Myo1c is the single-headed member of the myosin superfamily that plays roles in trafficking of GLUT4-containing vesicles to the plasma membrane in response to insulin stimulation (1
) and in compensatory endocytosis following regulated exocytosis (3
). Myo1c has also been proposed to play a key role in the process of adaptation in specialized sensory cells, where it is thought to dynamically adjust tension on mechanosensitive ion channels via its interaction with actin (4
Cell biological studies have shown that myo1c localizes and fractionates with cell membranes, and biochemical experiments have shown myosin-I isoforms bind directly to phosphoinositides (6
). Additionally, mechanical experiments have shown that a related myosin isoform (myo1b) acts as a tension sensor by responding to small resisting loads by increasing its actin-attachment lifetime, allowing it to generate and sustain tension for extended time periods (8
). Thus, evidence points to myo1c acting as a tension-sensing motor protein that links cellular membranes to the underlying actin cytoskeleton.
Consistent with the proposed role of myo1c acting as a tension-sensing adaptation motor in sensory cells, a myo1c point-mutation (R156W) has recently been identified in an individual with bilateral sensorineural hearing loss (; (9
)). R156 is highly conserved among the members of the myosin superfamily. It is located at the start of the switch-1 region in the motor domain, which is a crucial structural element involved in nucleotide binding, although R156 is not proposed to interact directly with the nucleotide (10
). Point mutations in switch-1 of myosins-II and -V affect nucleotide binding, ATP hydrolysis, phosphate release, and ADP release (11
). Thus, the R156W mutation likely affects the motile and tension-sensing properties of myo1c. Indeed, the role of myo1c in adaptation may be particularly sensitive to even minor alterations in the ATPase kinetics and load dependent mechanochemistry, since the processes of hearing and balance have stringent force and kinetic requirements (15
Fig. 1 A. Schematic of the expressed myo1b3IQ construct showing the relationship of the motor domain (large rectangle) to the IQ motifs (smaller numbered rectangles). The inset shows the positional relationship of the mutated residue (underlined) to switch-1 (more ...)
In this study, we determined the effect of the R156W mutation on key steps in the myo1c ATPase cycle () using transient and steady-state kinetic techniques. We also determined the effect of this mutation on the motile properties of the motor. Experiments were performed with wildtype (myo1c3IQ) and mutated (R156W-myo1c3IQ) myo1c constructs that consist of the motor domain, regulatory domain (contains three calmodulin-binding IQ motifs), and a C-terminal biotinylation tag for site-specific attachment of myosin for motility assays (). We found that the R156W mutation causes a reduction in the myosin duty cycle, likely by reducing the rate of phosphate release. Furthermore, the mutation appears to cause a reduction in the sensitivity of myo1c to resisting loads, possibly explaining the basis of the observed mutant phenotype.