Myosin VIIa is crucial in hearing and visual processes. We examined the kinetic and association properties of the baculovirus expressed, truncated mouse myosin VIIa construct containing the head, all 5IQ motifs and the putative coiled coil domain (myosin VIIa-5IQ). The construct appears to be monomeric as determined by analytical ultracentrifugation experiments, and only single headed molecules were detected by negative stain electron microscopy. The relatively high basal steady-state rate of 0.18 s ؊1 is activated by actin only by ϳ3.5-

Caffeine, which stimulates intracellular Ca2+ release channels known as ryanodine receptor (RyR) channels, induces contraction of individual muscle fibres dissociated from the trematode Schistosoma mansoni, and the turbellarians Dugesia tigrina and Procerodes littoralis. Caffeine is much more potent on S. mansoni fibres (EC50 0.7 mM) than those from D. tigrina or P. littoralis (3.2 mM and 4.6 mM, respectively). These caffeine-induced contractions are blocked by ryanodine, confirming the presence of functional RyR channels in these flatworm muscles. However, the contractions are not blocked by typical RyR channel blockers ruthenium red or neomycin, indicating that there may be important pharmacological differences between the RyR channels in this early-diverging phylum and those of later animals. These studies demonstrate that RyR channels are present in the muscle of these flatworms, and that the sarcoplasmic reticulum stores sufficient Ca2+ to support contraction.

Mutations in the gene, encoding the motor protein myosin VIIa, can cause Usher 1B, a deafness/blindness syndrome in humans, and the shaker-1 phenotype, characterized by deafness, head tossing, and circling behavior, in mice. Myosin VIIa is responsible for tension bearing and the transduction mechanism in the stereocilia and for melanosome transport in the retina, in line with the phenotypic outcomes observed in mice. However, the effect of the shaker-1 mutation, a R502P amino acid substitution, on the motor function is unclear. To explore this question, we determined the kinetic properties and the effect on the filopodial tip localization of the recombinant mouse myosin VIIa-5IQ-SAH R502P (myoVIIa-sh1) construct. Interestingly, although residue 502 is localized to a region thought to be involved in interacting with actin, the kinetic parameters for actin binding changed only slightly for the mutant construct. However, the rate constant for ATP hydrolysis ( + ) was reduced by ∼200-fold from 12 s to 0.05 s, making the hydrolysis step the rate-limiting step of the ATPase cycle in the presence and absence of actin. Given that wild-type mouse myosin VIIa is a slow, high-duty ratio, monomeric motor, this altered hydrolysis rate would reduce activity to extremely low levels. Indeed, the translocation to the filopodial tips was hampered by the diminished motor function of a dimeric construct of the shaker-1 mutant. We conclude that the diminished motor activity of this mutant is most likely responsible for impaired hearing in the shaker-1 mice.

Myosin V is a dimeric molecular motor, which transports organelles toward the barbed end of actin filaments in cells. Its highly efficient unidirectional motility requires the coordination of ATPase cycles in two head domains, to ensure that the rate-limiting ADP release almost exclusively occurs in the trailing head and the motor steps forward. Single-molecule measurements revealed that the directional loads modulate the kinetics of nucleotide binding to myosin V, suggesting that the head-head communication may be based on intramolecular load, generated when both heads are bound to actin. Here we directly tested the effect of the intramolecular load on the processive stepping of myosin V, using point mutations in the converter domain, which are inferred to reduce intramolecular load but do not affect the nucleotide binding or actin affinity. The converter is a compact structure, which transmits tiny conformational changes, induced at the nucleotide-binding site in the process of ATP hydrolysis, to the lever arm. To disturb the transmission mechanism, we replaced with alanines, one at a time, two phenylalanine residues that form a hydrophobic cluster with the C-terminus of the relay helix. The effects of the mutations on the myosin's V motility were tested by multiple kinetic and single-molecule assays. We found that the F749A mutation significantly increases the proportion of backward steps, whereas the F697A mutation completely abolishes the processive stepping of myosin V. These results provide strong experimental evidence that the efficient unidirectional processive stepping of myosin V is ensured by the head-head communication based on the intramolecular load, which coordinates ATPase cycles in two motor domains.

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