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

Mechanism and specificity of pentachloropseudilin-mediated inhibition of myosin motor activity.

Here, we report that the natural compound pentachloropseudilin (PClP) acts as a reversible and allosteric inhibitor of myosin ATPase and motor activity. IC(50) values are in the range from 1 to 5 μm for mammalian class-1 myosins and greater than 90 μm for class-2 and class-5 myosins, and no inhibition was observed with class-6 and class-7 myosins. We show that in mammalian cells, PClP selectively inhibits myosin-1c function. To elucidate the structural basis for PClP-induced allosteric coupling and isoform-specific differences in the inhibitory potency of the compound, we used a multifaceted approach combining direct functional, crystallographic, and in silico modeling studies. Our results indicate that allosteric inhibition by PClP is mediated by the combined effects of global changes in protein dynamics and direct communication between the catalytic and allosteric sites via a cascade of small conformational changes along a conserved communication pathway

Myosin 1b Regulates Amino Acid Transport by Associating Transporters with the Apical Plasma Membrane of Kidney Cells.

Amino acid transporters (AATers) in the brush border of the apical plasma membrane (APM) of renal proximal tubule (PT) cells mediate amino acid transport (AAT). We found that the membrane-associated class I myosin myosin 1b (Myo1b) localized at the apical brush border membrane of PTs. In opossum kidney (OK) 3B/2 epithelial cells, which are derived from PTs, expressed rat Myo1b-GFP colocalized in patched microvilli with expressed mouse V5-tagged SIT1 (SIT1-V5), which mediates neutral amino acid transport in OK cells. Lentivirus-mediated delivery of opossum Myo1b-specific shRNA resulted in knockdown (kd) of Myo1b expression, less SIT1-V5 at the APM as determined by localization studies, and a decrease in neutral AAT as determined by radioactive uptake assays. Myo1b kd had no effect on Pi transport or noticeable change in microvilli structure as determined by rhodamine phalloidin staining. The studies are the first to define a physiological role for Myo1b, that of regulating renal AAT by modulating the association of AATers with the APM

Myo1b kd reduces the number of SIT1 transporters at the apical plasma membrane.

<p>OK cells were transfected with SIT1-V5 and either collectrin or empty vector (pLenti-CMV Hygro) and stained with rhodamine phalloidin to visualize the apical microvilli. A, Immunoblot showing that Myo1b kd increased SIT1-V5 expression. Tubulin served as a loading control. <i>B-G</i>, B’-G’, Sample images showing that in control (Mock) OK 3B/2 cells (B-D; B’-D’), expressed SIT1-V5 (C, <i>green</i>) localized almost exclusively in actin-rich microvilli (B, <i>red; arrows</i>) on the apical surface; whereas in Myo1b kd-cells (shRNA 628) (E-G; E’-G’), SIT1-V5 localized to the cytoplasm. Cytoplasmic SIT1-V5 in Myo1b-kd cells is most evident in the lateral (<i>x-z</i>) view shown in F’. B-G are <i>x-y</i> images; B’-G’ are <i>x-z</i> images of B-G, respectively. In B’-G’, the position of the apical membrane is marked with <i>a</i>, and the position of the basal membrane is marked with <i>b</i>. D, D’, G, G’, Merge, <i>yellow</i>. <i>Bar</i>, 5 μm. H, Myo1b-kd decreased the number of cells expressing SIT1-V5 at the APM. The number of transfected cells with SIT1-V5 in apical microvilli was determined and expressed as a function of the total number of transfected cells. In each case 167–465 transfected cells were counted. The data are from three independent experiments and expressed as means ± s.e. *P<0.01, ** P<0.005. The expression of exogenous collectrin rescued the Myo1b-kd-induced decrease in SIT1-V5 at the APM. The number of cells expressing SIT1-V5 at the APM was not statistically different in control vs. Myo1b-kd cells when collectrin was overexpressed; P>0.05. I, [³H]isoleucine transport was not rescued in Myo1b-kd cells by collectrin. Overexpression of exogenous collectrin in Myo1b-kd cells did not rescue neutral amino acid transport. White filled bars, (-) collectrin; black-filled bars, (+) collectrin. Data are means ± s. d. for samples done in triplicate. * P<0.005, ** P<0.001.</p