CA2-C IN SKINNED SKELETAL-MUSCLE FIBERS ASSESSED WITH CASED CA2+ AND A CA2+ FLUOROPHORE - INVARIANCE OF CA2+ BINDING AS A FUNCTION OF SARCOMERE-LENGTH( BINDING TO TROPONIN)
Jr. Patel et al., CA2-C IN SKINNED SKELETAL-MUSCLE FIBERS ASSESSED WITH CASED CA2+ AND A CA2+ FLUOROPHORE - INVARIANCE OF CA2+ BINDING AS A FUNCTION OF SARCOMERE-LENGTH( BINDING TO TROPONIN), The Journal of biological chemistry, 272(9), 1997, pp. 6018-6027
Ca2+ sensitivity of tension varies with sarcomere length in both skele
tal and cardiac muscles, One possible explanation for this effect is t
hat the Ca2+ affinity of the regulatory protein troponin C decreases w
hen sarcomere length is reduced. To examine length dependence of Ca2binding to troponin C: in skeletal muscle, we developed a protocol to
simultaneously monitor changes in sarcomere length, tension, and Ca2concentration following sash photolysis of caged Ca2+. In this protoco
l, [Ca2+] was rapidly increased by flash photolysis of caged Ca2+, and
changes in [Ca2+] due to photolysis and the subsequent binding to tro
ponin C were assessed using a Ca2+ fluorophore. Small bundles of fiber
s from rabbit skinned psoas muscles were loaded with Ca2+ fluorophore
(Fluo-3) and caged Ca2+ (dimethoxynitrophenamine or o-nitrophenyl-EGTA
). The bundles were then transferred to silicone oil, where [Ca2+](fre
e), tension, and sarcomere length were monitored before and after phot
olysis of caged Ca2+. Upon photolysis of caged Ca2+, fluorescence incr
eased and then decayed to a new steady-state level within similar to 1
s, while tension increased to a new steady-state level within similar
to 1.5 s, After extracting troponin C, fibers did not generate tensio
n following the flash, but steady-state post-flash fluorescence was si
gnificantly greater than when troponin C was present, The difference i
n [Ca2+](free) represents the amount of Ca2+ bound to troponin C. In f
ibers that were troponin C-replete, Ca2+ binding to troponin C did not
differ at short (similar to 1.97 mu m) and long (similar to 2.51 mu m
) sarcomere length, yet tension was similar to 50% greater at the long
sarcomere length, These results show that the affinity of troponin C
for Ca2+ is not altered by changes in sarcomere length, indicating tha
t length-dependent changes in Ca2+ sensitivity of tension in skeletal
muscle are not related to length-dependent changes in Ca2+ binding aff
inity of troponin C.