No abstract

The class I myosin Myo1c is a mediator of adaptation of mechanoelectrical transduction in the stereocilia of the inner ear. Adaptation, which is strongly affected by Ca 2؉ , permits hair cells under prolonged stimuli to remain sensitive to new stimuli. Using a Myo1c fragment (motor domain and one IQ domain with associated calmodulin), with biochemical and kinetic properties similar to those of the native molecule, we have performed a thorough analysis of the biochemical cross-bridge cycle. We show that, although the steady-state ATPase activity shows little calcium sensitivity, individual molecular events of the cross-bridge cycle are calcium-sensitive. Of significance is a 7-fold inhibition of the ATP hydrolysis step and a 10-fold acceleration of ADP release in calcium. These changes result in an acceleration of detachment of the cross-bridge and a lengthening of the lifetime of the detached M-ATP state. These data support a model in which slipping adaptation, which reduces tip-link tension and allows the transduction channels to close after an excitatory stimulus, is mediated by Myo1c and modulated by the calcium transient. molecular motor ͉ myosin M yosins are actin-based molecular motors that support a variety of cellular functions including muscle contraction and cell migration. There are at least two dozen classes in the myosin superfamily based on analysis of sequences in the catalytic domain (1). Myo1c is a class I myosin that is widely expressed in vertebrate tissues (2, 3). It consists of a motor domain, a neck or lever arm domain (three or four IQ repeats each of which binds a calmodulin), and a cargo-binding domain (4-6). In adipocytes Myo1c facilitates glucose transporter recycling in response to insulin (7,8), and in amphibian oocytes Myo1c mediates exocytosis (9). In the specialized cells of the inner ear, there is considerable evidence that Myo1c acts as a mediator of adaptation of mechanoelectrical transduction in stereocilia (10, 11).Neighboring stereocilia on the hair cells of the inner ear are connected by extracellular tip links that are attached to transduction channels, which in turn are attached to an adaptation-motor complex consisting of Myo1c molecules. The prevailing model for mechanotransduction is that deflection of the stereocilia by sound or motion affects tip-link tension and causes opening or closing of transduction channels through which potassium and calcium ions pass (5, 12, 13). Myo1c sets the transducer sensitivity by moving along the core bundle of actin filaments in the stereocilia until the resting tension in the tip link is at a point where the channels are poised just below the threshold tension required to open the channels (Fig. 1). During an excitatory stimulus, movement of the stereocilia increases tip-link tension, opening the channels. Fig. 1 illustrates how in adaptation the motor/transducer complex responds to increased tension by slipping down the actin cytoskeleton thereby reducing tip-link tension. Alternatively, reduced tension causes Myo1c to ascend the ac...

We have previously demonstrated that substitution of ATP with 2 deoxy-ATP (dATP) increased the magnitude and rate of force production at all levels of Ca2+-mediated activation in demembranated cardiac muscle. In the current study we hypothesized that cellular [dATP] could be increased by viral-mediated over expression of the ribonucleotide reductase (Rrm1 and Rrm2) complex, which would increase contractility of adult rat cardiomyocytes. Cell length and ratiometric (fura2) Ca2+ fluorescence were monitored by video microscopy. At 0.5 Hz stimulation, the extent of shortening was increased ~40% and maximal rate of shortening was increased ~80% in cardiomyocytes overexpressing Rrm1+Rrm2 as compared to non-transduced cardiomyocytes. The maximal rate of relaxation was also increased ~150% with Rrm1+Rrm2 over expression, resulting in decreased time to 50% relaxation over non-transduced cardiomyocytes. These differences were even more dramatic when compared to cardiomyocytes expressing GFP-only. Interestingly, Rrm1+Rrm2 over expression had no effect on minimal or maximal intracellular [Ca2+] (Fura2 fluorescence), indicating increased contractility is primarily due to increased myofilament activity without altering Ca2+ release from the sarcoplasmic reticulum. Additionally, functional potentiation was maintained with Rrm1+Rrm2 over expression as stimulation frequency was increased (1 Hz and 2 Hz). HPLC analysis indicated cellular [dATP] was increased by approximately 10-fold following transduction, becoming ~1.5% of the adenine nucleotide pool. Furthermore, 2% dATP was sufficient to significantly increase crossbridge binding and contractile force during sub-maximal Ca2+ activation in demembranated cardiac muscle. These experiments demonstrate the feasibility of directly targeting the actin-myosin chemomechanical crossbridge cycle to enhance cardiac contractility and relaxation without affecting minimal or maximal Ca2+.

