Ionic interventions that alter the association of troponin C C-domain with the thin filaments of vertebrate striated muscle.
The regulatory complex of vertebrate skeletal muscle integrates information about cross-bridge binding, divalent cations and other intracellular ionic conditions to control activation of muscle contraction. Relatively little is known about the role of the troponin C (TnC) C-domain in the absence of Ca2+. Here, we use a standardized condition for measuring isometric tension in rabbit psoas skinned fibers to track TnC attachment and detachment in the absence of Ca2+ under different conditions of ionic strength, pH and MgATP. In the presence of MgATP and Mg2+, TnC detaches more readily and has a 1.5- to 2-fold lower affinity for the intact thin filament at pH 8 and 250 mM K+ than at pH 6 or in 30 mM K+; changes in affinity are fully reversible. The response to ionic strength is lost when Mg2+ and MgATP are absent, whereas the response to pH persists, suggesting that weaker electrostatic TnC-TnI-TnT interactions can be overridden by strongly bound cross-bridges. In solution, titration of a fluorescent C-domain mutant (F154W TnC) with Mg2+ reveals no significant changes in Mg2+ affinity with pH or ionic strength, suggesting that these parameters influence TnC binding by acting directly on electrostatic forces between TnC and TnI rather than by changing Mg2+ binding to C-domain sites III and IV.
Sousa
Valeria P
VP
Instituto de Bioquímica Médica, Centro de Ciências da Saúde, Universidade Federal do Rio de Janeiro, Cidade Universitária, Rio de Janeiro, RJ 21941-590, Brazil.
Pinto
José R
JR
Sorenson
Martha M
MM
D000818:Animals; D002645:Chickens; D006863:Hydrogen-Ion Concentration; D008274:Magnesium / Q000378:metabolism / Q000494:pharmacology; D018485:Muscle Fibers, Skeletal / Q000187:drug effects / Q000378:metabolism*; D018482:Muscle, Skeletal / Q000187:drug effects / Q000378:metabolism*; D009994:Osmolar Concentration; D017434:Protein Structure, Tertiary; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D019209:Troponin C / Q000737:chemistry*; D019210:Troponin I / Q000737:chemistry; D020107:Troponin T / Q000378:metabolism
D019209:Troponin C; D019210:Troponin I; D020107:Troponin T; D008274:Magnesium