Orientation of the Cardiac Myosin Regulatory Light Chain Determined by Polarized Fluorescence

The regulatory light chain (RLC) of myosin is a component of the lever arm of the<br/>myosin motor in muscle thick filaments. Phosphorylation of RLC by myosin light<br/>chain kinase (MLCK) modulates the force and speed of muscle contraction, but the mechanisms of RLC‐mediated regulation in striated muscles are less well<br/>understood than those of Ca2+‐dependent regulation via troponin in the thin<br/>filament.<br/>To help elucidate the role of RLC in muscle regulation, its orientation in the native<br/>environment of cardiac muscle cells was measured. Pairs of cysteine residues were genetically introduced into the N‐ and C‐terminal lobe of the human cardiac RLC.<br/>Each pair of cysteines was crosslinked with a bifunctional‐rhodamine (BSR). The<br/>pure BSR‐RLC conjugates were exchanged into demembranated trabeculae from rat ventricle, and the orientation of the BSR fluorescence dipole determined by<br/>polarized fluorescence.<br/>The orientations of the N‐lobe were similar to those determined from chicken<br/>gizzard RLC probes exchanged into skeletal muscle fibers indicating a conserved<br/>domain orientation. The orientation of the RLC C ‐ lobe was similar in relaxation,<br/>active isometric contraction and rigor, suggesting that either the orientation of the<br/>RLC is relatively insensitive to strong binding of myosin heads to actin, or that only a small fraction of myosin heads are strongly bound to the thin filament in both<br/>active isometric contraction and rigor. Bending between the two RLC lobes occurs<br/>in ‐ situ and may have a functional significance in cardiac muscle contraction and<br/>regulation.<br/>Expressed and purified catalytic subunit of human cardiac MLCK efficiently mono ‐<br/>phosphorylates cardiac RLC on serine 15 in a calcium/calmodulin dependent<br/>manner. Exchange of in ‐ vitro phosphorylated BSR‐RLCs into demembranated<br/>trabeculae to replace 10‐15% of native RLC showed that the orientation of<br/>phosphorylated RLC C ‐ lobe is similar to that of unphosphorylated RLCs.

Name dc:type.qualificationname

Doctor of Philosophy

Level dc:type.qualificationlevel

Doctoral Thesis

Grantor dc:publisher.institution

King's College London

Year dc:date.issued

2012

Author dc:creator

• Kampourakis, Thomas

Advisors dc:contributor.advisor

• Irving, Malcolm
• Gautel, Mathias Sebastian

Language dc:language

eng

Identifier

oai:kclpure.kcl.ac.uk:studenttheses/f04760ad-8fc6-4926-b3c7-9c2a1bab7799

OAI identifier oai:identifier

oai:kclpure.kcl.ac.uk:studenttheses/f04760ad-8fc6-4926-b3c7-9c2a1bab7799