Modification of Loop 1 Affects the Nucleotide Binding Properties of Myo1c, the Adaptation Motor in the Inner Ear

Myo1c is one of eight members of the mammalian myosin I family of actin-associated molecular motors. In stereocilia of the hair cells in the inner ear, Myo1c presumably serves as the adaptation motor, which regulates the opening and closing of transduction channels. Although there is conservation of sequence and structure among all myosins in the N-terminal motor domain, which contains the nucleotide- and actin-binding sites, some differences include the length and composition of surface loops, including loop 1, which lies near the nucleotide-binding domain. To investigate the role of loop 1, we expressed in insect cells mutants of a truncated form of Myo1c, Myo1c1IQ, as well as chimeras of Myo1c1IQ with the analogous loop from other myosins. We found that replacement of the charged residues in loop 1 with alanines or the whole loop with a series of alanines did not alter the ATPase activity, transient kinetics properties, or Ca2+ sensitivity of Myo1c1IQ. Substitution of loop 1 with that of the corresponding region from tonic smooth muscle myosin II (Myo1c1IQ-tonic) or replacement with a single glycine (Myo1c1IQ-G) accelerated the release of ADP from A.M 2?3-fold in Ca2+, whereas substitution with loop 1 from phasic muscle myosin II (Myo1c1IQ-phasic) accelerated the release of ADP 35-fold. Motility assays with chimeras containing a single ?-helix, or SAH, domain showed that Myo1cSAH-tonic translocated actin in vitro twice as fast as Myo1cSAH-WT and 3-fold faster than Myo1cSAH-G. The studies show that changes induced in Myo1c via modification of loop 1 showed no resemblance to the behavior of the loop donor myosins or to the changes previously observed with similar Myo1b chimeras

Modulators of actin-myosin dissociation: basis for muscle type functional differences during fatigue

The muscle types present with variable fatigue tolerance, in part due to the myosin isoform expressed. However, the critical steps that define 'fatigability' in vivo of fast vs slow myosin isoforms, at the molecular level, are not yet fully understood. We examined the modulation of the ATP-induced myosin sub-fragment 1 (S1) dissociation from pyrene-actin by inorganic phosphate (Pi), pH and temperature using a specially modified stopped-flow system that allowed fast kinetics measurements at physiological temperature. We contrasted the properties of rabbit psoas (fast) and bovine masseter (slow) myosins (obtained from samples collected from New Zealand rabbits and from a licensed abattoir, respectively, according to institutional and national ethics permits). To identify ATP cycling biochemical intermediates, we assessed ATP binding to a pre-equilibrated mixture of actomyosin and variable [ADP], pH (pH 7 vs pH 6.2) and Pi (zero, 15 or 30 added mM Pi) in a range of temperatures (5 to 45°C). Temperature and pH variations had little, if any, effect on the ADP dissociation constant (KADP) for fast S1 but for slow S1 KADP was weakened with increasing temperature or low pH. In the absence of ADP, the dissociation constant for phosphate (KPi) was weakened with increasing temperature for fast S1. In the presence of ADP, myosin type differences were revealed at the apparent phosphate affinity, depending on pH and temperature. Overall, the newly revealed kinetic differences between myosin types could help explain the in vivo observed muscle type functional differences at rest and during fatigue

Kinetic properties of slow isoforms of mammalian muscle and non-muscle myosin

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The Future of Social Work in Aging: “Everything Old is New Again”

With the aging of the baby boom generation, the number of older adults in the US will increase substantially. Using a biopsychosocial framework, this article presents cutting-edge of older adulthood and considers emerging roles of social workers with older adults and their families. Research, education, and policy perspectives that will advance social work knowledge, skills and resources in aging are proposed. Social work as a profession is challenged to lead the way in making “everything old new again.

The Future of Aging in Social Work: Everything Old is New Again

With the aging of the baby boom generation, the number of older adults in the U.S. will increase substantially. Using a biopsychosocial framework, this article presents cutting-edge issues of older adulthood and considers emerging roles of social workers with older adults and their families. Research, education, and policy perspectives that will advance social work knowledge, skills and resources in aging are proposed. Social work as a profession is challenged to lead the way in making everything old new again

The slow skeletal muscle isoform of myosin shows kinetic features common to smooth and non-muscle myosins

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,beta-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 actin-myosin dissociates at up to similar to 1000 s(-1), a very similar rate constant to MHC-2, but 10-15% of the complex must go through a slow isomerization (similar to 20 s(-1)) before ATP can dissociate it. Similar slow isomerizations were seen in the displacement of ADP from actinmyosin(.)ADP and provide evidence of three closely related actinmyosin(.)ADP 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 (similar to 20 s(-1)), and a third complex that releases ADP too slowly (similar to 6 s(-1)) to be on the main ATPase pathway. The role of these actin-myosin(.)ADP complexes in the mechanochemistry of slow muscle contraction is discussed in relation to the load dependence of ADP releas

What Limits the Velocity of Fast-skeletal Muscle Contraction in Mammals?

In rat skeletal muscle the unloaded shortening velocity (V(o)) is defined by the myosin isoform expressed in the muscle fibre. In 2001 we suggested that ADP release from actomyosin in solution (controlled by k(-AD)) was of the right size to limit V(o). However, to compare mechanical and solution kinetic data required a series of corrections to compensate for the differences in experimental conditions (0.5M KCl, 22 degrees C for kinetic assays of myosin, 200mM ionic strength, 12 degrees C to measure V(o)). Here, a method was developed to prepare heavy meromyosin (HMM) from pure myosin isoforms isolated from single muscle fibres and to study k(-AD) (determined from the affinity of the acto-myosin complex for ADP, K(AD)) and the rate of ATP-induced acto-HMM dissociation (controlled by K(1)k(+2)) under the same experimental condition used to measure V(o). In fast-muscle myosin isolated from a wide range of mammalian muscles, k(-AD) was found to be too fast to limit V(o), whereas K(1)k(+2) was of the right magnitude for ATP-induced dissociation of the cross-bridge to limit shortening velocity. The result was unexpected and prompted further experiments using the stopped-flow approach on myosin subfragment-1 (S1) and HMM obtained from bulk preparations of rabbit and rat muscle. These confirmed that the rate of cross-bridge dissociation by ATP limits the velocity of contraction for fast myosin II isoforms at 12 degrees C, while k(-AD) limits the velocity of slow myosin II isoforms. Extrapolating our data to 37 degrees C suggests that at physiological temperature the rate of ADP dissociation may limit V(o) for both isoforms

Structural Basis for the Allosteric Interference of Myosin Function by Reactive Thiol Region Mutations G680A and G680V*

Background: Cold-sensitive mutations in the reactive thiol region of myosin interfere with motor function