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The Calcium-Calmodulin System
Published in Enrique Pimentel, Handbook of Growth Factors, 2017
Calmodulin is a 17-kDa monomeric protein composed of a single peptide chain of 148 amino acids containing four Ca2+-binding domains. The three-dimensional structure of the calmodulin molecule has been determined by crystallography at 3.0 Å resolution.84 The molecule consists of two globular lobes connected by a long exposed α-helix. Each lobe binds two calcium ions through helix-loop-helix domains. The long helix between the lobes may be involved in interactions of calmodulin with drugs and various proteins. The molecule is stabilized by multiple interactions between the helices. It is possible that the regulation of different enzymes by calmodulin is partially a function of the number of Ca2+ bound, but controversy exists regarding the order in which the four sites are filled and the discrete structural changes associated with Ca2+ binding. The three-dimensional solution structure of the complex between calcium-bound calmodulin and a 26-residue synthetic peptide comprising the calmodulin-binding domain (residues 577 to 602) of skeletal muscle myosin light chain kinase has been determined by nuclear magnetic resonance spectroscopy.85 Calmodulin-binding peptides may share common structural features.
Molecular Biology of Calcium Pumps in Myometrium
Published in Robert E. Garfield, Thomas N. Tabb, Control of Uterine Contractility, 2019
Joanne O’Reilly, Ashok Kumar Grover
The products of the PMCA transcripts have all been found to contain a calmodulin binding domain as well as binding sites for ATP (FITC), and Ca2+ (putative). The proteins also have the ability to form high-energy acylphosphates. The calmodulin binding domain is characterized by a predominance of basic amino acids with hydrophobic residues and a high probability of forming an amphipathic helix upon binding to calmodulin.17,20,43,65,70,71 The protein sequence in this domain is not highly conserved between calmodulin-regulated pumps. There is, however, a highly conserved tryptophan residue in the N terminal half of the domain that seems to be involved in calmodulin binding. Using point mutation studies and proteolysis, this region has been studied extensively. Verma et al. demonstrated that residues 1100 to 1127 in PMCAlb and its 15 to 20 N-terminal amino acids inhibit an ATPase fragment that had been activated by proteolytic removal of its own endogenous calmodulin binding domain.71 The primary structure of this domain has been reported by James et al., who digested the purified PM Ca2+ pump protein with chymotrypsin to release a 12-kDa fragment that bound to calmodulin.43 To isolate the domain, calmodulin linked to a bifunctional, photoactivatable, radioactive, cleavable cross linker (Denny Jaffe reagent) was reacted with whole pump, illuminated to allow cross reaction of the reagent to the pump, and then calmodulin was cleaved. A chymotrypsin-digested, HPLC purified 12.4-kD product was solubilized with CNBr and the fragment was sequenced to give E-L-R-R-G-Q-I-L-W-F-R-G-L-N-R-I-Q-T-Q-I-K-V-V-N-A-F-S-S-S-C-H-E-F. This sequence shows a predominance of basic amino acids common to other putative binding domains, as well as a tryptophan residue in the NH2 terminal portion. Secondary structure predicts helix formation and beta-pleated sheets in analogy with the structure of other calmodulin-regulated proteins. The location of the domain in the C terminus has been confirmed by sequencing of the PM Ca2+ pump from rat brain PMCA1 cDNA clones from a number of tissues.
Investigating potential TRPV1 positive feedback to explain TRPV1 upregulation in airway disease states
Published in Drug Development and Industrial Pharmacy, 2021
Jesse Xu, Maliheh Ghadiri, Maree Svolos, Brent McParland, Daniela Traini, Hui Xin Ong, Paul M. Young
One of the hypothesized mechanisms of TRPV1 upregulation in airway disease states is that airway stress events such as viral infections or lung cancer can acidify the airways and lead to the endogenous build-up of bradykinin to cause activation of TRPV1 receptors. This action then leads to further cellular damage [20]. Bradykinin is produced locally in the airways by cleavage of active kinins [21], and the binding of bradykinin onto bradykinin-2 (B2R) receptors will trigger the release of inflammatory mediators including interleukin-1 (22), interleukin-6 (IL6) and interleukin-8 (IL8) [23]. In addition, in sensory neurons, bradykinin is known to catalyze the phosphorylation of TRPV1 channels via activation of Protein Kinase C (PKC), and PKC activity leads to modification of the TRPV1 channel by associating with the calcium-calmodulin binding domain [24]; a conformational change in TRPV1 channels ultimately leads to an increase in responsiveness to TRPV1 agonists. This is particularly notable in over-reactivity amongst patients with a chronic cough to the capsaicin challenge [22].
