Thin filament activation probed by fluorescence of N-((2-(iodoacetoxy)ethyl)-N-methyl)amino-7-nitrobenz-2-oxa-1,3-diazole-labeled troponin I incorporated into skinned fibers of rabbit psoas muscle.
A method is described for the exchange of native troponin of single rabbit psoas muscle fibers for externally applied troponin complexes without detectable impairment of functional properties of the skinned fibers. This approach is used to exchange native troponin for rabbit skeletal troponin with a fluorescent label (N-((2-(iodoacetoxy)ethyl)-N-methyl)amino-7-nitrobenz-2-oxa-1, 3-diazole, IANBD) on Cys(133) of the troponin I subunit. IANBD-labeled troponin I has previously been used in solution studies as an indicator for the state of activation of reconstituted actin filaments (. Proc. Natl. Acad. Sci. USA. 77:7209-7213). In the skinned fibers, the fluorescence of this probe is unaffected when cross-bridges in their weak binding states attach to actin filaments but decreases either upon the addition of Ca(2+) or when cross-bridges in their strong binding states attach to actin. Maximum reduction is observed when Ca(2+) is raised to saturating concentrations. Additional attachment of cross-bridges in strong binding states gives no further reduction of fluorescence. Attachment of cross-bridges in strong binding states alone (low Ca(2+) concentration) gives only about half of the maximum reduction seen with the addition of calcium. This illustrates that fluorescence of IANBD-labeled troponin I can be used to evaluate thin filament activation, as previously introduced for solution studies. In addition, at nonsaturating Ca(2+) concentrations IANBD fluorescence can be used for straightforward classification of states of the myosin head as weak binding (nonactivating) and strong binding (activating), irrespective of ionic strength or other experimental conditions. Furthermore, the approach presented here not only can be used as a means of exchanging native skeletal troponin and its subunits for a variety of fluorescently labeled or mutant troponin subunits, but also allows the exchange of native skeletal troponin for cardiac troponin.
Brenner
B
B
Department of Molecular and Cell Physiology, Medical School Hannover, D-30623 Hannover, Germany. brenner.bernhard@mh.hannover.de
Kraft
T
T
Yu
L C
LC
Chalovich
J M
JM
D008841:Actin Cytoskeleton / Q000378:metabolism*; D000818:Animals; D001696:Biomechanical Phenomena; D004591:Electrophoresis, Polyacrylamide Gel; D005456:Fluorescent Dyes / Q000378:metabolism*; D018613:Microscopy, Confocal; D018485:Muscle Fibers, Skeletal / Q000166:cytology / Q000378:metabolism*; D010069:Oxadiazoles / Q000378:metabolism*; D016658:Psoas Muscles / Q000378:metabolism; D011817:Rabbits; D015203:Reproducibility of Results; D012996:Solutions; D013050:Spectrometry, Fluorescence / Q000379:methods*; D019210:Troponin I / Q000378:metabolism*; D014961:X-Ray Diffraction
D005456:Fluorescent Dyes; D010069:Oxadiazoles; D012996:Solutions; D019210:Troponin I; C035573:4-(N-(iodoacetoxy)ethyl-N-methyl)amino-7-nitrobenz-2-oxa-1,3-diazole