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Variability in spontaneous subcellular calcium release in guinea-pig ileum smooth muscle cells. J Physiol507: 707–720 [PMC free article][PubMed] [Google Scholar]
Smooth muscle contraction is driven by Ca2+-calmodulin activation of myosin light chain kinase, which has a Ca2+half-activation of ∼400 nm(Stull et al. 1998). The gain of smooth muscle contraction to Ca2+can be adjusted through regulation of myosin light chain phosphatase (Somlyo and Somlyo 2003; Mizuno et al. 2008). Global Ca2+
S1P activates Store-operated calcium entry via receptor and non receptor-mediated pathways in vascular smooth muscle cells. Am J Physiol Cell Physiol300: 919–926 [PMC free article][PubMed] [Google Scholar]
In arterial smooth muscle, this membrane potential is sufficient to increase the steady-state open probability of VDCCs, elevate global intracellular Ca2+from ∼100 nmto ∼200 nm, and cause a tonic constriction (Knot and Nelson 1998).
Calmodulin, the ubiquitous and multifunctional Ca(2+)-binding protein, mediates many of the regulatory effects of Ca2+, including the contractile state of smooth muscle.
Regulatory calcium-binding proteins such as calmodulin (CaM) and troponin C (TnC) expose a hydrophobic surface upon binding calcium. This property allows them to bind in a calcium dependent manner to their target proteins (1), but also to hydrophobic sites on phenyl-Sepharose, for example (2, 3, 4).
Troponin plays a central role in the calcium-regulation of muscle contraction: Troponin is the sole calcium-binding component of thin filaments (actin-tropomyosin-troponin complex) of striated muscles.
Calcium-binding proteins (CBPs) not only control cytoplasmic Ca2+ concentration—by means of a multiple calcium pumps, channels, sequestering agents, and buffers—but also function as Ca2+ transporters and calcium-sensors with regulatory potential, including transcription factors and enzymes, which elicit the appropriate ...
The addition of components known to strongly bind calcium ions, such as EDTA or alginate, was found to substantially decrease the lipid digestion rate. These results have important implications for understanding and controlling the digestion of lipids in the human diet.
Calmodulin acts as an intermediary protein that senses calcium levels and relays signals to various calcium-sensitive enzymes, ion channels and other proteins. Calmodulin is a small dumbbell-shaped protein composed of two globular domains connected together by a flexible linker.
Troponin is a calcium-regulatory protein for the calcium regulation of contractile function in skeletal and cardiac muscles. Troponin is distributed regularly along the entire length of thin filaments and forms an ordered complex with tropomyosin and actin.
Calcium's positive molecule is important to the transmission of nerve impulses to the muscle fiber via its neurotransmitter triggering release at the junction between the nerves (2,6). Inside the muscle, calcium facilitates the interaction between actin and myosin during contractions (2,6).
cytoplasmCalmodulin is expressed in many cell types and can have different subcellular locations, including the cytoplasm, within organelles, or associated with the plasma or organelle membranes, but it is always found intracellularly.
The calcium-binding site in α-lactalbumin consists of a short four-residue N-terminal side helix, a four-residue loop, and a longer (at least 12-residue) C-terminal side helix (PDB ID 1hml).
Ca2+ binding to troponin initiates thin filament activation exposing myosin binding sites on actin. However, unlike in skeletal muscle, thin filament activation in cardiac muscle is not achieved primarily by Ca2+ binding alone (16, 17, 37).
Intracellular storage and release of Ca2+ from the sarcoplasmic reticulum is associated with the high-capacity, low-affinity calcium-binding protein calsequestrin. Calretinin is another type of Calcium binding protein weighing 29kD. It is involved in cell signaling and shown to exist in neurons.
