions of rhombomeres 3/4. Despite their ectopic position, both rhombomeres continued to express DBHR at the level appropriate for their original location, indicating that DBHR is regulated autonomously within rhombomeres. We conclude that DBHR is a divergent member of a growing family of DBH-related genes; thus, DBHR represents a completely new type of neural crest marker, expressed throughout the development of the neural crest, with possible functions in cell-cell signaling.     

Role of CCN,a vertebrate specific gene family,in development

The CCN family of genes constitutes six members of small secreted cysteine rich proteins, which exists only in vertebrates. The major members of CCN are CCN1 (Cyr61), CCN2 (CTGF), and CCN3 (Nov). CCN4, CCN5, and CCN6 were formerly reported to be in the Wisp family, but they are now integrated into CCN due to the resemblance of their four principal modules: insulin like growth factor binding protein, von Willebrand factor type C, thrombospondin type 1, and carboxy‐terminal domain. CCNs show a wide and highly variable expression pattern in adult and in embryonic tissues, but most studies have focused on their principal role in osteo/chondrogenesis and vasculo/angiogenesis from the aspect of migration, growth, and differentiation of mesenchymal cells. CCN proteins simultaneously integrate and modulate the signals of integrins, bone morphogenetic protein, vascular endothelial growth factor, Wnt, and Notch by direct binding. However, the priority in the use of the signals is different depending on the cell status. Even the equivalent counterparts show a difference in signal usage among species. It may be that the evolution of the CCN family continues to keep pace with vertebrate evolution itself.     

Ryanodine receptors: structure, expression, molecular details, and function in calcium release

Ryanodine receptors (RyRs) are located in the sarcoplasmic/endoplasmic reticulum membrane and are responsible for the release of Ca(2+) from intracellular stores during excitation-contraction coupling in both cardiac and skeletal muscle. RyRs are the largest known ion channels (> 2MDa) and exist as three mammalian isoforms (RyR 1-3), all of which are homotetrameric proteins that interact with and are regulated by phosphorylation, redox modifications, and a variety of small proteins and ions. Most RyR channel modulators interact with the large cytoplasmic domain whereas the carboxy-terminal portion of the protein forms the ion-conducting pore. Mutations in RyR2 are associated with human disorders such as catecholaminergic polymorphic ventricular tachycardia whereas mutations in RyR1 underlie diseases such as central core disease and malignant hyperthermia. This chapter examines the current concepts of the structure, function and regulation of RyRs and assesses the current state of understanding of their roles in associated disorders.     

Orchestration of neuronal migration by activity of ion channels,neurotransmitter receptors,and intracellular Ca2+ fluctuations

The real-time observation of cell movement in acute cerebellar slices reveals that granule cells alter their shape concomitantly with changes in the mode and rate of migration as they traverse different cortical layers. Although the origin of local environmental cues responsible for these position-specific changes in migratory behavior remains unclear, several signaling mechanisms involved in controlling granule cell movement have emerged. The onset of one such mechanism is marked by the expression of voltage-gated ion channels and neurotransmitter receptors in postmitotic cells prior to the initiation of their migration. Granule cells start their radial migration after the expression of N-type Ca²⁺ channels and the N-methyl-D -aspartate subtype of glutamate receptors on the plasmalemmal surface. Blockade of the channel or receptor activity significantly decreases the rate of cell movement, indicating that the activation of these membrane constituents provides an essential signal for the translocation of granule cells. Another signal that controls the rate of cell migration is embedded in the combined amplitude and frequency components of Ca²⁺ fluctuations in the somata of migrating granule cells. Interestingly, each phase of Ca²⁺ fluctuation controls a separate phase of saltatory movement in the granule cells: The cells move forward during the phase of transient Ca²⁺ elevation and remain stationary during the troughs. Consequently, the changes in the amplitude and frequency components of Ca²⁺ fluctuations directly affect granule cell movement: Reducing the amplitude or frequency of Ca²⁺ fluctuations slows down the speed of cell movement, while the enhancement of these components accelerates migration. These findings suggest that signaling molecules present in the local cellular milieu encountered on the migratory route control the shape and motility of granule cells by modifying Ca²⁺ fluctuations in the soma through the activation of specific ion channels and neurotransmitter receptors. © 1998 John Wiley & Sons, Inc. J Neurobiol 37: 110–130, 1998     

