Modification Of Loop 1 Affects The Nucleotide-Binding Properties Of Myo1c, The Adaptation Motor In The Inner Ear

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Myo1c is a ubiquitously expressed mammalian class I myosin that serves as a component of the hair cell's adaptation-motor complex in the inner ear. We have recently shown that a truncated form of Myo1c consisting of the motor domain and a single IQ domain, Myo1c1IQ, has kinetic properties similar to full-length Myo1c (Adamek et al, 2008). We also showed that the ATPase cycle of Myo1c shows a unique response to Ca2+, inhibiting the ATP hydrolysis step 7-fold and accelerating ADP release by 10-fold. Here we probed the role of loop 1, a flexible loop near the nucleotide-binding region, in defining the properties of Myo1c by creating six chimeras. 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, transient kinetics properties and 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 ADP release 2-3-fold from A.M in Ca2+, whereas substitution with loop 1 from phasic muscle myosin II (Myo1c1IQ-phasic) accelerated ADP release 35-fold. Myo1c1IQ-tonic translocated actin in vitro twice as fast as wild type and Myo1c1IQ-G 3-fold faster. The changes induced in Myo1c showed no resemblance to the behaviour of the loop donor myosins or to the changes observed with similar Myo1b chimeras (Clark et al, 2005).