The nature of the Ca2+signal depends to a large extent on the molecular mechanism responsible for generating it. Broadly speaking, there are two major mechanisms by which [Ca2+]iis raised in smooth muscle: (1) entry of Ca2+from the extracellular space, and (2) release of Ca2+from intracellular stores. Influx of extracellular Ca2+is mediated by ion channels in the plasmalemmal membrane, the most prominent of which is the voltage-dependent Ca2+channel (VDCC). Nonselective cation channels, such as transient receptor potential (TRP) channels and ionotropic purinergic (P2X) receptors, are also potentially important extracellular Ca2+entry pathways in smooth muscle cells. Although a number of intracellular organelles take up and release Ca2+, the sarcoplasmic reticulum (SR) represents the largest pool of releasable Ca2+in smooth muscle cells. In response to a variety of stimuli, Ca2+-release channels in the SR, namely ryanodine receptors (RyR) and inositol trisphosphate receptors (IP3Rs), mediate efflux of Ca2+from the SR into the cytoplasm of the cell.
Changes in intracellular Ca2+are central to the function of smooth muscle, which lines the walls of all hollow organs. These changes take a variety of forms, from sustained, cell-wide increases to temporally varying, localized changes. The nature of the Ca2+signal is a reflection of the source of Ca2+(extracellular or intracellular) and the molecular entity responsible for generating it. Depending on the specific channel involved and the detection technology employed, extracellular Ca2+entry may be detected optically as graded elevations in intracellular Ca2+, junctional Ca2+transients, Ca2+flashes, or Ca2+sparklets, whereas release of Ca2+from intracellular stores may manifest as Ca2+sparks, Ca2+puffs, or Ca2+waves. These diverse Ca2+signals collectively regulate a variety of functions. Some functions, such as contractility, are unique to smooth muscle; others are common to other excitable cells (e.g., modulation of membrane potential) and nonexcitable cells (e.g., regulation of gene expression).
The pore-forming α1 subunit is expressed as multiple splice variants with different regulatory and biophysical properties. Additional molecular diversity is provided by four different, variably spliced β subunits (Birnbaumer et al. 1998), which further modify VDCC biophysical properties and regulate surface expression of the α1 subunit; properties of the VDCC complex may be additionally modulated by splice variants of the α2δ regulatory subunit (Angelotti and Hofmann 1996).
To record very rapid events or determine the kinetics of a Ca2+event by confocal microscopy, researchers have typically measured Ca2+fluxes using a line-scanning procedure. In line-scan mode, a single line is repeatedly scanned across the cell for a period of time. Each line is then aligned to form an image that is a plot of fluorescence along the scanned line versus time (Fig. 1). Although line scans are still used, the development of very sensitive CCD (charge-coupled device) cameras and rapid Ca2+-sensitive dyes allows laser-scanning confocal systems to routinely achieve detailed spatial and temporal resolution, which is crucial for determining the origin of a Ca2+event.
Ca2+signals are typically imaged using laser-scanning confocal microsopes. In its simplest form, a confocal microscope system comprises three main components: (1) a light source; (2) optical and electronic components to manipulate, display, and analyze signals; and (3) a microscope. Lasers, coupled to either upright or inverted microscopes, are commonly used as the light source for confocal microscopes. Although gas lasers (e.g., Ar-ion, Kr-ion, HeNe) provide numerous lasing lines from UV to red, and are well suited for optimal excitation of fluorescent probes, solid-state lasers are increasingly being used because they offer the advantages of longer lifetime, lower power consumption, and compact size.
Ca2+SIGNALS FROM INNER SPACE: RELEASE OF Ca2+FROM INTRACELLULAR STORES
Smooth muscle cells express a number of nonselective cation channels of the TRP family. Although no signature signaling event associated with Ca2+influx through TRP channels has been reported in the literature, given the relative selectivity of these channels for Ca2+(and the high Ca2+permeability and single-channel conductance of some TRP family members, notably TRPV), imaging methods that have been used to examine jCaT-like events (confocal microscopy) and/or VDCC-mediated sparklets (TIRF) may ultimately provide a means to optically detect Ca2+influx through these channels.