Optical stimulation has enabled important advances in the study of brain function and other biological processes, and holds promise for medical applications ranging from hearing restoration to cardiac pace making. In particular, pulsed laser stimulation using infrared wavelengths >1.5 μm has therapeutic potential based on its ability to directly stimulate nerves and muscles without any genetic or chemical pre-treatment. However, the mechanism of infrared stimulation has been a mystery, hindering its path to the clinic. Here we show that infrared light excites cells through a novel, highly general electrostatic mechanism. Infrared pulses are absorbed by water, producing a rapid local increase in temperature. This heating reversibly alters the electrical capacitance of the plasma membrane, depolarizing the target cell. This mechanism is fully reversible and requires only the most basic properties of cell membranes. Our findings underscore the generality of pulsed infrared stimulation and its medical potential.

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Myosin VI is an unconventional motor protein with unusual motility properties such as its direction of motion and path on actin and a large stride relative to its short lever arms. To understand these features, the rotational dynamics of the lever arm were studied by single-molecule polarized total internal reflection fluorescence (polTIRF) microscopy during processive motility of myosin VI along actin. The axial angle is distributed in two peaks, consistent with the hand-over-hand model. The changes in lever arm angles during discrete steps suggest that it exhibits large and variable tilting in the plane of actin and to the sides. These motions imply that, in addition to the previously suggested flexible tail domain, there is a compliant region between the motor domain and lever arm that allows myosin VI to accommodate the helical position of binding sites while taking variable step sizes along the actin filament.

Abstract-Nitric oxide/cGMP pathway induces vasodilatation, yet the underlying mechanism is obscure. In the present study, we studied the mechanism of cGMP-induced relaxation of the smooth muscle contractile apparatus using permeabilized rabbit femoral arterial smooth muscle. 8-Br-cGMP-induced relaxation was accompanied with a decrease in myosin light chain (MLC) phosphorylation. MLC phosphatase (MLCP) activity, once decreased by agoniststimulation, recovered to the resting level on addition of 8-Br-cGMP. Because MLCP activity is regulated by the phosphorylation of a MLCP-specific inhibitor, CPI17 at Thr38 and MBS (myosin binding subunit of MLCP) at Thr696, we examined the effect of 8-Br-cGMP on the phosphorylation of these MLCP modulators. Whereas CPI17 phosphorylation was unchanged after addition of 8-Br-cGMP, MBS phosphorylation at Thr696 was significantly decreased by 8-Br-cGMP. We found that 8-Br-cGMP markedly increased MBS phosphorylation at Ser695 in the fiber pretreated with phenylephrine. MBS phosphorylation of Thr696 phosphorylated MBS at Ser695 partially resumed MLCP activity inhibited by Thr696 phosphorylation. Whereas Ser695 phosphorylation was markedly increased, the extent of diphosphorylated MBS at Ser695 and Thr696 in fibers was unchanged after cGMP-stimulation. We found that MBS phosphatase activity in arteries for both diphosphorylated MBS and monophosphorylated MBS at Thr696 significantly increased by 8-Br-cGMP, whereas MBS kinase activity was unchanged. These results suggest that the phosphorylation at Ser640 induced by cGMP shifted the equilibrium of the Thr641 phosphorylation toward dephosphorylation, thus increasing MLCP activity. This results in the decrease in MLC phosphorylation and smooth muscle relaxation. Key Words: cGMP Ⅲ myosin light chain phosphatase Ⅲ vasodilation Ⅲ phosphorylation Ⅲ smooth muscle I t has been known that endothelial-derived nitric oxide (NO) acts as a vasodilator, 1 and the pharmacological NO producing drugs have been used to prevent acute heart failure. NO has been defined as the activator of soluble guanylate cyclase, 2 thus increasing cGMP. A number of studies have indicated that cGMP induces relaxation of various smooth muscles contracted either by receptor-coupling agonists or depolarization 3 ; therefore, NO-induced vasodilation is thought to be attributable to the cGMP-induced relaxation of vascular smooth muscle. The key question is how cGMP triggers the vascular smooth muscle relaxation.Smooth muscle contraction is controlled by the phosphorylation of the regulatory light chain (RLC) of myosin at Ser19 4,5 by Ca 2ϩ /calmodulin-dependent protein kinase, called myosin light chain kinase (MLCK). 4 -6 On the other hand, MLC phosphatase (MLCP) activity is also regulated during the agonist-induced contraction of smooth muscle, thus contributing to the increase in RLC phosphorylation, but in contrast to the regulation of MLCK, the mechanism is Ca 2ϩ independent. 6 MLCP consists of 3 subunits, a myosin binding large subunit (MBS), 7,8 a 20-kDa small subunit (M2...

