The cold-and menthol-sensitive transient receptor potential melastatin 8 (TRPM8) channel is important for both physiological temperature detection and cold allodynia. Activation of G-protein-coupled receptors (GPCRs) by proinflammatory mediators inhibits these channels. It was proposed that this inhibition proceeds via direct binding of G ␣q to the channel. TRPM8 requires the plasma membrane phospholipid phosphatidylinositol 4,5-bisphosphate [PI(4,5)P 2 or PIP 2 ] for activity. However, it was claimed that a decrease in cellular levels of this lipid upon receptor activation does not contribute to channel inhibition. Here, we show that supplementing the whole-cell patch pipette with PI(4,5)P 2 reduced inhibition of TRPM8 by activation of G ␣q -coupled receptors in mouse dorsal root ganglion (DRG) neurons isolated from both sexes. Stimulating the same receptors activated phospholipase C (PLC) and decreased plasma membrane PI(4,5)P 2 levels in these neurons. PI(4,5)P 2 also reduced inhibition of TRPM8 by activation of heterologously expressed muscarinic M1 receptors. Coexpression of a constitutively active G ␣q protein that does not couple to PLC inhibited TRPM8 activity, and in cells expressing this protein, decreasing PI(4,5)P 2 levels using a voltage-sensitive 5Ј-phosphatase induced a stronger inhibition of TRPM8 activity than in control cells. Our data indicate that, upon GPCR activation, G ␣q binding reduces the apparent affinity of TRPM8 for PI(4,5)P 2 and thus sensitizes the channel to inhibition induced by decreasing PI(4,5)P 2 levels.Increased sensitivity to heat in inflammation is partially mediated by inhibition of the cold-and menthol-sensitive transient receptor potential melastatin 8 (TRPM8) ion channels. Most inflammatory mediators act via G-protein-coupled receptors that activate the phospholipase C pathway, leading to the hydrolysis of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P 2 ]. How receptor activation by inflammatory mediators leads to TRPM8 inhibition is not well understood. Here, we propose that direct binding of G ␣q both reduces TRPM8 activityandsensitizesthechanneltoinhibitionbydecreasedlevelsofitscofactor,PI(4,5)P 2 .Ourdatademonstratetheconvergenceoftwo downstream effectors of receptor activation, G ␣q and PI(4,5)P 2 hydrolysis, in the regulation of TRPM8.

Force production in muscle results from ATP-driven cyclic interactions of myosin with actin. A myosin cross bridge consists of a globular head domain, containing actin and ATP-binding sites, and a neck domain with the associated light chain 1 (LC1) and the regulatory light chain (RLC). The actin polymer serves as a "rail" over which myosin translates. Phosphorylation of the RLC is thought to play a significant role in the regulation of muscle relaxation by increasing the degree of skeletal cross-bridge disorder and increasing muscle ATPase activity. The effect of phosphorylation on skeletal cross-bridge kinetics and the distribution of orientations during steady-state contraction of rabbit muscle is investigated here. Because the kinetics and orientation of an assembly of cross bridges (XBs) can only be studied when an individual XB makes a significant contribution to the overall signal, the number of observed XBs was minimized to ∼20 by limiting the detection volume and concentration of fluorescent XBs. The autofluorescence and photobleaching from an ex vivo sample was reduced by choosing a dye that was excited in the red and observed in the far red. The interference from scattering was eliminated by gating the signal. These techniques decrease large uncertainties associated with determination of the effect of phosphorylation on a few molecules ex vivo with millisecond time resolution. In spite of the remaining uncertainties, we conclude that the state of phosphorylation of RLC had no effect on the rate of dissociation of cross bridges from thin filaments, on the rate of myosin head binding to thin filaments, and on the rate of power stroke. On the other hand, phosphorylation slightly increased the degree of disorder of active cross bridges.

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J. Effect of a myosin regulatory light chain mutation K104E on actin-myosin interactions. Am J Physiol Heart Circ Physiol 308: H1248 -H1257, 2015. First published March 13, 2015; doi:10.1152/ajpheart.00834.2014 is the most common cause of sudden cardiac death in young individuals. Molecular mechanisms underlying this disorder are largely unknown; this study aims at revealing how disruptions in actin-myosin interactions can play a role in this disorder. Cross-bridge (XB) kinetics and the degree of order were examined in contracting myofibrils from the ex vivo left ventricles of transgenic (Tg) mice expressing FHC regulatory light chain (RLC) mutation K104E. Because the degree of order and the kinetics are best studied when an individual XB makes a significant contribution to the overall signal, the number of observed XBs in an ex vivo ventricle was minimized to ϳ20. Autofluorescence and photobleaching were minimized by labeling the myosin lever arm with a relatively long-lived red-emitting dye containing a chromophore system encapsulated in a cyclic macromolecule. Mutated XBs were significantly better ordered during steady-state contraction and during rigor, but the mutation had no effect on the degree of order in relaxed myofibrils. The K104E mutation increased the rate of XB binding to thin filaments and the rate of execution of the power stroke. The stopped-flow experiments revealed a significantly faster observed dissociation rate in Tg-K104E vs. Tg-wild-type (WT) myosin and a smaller second-order ATP-binding rate for the K104E compared with WT myosin. Collectively, our data indicate that the mutation-induced changes in the interaction of myosin with actin during the contractionrelaxation cycle may contribute to altered contractility and the development of FHC. left ventricle; mutation of regulatory light chain; polarization of fluorescence INTERACTIONS OF MYOSIN WITH actin and associated proteins (sarcomeric proteins) are responsible for muscle contraction (19,24,48). These interactions include the mode of attachment of actin to myosin during different physiological states and the kinetics of conformational changes of myosin when interacting with actin. Familial hypertrophic cardiomyopathy (FHC) has been recognized to be caused by mutations in all major sarcomeric proteins of the heart, including the myosin regulatory light chain (RLC) (1). Compared with the myosin heavy chain, mutations in the RLC are rare, but often associated with malignant outcomes (18,27,41). It is therefore important to find out whether these FHC-linked mutations result in alterations of the mode of attachment of myosin to actin, and/or whether they lead to alterations of kinetics of acto-myosin interactions. These changes may ultimately contribute to the development of FHC. In this report we have focused on the K104E (lysine to glutamic acid) substitution in the myosin RLC expressed in the heart of transgenic (Tg) mice (25). The mutation was identified in a Danish population by Andersen et al. (2) and was associated with left ventricu...

The systemic circulation offers larger resistance to the blood flow than the pulmonary system. Consequently, the left ventricle (LV) must pump blood with more force than the right ventricle (RV). The question arises whether the stronger pumping action of the LV is due to a more efficient action of left ventricular myosin, or whether it is due to the morphological differences between ventricles. Such a question cannot be answered by studying the entire ventricles or myocytes because any observed differences would be wiped out by averaging the information obtained from trillions of myosin molecules present in a ventricle or myocyte. We therefore searched for the differences between single myosin molecules of the LV and RV of failing hearts In-situ. We show that the parameters that define the mechanical characteristics of working myosin (kinetic rates and the distribution of spatial orientation of myosin lever arm) were the same in both ventricles. These results suggest that there is no difference in the way myosin interacts with thin filaments in myocytes of failing hearts, and suggests that the difference in pumping efficiencies are caused by interactions between muscle proteins other than myosin or that they are purely morphological.