Fundamental Mechanism and Impact of Post Translational Modifications on Calsequestrin: Implications for Function

Emiliano J. Sanchez

Calsequestrin, the major resident protein found within the sarcoplasmic reticulum (SR), is a critical component required for proper muscle function. Exicitation-contraction coupling in muscle is regulated through fine control of the cytosolic Ca2+ level. During muscle contraction, Ca2+ is released out of the SR through the Ca2+ release channel, a multiprotein complex composed of the ryanodine receptor, calsequestrin, junctin and triadin. This flux of Ca2+ causes regulatory subunits of troponin to reveal myosin binding sites and through ATP-dependent activity, muscle contraction occurs. During relaxation, Ca2+ must be rapidly cleared from the cytosol and redeposited back into the SR. This is accomplished through active transport mediated by the sarco(endo) plasmic reticulum Ca2+ ATPase (SERCA) pump. The ATPase activity is affected in part by the free luminal concentration of Ca2+ within the SR, and upon reaching ~ 1 mM free Ca2+, the ATPase cannot transport against the electrochemical gradient of the SR. Calsequestrin serves to coordinate Ca2+ as it is transported into the SR, and actively remove it from the free concentration. Calsequestrin will then co-localize to the Ca2+ release channel and allow for one-dimensional diffusion out of the SR in anticipation for the next contractile event. While much is known about the overall process of Ca2+ cycling in muscle tissue, many of the regulatory mechanisms that govern this process remain unknown. This dissertation is composed of three studies that seek to characterize how these regulatory mechanisms directly affect calsequestrin and its function within the SR. The first study investigated the effects of phosphorylation on calsequestrin SR function. What we determined is that in a fully phosphorylated state, calsequestrin has a higher Ca2+-binding capacity, has a higher degree of solubility and forms a more compact polymer. The second study investigated the effect that glycosylation has on calsequestrin in both intracellular trafficking and SR function. What we determined is that glycosylation of calsequestrin prevents premature oligomerization during transit from the endoplasmic reticulum / golgi intermediate complex (ERGIC) and plays an active role in oligomeric behavior within the SR. We also proposed a mode of action for a newly characterized mutation that incorporates an additional glycosylation site and has a severe impact on Ca2+ cycling and cardiac function. The final study investigated the structural changes induced by Ca2+ coordination and allows us to propose a modified model that can link our in vitro results to observed in vivo behaviors.

Fundamental Mechanism and Impact of Post Translational Modifications on Calsequestrin: Implications for Function

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