Mouse myosin V constructs were produced that consisted of the myosin motor domain plus either one IQ motif (M5IQ1), two IQ motifs (M5IQ2), a complete set of six IQ motifs (SHM5), or the complete IQ motifs plus the coiled-coil domain (thus permitting formation of a double-headed structure, DHM5) and expressed in Sf9 cells. The actin-activated ATPase activity of all constructs except M5IQ1 was inhibited above pCa 5, but this inhibition was completely reversed by addition of exogenous calmodulin. At the same Ca 2؉ concentration, 2 mol of calmodulin from SHM5 and DHM5 or 1 mol of calmodulin from M5IQ2 were dissociated, suggesting that the inhibition of the ATPase activity is due to dissociation of calmodulin from the heavy chain. However, the motility activity of DHM5 and M5IQ2 was completely inhibited at pCa 6, where no dissociation of calmodulin was detected. Inhibition of the motility activity was not reversed by the addition of exogenous calmodulin. These results indicate that inhibition of the motility is due to conformational changes of calmodulin upon the Ca 2؉ binding to the high affinity site but is not due to dissociation of calmodulin from the heavy chain.Myosins are motor proteins that translocate actin filaments upon hydrolysis of ATP, and thus they play a critical role in diverse forms of cell contractility and motility. During the last decade a number of myosin-like proteins have been found, and the myosins are currently organized into 15 classes based upon phylogenetic sequence comparisons of the motor domains (1-5). Class V myosin was originally identified in brain as a calmodulin-binding protein that had actin-dependent ATPase activity (6). Myosin V is a member of the myosin superfamily that is expressed in variety of cell types and is involved in a variety of membrane trafficking and organelle transport functions (1-5). Myosin V has two heads that are connected with a long coiledcoil domain; however, in contrast to conventional myosin, it contains a globular C-terminal domain and does not form thick filaments (7). The head domain is composed of a globular motor domain and an elongated neck domain that is associated with a number of light chains. The sequence at the neck region contains six IQ motifs that have been implicated as calmodulin or myosin light chain binding consensus motifs as found in a variety of calmodulin-binding proteins and myosins (7). Since light chains play a critical role in the regulation of various conventional myosins, it has been proposed that the IQ domain serves as a regulatory component of myosin V. The role of the IQ motif and bound calmodulin serving as a regulatory component of unconventional myosins is best studied for mammalian myosin Is. For both brush border myosin I (8 -10) and myosin I␤ (11-13), high Ca 2ϩ inhibits motor activity due to Ca 2ϩ binding to the calmodulin light chain. Since 1 mol of bound calmodulin dissociates from myosin I at high Ca 2ϩ , it was originally thought that this dissociation of calmodulin was responsible for the inhibition of myosin I...

Class-V myosin proceeds along actin filaments with large ( approximately 36 nm) steps. Myosin-V has two heads, each of which consists of a motor domain and a long (23 nm) neck domain. In accordance with the widely accepted lever-arm model, it was suggested that myosin-V steps to successive (36 nm) target zones along the actin helical repeat by tilting its long neck (lever-arm). To test this hypothesis, we measured the mechanical properties of single molecules of myosin-V truncation mutants with neck domains only one-sixth of the native length. Our results show that the processivity and step distance along actin are both similar to those of full-length myosin-V. Thus, the long neck domain is not essential for either the large steps or processivity of myosin-V. These results challenge the lever-arm model. We propose that the motor domain and/or the actomyosin interface enable myosin-V to produce large processive steps during translocation along actin.