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 detec table impairment of functional properties of the skinned fibers. This appro ach 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-label ed troponin I has previously been used in solution studies as an indicator for the state of activation of reconstituted actin filaments (Trybus and Ta ylor, 1980, 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 Ca2+ or when cross-bridges in their strong binding states atta ch to actin. Maximum reduction is observed when Ca2+ is raised to saturatin g concentrations. Additional attachment of cross-bridges in strong binding states gives no further reduction of fluorescence. Attachment of cross-brid ges in strong binding states alone (low Ca2+ concentration) gives only abou t half of the maximum reduction seen with the addition of calcium. This ill ustrates that fluorescence of IANBD-labeled troponin I can be used to evalu ate thin filament activation, as previously introduced for solution studies . In addition, at nonsaturating Ca2+ concen; trations IANBD fluorescence ca n be used for straightforward classification of states of the myosin head a s weak binding (nonactivating) and strong binding (activating), irrespectiv e of ionic strength or other experimental conditions. Furthermore, the appr oach presented here not only can be used as a means of exchanging native sk eletal troponin and its subunits for a variety of fluorescently labeled or mutant troponin subunits, but also allows the exchange of native skeletal t roponin for cardiac troponin.