Myosin light chain kinase regulates intestinal permeability of mucosal homeostasis in Crohn’s disease
Published in Expert Review of Clinical Immunology, 2020
The PKC regulates the MLCK activation in two ways (Figure 4(b)) [124]. First, researchers have noticed that at the same time as PKC activation, MLCK phosphorylation increases acutely and precedes the decrease in MLC2 phosphorylation. This phenomenon indicated that PKC activation triggers MLCK phosphorylation. The interactions between the calmodulin-binding inhibitory and catalytic domains inhibit MLCK activity. However, Ca2+ and calmodulin can block this intramolecular interaction and release MLCK inhibition. However, if PKC and PKA target sites present in the calmodulin-binding domain of MLCK are phosphorylated, calmodulin-dependent enzyme activation will interfere with the MLCK and inhibit MLCK activation. Therefore, PKC phosphorylates MLCK, and inactivated MLCK leads to decreased MLC2 phosphorylation. Second, PKC phosphorylation of myristoylated alanine-rich C kinase substrates (MARCKs) releases calmodulin, which is bound to MARCKs. Then, calmodulin is available for MLCK activation. Therefore, PKC also regulates the MLCK activity by modulating the availability of calmodulin related to the MARCKs phosphorylation. Furthermore, the PKC pathway could possibly regulate MLCK activation by influencing NF-κB signaling.
The light-activated TRP channel: the founding member of the TRP channel superfamily
Published in Journal of Neurogenetics, 2022
Application of the patch clamp technique to Drosophila photoreceptors (Hardie, 1991; Ranganathan, Harris, Stevens, & Zuker, 1991) led to the evidence that unlike the Ca2+ impermeable light activated channels of Limulus photoreceptors ((Brown & Blinks, 1974), the dominant model of invertebrate phototransduction at that time), the light activated channels of Drosophila are highly Ca2+ permeable. Subsequent whole-cell patch clamp recording showed that the trp gene product generates the large Ca2+ permeability of light-activated channels of Drosophila (Hardie & Minke, 1992). Ion selective electrode and micro-fluorimetric measurements led to the direct demonstration of TRP-dependent Ca2+ influx. Importantly, the study of Hardie and Minke demonstrated that the primary defect in trp mutants or in La3+-treated Drosophila eye (Figure 2(B)) is a drastic reduction in Ca2+ permeability of the light-sensitive channels themselves (Hardie & Minke, 1992). These authors concluded that the light response of a wild-type photoreceptor consists of two distinct conductances, one is highly Ca2+ permeable and is encoded by the trp gene and the other encoded by another gene that is responsible for the residual transient current in trp mutants. The nature of the second conductance was clarified by the work of Kelly and colleagues (Phillips, Bull, & Kelly, 1992). While searching for calmodulin-binding Drosophila proteins, they discovered a novel calmodulin binding membrane protein, with homology to TRP (∼40% overall identity and 74% identity within the transmembrane segments), which they called TRP-like (TRPL, (Phillips et al., 1992)). They showed that it is slightly smaller than TRP (900 vs. 1275 amino acids), has two calmodulin binding domain in the C-terminal region, and 4 ankyrin repeats in the N-terminal region (see Figure 3(C)). Their analysis further revealed a homo tetrameric assembly with transmembrane topology reminiscent of voltage-gated channels: six transmembrane segments S1-S6 and a putative pore region between S5 and S6 (Figure 3(C)), except that the charged residues in S4 of voltage-gated channels are replace by non-polar ones. Moreover, within S5 and S6, they found several short stretches of amino acid identity with the mammalian voltage-gated Ca2+ channels. They suggested that both trp and trpl genes encode light-activated channels.