A novel family of ancient vertebrate odorant receptors

Vertebrate odorant receptor (OR) genes have been isolated and characterized in several taxa, including bony fish and mammals. However, the search for more ancient vertebrate OR genes has been unsuccessful to date, indicating that these ancient genes share little sequence identity with previously isolated ORs. The lamprey (Lampetra fluviatilis) olfactory epithelium does not appear to express any of the modern vertebrate ORs previously identified in bony fish and mammals. We have isolated and characterized an ancient family of vertebrate membrane receptors from the olfactory epithelium of the lamprey. Sequence analysis reveals similarities with other Class A (rhodopsin-like) G protein-coupled receptors such as serotonin, dopamine, and histamine receptors, but the expression patterns of members of the new family, as well as certain conserved motifs, strongly suggest that the sequences encode ORs. Sequence similarity within the lamprey OR family is low, and Southern blot analysis suggests reduced-sized subfamilies. This novel vertebrate OR gene family, the most ancient isolated to date, is proposed to be involved in the detection of water-borne molecules in jawless fishes. Lamprey OR genes therefore represent a new level of diversity within the vertebrate OR gene family, but also provide clues as to how vertebrate ORs might have emerged. © 1998 John Wiley & Sons, Inc. J Neurobiol 37: 383–392, 1998     

Depolarization, intracellular calcium and exocytosis in single vertebrate nerve endings.

We have investigated the temporal relationship between depolarization, elevation of [Ca2+]i and exocytosis in single vertebrate neuroendocrine nerve terminals. The change of [Ca2+]i and vasopressin release were measured with a time resolution of less than 1 s in response to K(+)-induced depolarization. Exocytosis was also monitored in the whole-terminal patch-clamp configuration by time resolved capacitance measurements while [Ca2+]i was simultaneously followed by fura-2 fluorescence measurements. In intact as well as patch-clamped nerve terminals sustained depolarization leads to a sustained rise of [Ca2+]i. The rate of vasopressin release from intact nerve terminals rises in parallel with [Ca2+]i but then declines rapidly towards basal (t1/2 approximately 15 s) despite the maintained high [Ca2+]i indicating that only a limited number of exocytotic vesicles can be released. We demonstrate that in nerve terminals exocytosis can be followed during step depolarization by capacitance measurements. The capacitance increase starts instantaneously whereas [Ca2+]i rises with a half time of several hundred milliseconds. An instantaneous steep capacitance increase is followed by a slow increase with a slope of 25-50 fF/s indicating the sequential fusion of predocked and cytoplasmic vesicles. During depolarization the capacitance slope declines to zero with a similar time course as the vasopressin release indicating a decrease in exocytotic activity. Depolarization per se in the absence of a sufficient rise of [Ca2+]i does not induce exocytosis but elevation of [Ca2+]i in the absence of depolarization is as effective as in its presence. The experiments suggest that a rapid rise of [Ca2+]i in a narrow region beneath the plasma membrane induces a burst of exocytotic activity preceding the elevation of bulk [Ca2+]i in the whole nerve terminal.     

Dynamic regulation of intracellular calcium signals through calcium release channels

After the seminal work of Ebashi and coworkers which established the essential role of the intracellular Ca2+ concentration ([Ca2+]i) in the regulation of skeletal muscle contraction, we have witnessed an explosive elongation of the list of cell functions that are controlled by the [Ca2+]i. In numerous instances, release of intracellular Ca2+ stores plays important roles in Ca2+ signalling which displays significant variation in spatio-temporal pattern. There are two families of Ca2+ release channels, ryanodine receptors and inositol 1,4,5-trisphosphate (IP3) receptors. These Ca2+ release channels are structurally and functionally similar. In particular, the activity of both types of channels is regulated by the [Ca2+]i. The [Ca2+]i dependence of the Ca2+ release channel activity provides both types of channels with properties of a Ca2+ signal amplifier. This function of the ryanodine receptor is important in striated muscle excitation-contraction coupling, whereas that of the IP3 receptor seems to be the basis of the generation of Ca2+ waves. Thus the wide variety of Ca2+ signalling patterns seem to be critically dependent on the [Ca2+]i dependence of the Ca2+ release channels.     

Studies of ion channels using expressed protein ligation

Expressed protein ligation (EPL) is a semisynthetic technique for the chemoselective ligation of a synthetic peptide to a recombinant peptide that results in a native peptide bond at the ligation site. EPL therefore allows us to engineer proteins with chemically defined, site-specific modifications. While EPL has been used mainly in investigations of soluble proteins, in recent years it has been increasingly used in investigations of integral membrane proteins. These include studies on the KcsA K(+) channel, the non-selective cation channel NaK, and the porin OmpF. These studies are discussed in this review.