Myosin IXb (Myo9b) was reported to be a single-headed, processive myosin. In its head domain it contains an N-terminal extension and a large loop 2 insertion that are specific for class IX myosins. We characterized the kinetic properties of purified, recombinant rat Myo9b, and we compared them with those of Myo9b mutants that had either the N-terminal extension or the loop 2 insertion deleted. Unlike other processive myosins, Myo9b exhibited a low affinity for ADP, and ADP release was not rate-limiting in the ATPase cycle. Myo9b is the first myosin for which ATP hydrolysis or an isomerization step after ATP binding is rate-limiting. Myo9b-ATP appeared to be in a conformation with a weak affinity for actin as determined by pyrene-actin fluorescence. However, in actin cosedimentation experiments, a subpopulation of Myo9b-ATP bound F-actin with a remarkably high affinity. Deletion of the N-terminal extension reduced actin affinity and increased the rate of nucleotide binding. Deletion of the loop 2 insertion reduced the actin affinity and altered the communication between actin and nucleotide-binding sites.The myosins represent a large superfamily of motor molecules that convert the chemical energy liberated by ATP hydrolysis into mechanical force along actin filaments. They have been subdivided into 18 different classes based on homologous myosin head domain sequences (1). In addition to a characteristic head domain, all myosins contain a light chain binding domain and a tail domain. The tail domains of some myosins dimerize giving rise to two-headed myosins. Motor properties like direction of movement, speed, step size, duty ratio, processivity, and regulation can vary greatly between various myosins. Movement on actin involves the repeated hydrolysis of ATP by myosins, leading to an ordered cycling between different nucleotide binding states that exhibit different actin binding affinities. Cycling rates and relative time spent in these nucleotide states vary in different myosins, such that the fraction of time a given myosin remains strongly attached to the actin filament during its ATPase cycle differs substantially. Generally, myosin-ATP and myosin-ADP-P i bind weakly to actin filaments, whereas myosin-ADP and myosin alone bind strongly (2). In many myosins, e.g. skeletal muscle myosin II, the release of P i is the rate-limiting step, which means that they spend a large fraction of the total cycling time in a weak actinbinding state. On the other hand, in class V and class VI myosins, the cycle is modified in a way that ADP release is slow and rate-limiting, so these myosins spend most of their cycling time strongly attached to actin (3, 4). Myosin V is two-headed and the two heads cooperate to allow for continuous, processive, hand over hand movement along actin filaments (5).The class IX myosin, myosin 9b (Myo9b), 5 has been reported to exhibit unique motor properties. Despite being a single-headed myosin, it demonstrated typical characteristics of a processive myosin in in vitro motility assays, meaning tha...

Fast and slow mammalian muscle myosins differ in the heavy chain sequences (MHC-2, MHC-1) and muscles expressing the two isoforms contract at markedly different velocities. One role of slow skeletal muscles is to maintain posture with low ATP turnover, and MHC-1 expressed in these muscles is identical to heavy chain of the ␤-myosin of cardiac muscle. Few studies have addressed the biochemical kinetic properties of the slow MHC-1 isoform. We report here a detailed analysis of the MHC-1 isoform of the rabbit compared with MHC-2 and focus on the mechanism of ADP release. We show that MHC-1, like some non-muscle myosins, shows a biphasic dissociation of actin-myosin by ATP. Most of the actinmyosin dissociates at up to ϳ1000 s ؊1, a very similar rate constant to MHC-2, but 10 -15% of the complex must go through a slow isomerization (ϳ20 s ؊1 ) before ATP can dissociate it. Similar slow isomerizations were seen in the displacement of ADP from actinmyosin⅐ADP and provide evidence of three closely related actinmyosin⅐ADP complexes, a complex in rapid equilibrium with free ADP, a complex from which ADP is released at the rate required to define the maximum shortening velocity of slow muscle fibers (ϳ20 s ؊1 ), and a third complex that releases ADP too slowly (ϳ6 s ؊1 ) to be on the main ATPase pathway. The role of these actin-myosin⅐ADP complexes in the mechanochemistry of slow muscle contraction is discussed in relation to the load dependence of ADP release.Myosins comprise a family of ATP-dependent motor proteins and are best known for their role in muscle contraction and their involvement in a wide range of other eukaryotic motility processes (1-6). During the myosin ATPase cycle, the myosin motor domain (or cross-bridge) undergoes a series of conformational changes coupled to the binding of nucleotide and actin, which results in a translocation of the myosin cargo domain relative to the actin track (7). The sequence of molecular events in the actin-myosin cross-bridge cycle appears essentially the same for all myosins so far studied. The different mechanochemical properties of each myosin are attributed to a modulation of the rates and equilibrium constants of individual molecular events to match each myosin to its physiological role.The most widely studied myosins are the vertebrate, striatedmuscle myosins, which define the class II myosins. This class of myosins have a dimerization domain that forms a long coiled-coil and which can assemble further to form the backbone of the bipolar thick myosin filament. Of the class II myosin, the myosin heavy chain 2 (MHC-2) 4 isoform found primarily in white, anaerobic, fast-contracting, skeletal muscle has been most thoroughly described at both the molecular and physiological levels. In adult, mammalian, muscle tissue the MHC-2 is found as various isoforms (e.g. 2a, 2b, 2x) and it has been established that the essential mechanical properties of the muscle fiber contraction (e.g. maximum velocity of shortening, force per cross-bridge) are properties of the MHC present in the t...