• Calcium transport in intact mammalian fast-twitch and slow-twitch skeletal muscle fibers

      Carroll, Stefanie Leigh; Schneider, Martin F. (1996)
      Calcium ions that are released from the Sarcoplasmic Reticulum (SR) upon electrical stimulation bind to troponin C of the thin filament structure, and initiate contraction of the skeletal muscle fiber. In order for the muscle fiber to relax, calcium must dissociate from troponin C and be removed from the cytoplasm by reuptake via the SR Calcium ATPase or by binding to cytoplasmic proteins. The intricacies of the calcium removal system in intact mammalian fibers have not been elucidated. The goal of my thesis project was to characterize the calcium removal system in mammalian intact skeletal muscle fibers, and determine the contribution of the individual components involved, such as the SR calcium ATPase, troponin C, and parvalbumin. Rat fast-twitch flexor digitorum brevis fibers (FDB) and slow-twitch soleus fibers are enzymatically dissociated and suspended in low melting temperature agarose gel to minimize fiber movement during fluorescence recordings. FDB fibers and soleus fibers are loaded with fura-2 (cell permeant form) and electrically stimulated by 1 to 40 pulses. Florescence signals are recorded at 380 (calcium sensitive) and 358 (calcium insensitive) nm excitation. Ca2+ is calculated assuming non-instantaneous equilibrium with fura-2. The rate constant of calcium decay decreased significantly with increasing stimulation duration in the FDB fibers, but remained relatively constant in the soleus fibers. This is due to expected differences in parvalbumin concentration between fast-twitch and slow-twitch fibers. In fast-twitch fibers parvalbumin becomes increasing saturated by calcium with increasing stimulation durations and can no longer contribute to the decay of calcium. However, there is negligible amounts of parvalbumin in slow-twitch fibers, therefore they do not exhibit this slowing of calcium decay effect. Quantification of the SR calcium ATPase, troponin C and parvalbumin content, using SDS page and immunoblotting techniques confirmed that there was a significant difference in the concentration of parvalbumin between rat FDB (1.2 mM calcium binding site concentration) and soleus fibers ({dollar}<{dollar}50 {dollar}\mu{dollar}M calcium binding site concentration). Unexpectedly there was no significant difference in the concentration of SR calcium ATPase, and troponin C. The values determined by the gel and immunoblot Quantification were well supported by preliminary modeling analysis of the Ca2+ decay. In conclusion, there are significant differences in the decay of Ca2+ in rat fast-twitch and slow-twitch muscle, which is due to differences in parvalbumin concentration. This indicates that parvalbumin has a significant role in the decay of calcium in mammalian skeletal muscle fibers.
    • Detailed characterization of the cooperative mechanism of calcium(2+) binding and catalytic activation in the sarcoplasmic reticulum calcium(2+) transport (SERCA) ATPase

      Zhang, Zhongsen; Inesi, Giuseppe (2001)
      Occupation of two calcium-binding sites is required for catalytic activation of the sarcoplasmic reticulum Ca2+ ATPase (SERCA). The residues in the transmembrane domain, the cytoplasmic phosphorylation domain, and the L67 and L89 loops were subjected to mutational analysis. Direct measurements of Ca2+ binding and measurements of various enzymatic functions clarified the cooperative mechanism of calcium binding and catalytic activation of SERCA. The functional characterization and high-resolution structure of ATPase suggested cooperative and sequential calcium binding in which side chains of Glu771, Thr799, Asp800 and Glu908 contribute to site I, while Glu309, Asn796 and Asp800 contribute to site II. Mutational analysis of the L67 loop indicated its importance in protein folding and stabilization of the Ca 2+ ATPase. Single mutation of Pro820 to Ala resulted in negligible protein recovery while transcription occurred at normal levels. Single mutations of Lys819 or Arg822 interfered significantly with the formation of the phosphoenzyme intermediates. A triple conservative mutation of Asp813, 815 and 818 to Asn interfered mainly with the Ca2+-dependent activation of the ATPase but not Ca2+-independent phosphorylation by Pi. The effect of the triple mutation could be reproduced by a single mutation of Asp813 (but not of Asp815 or Asp818) to Asn. Functional and structural analysis of the experimental data demonstrates that the L67 loop plays an important role in protein folding and stabilization by linking the cytosolic catalytic domain and the transmembrane Ca2+ binding domain through a network of hydrogen bonds.
    • Identification of Small Ankyrin 1 as a novel SERCA1 regulatory protein in Skeletal Muscle

      Desmond, Patrick Francis; Bloch, Robert J.; 0000-0002-2006-6280 (2016)
      Small Ankyrin 1 (sAnk1) is a ~20 kDa transmembrane (TM) protein that binds to the cytoskeletal protein, obscurin, and stabilizes the network sarcoplasmic reticulum (nSR) in skeletal muscle. Previous reports from out lab show that sAnk1 knock down results in loss of network SR integrity, along with a decrease in SR Ca2+ load and Ca2+ re-uptake rates. Upon closer examination of the sAnk1 transmembrane domain, we discovered that sAnk1 shares sequence similarity with sarcolipin (SLN), a small protein that inhibits activity of the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA). The goal of the current study was to determine if sAnk1 interacts with SERCA1 or SLN directly in skeletal muscle, and elucidate the consequences such interactions pose on SERCA1 activity. Our results indicate that sAnk1 interacts specifically with SERCA1 in SR vesicles isolated from rabbit skeletal muscle, and in COS7 cells transfected to express these proteins. This interaction was demonstrated by co-immunoprecipitation and an anisotropy-based FRET method (AFRET). Binding was significantly reduced by the replacement of all the TM amino acids of sAnk1 to leucines by mutagenesis. This suggests that, like SLN, sAnk1 interacts with SERCA1 via its TM domain. Assays of ATPase activity show that co-expression of sAnk1 with SERCA1 leads to a reduction of SERCA1's apparent Ca2+ affinity, but that sAnk1's effect is less than that of SLN. Interestingly, the sAnk1 TM mutant has no effect on SERCA1 activity. Our results suggest that sAnk1 interacts with SERCA1 through its TM domain to regulate SERCA1 activity and thereby modulate the sequestration of Ca2+ in the lumen of the ER and SR. Additionally, we determined that sAnk1 can also interact with SLN using the same analytical methods. Unexpectedly, ATPase assays in which all three proteins were co-expressed showed that sAnk1 was able to limit SLN's ability to inhibit SERCA1 activity. Furthermore, coIP and AFRET experiments demonstrate that SLN promotes the interaction between SERCA1 and sAnk1. The identification of sAnk1 as a novel regulator of SERCA1 activity has significant implications for the physiology of muscle and the development of therapeutic approaches to treat heart failure and muscular dystrophies linked to Ca2+ misregulation.
    • Mutational study on the coupling mechanism of catalysis and transport functions in the sarcoplasmic reticulum Ca(2+)-ATPase

      Zhang, Ziyu; Inesi, Giuseppe (1995)
      ATP utilization by P-type cation transport ATPases includes a phosphorylated intermediate which is formed by transfer of the ATP terminal phosphate onto an aspartyl residue at the catalytic site. The phosphorylation site and cation binding site of the ATPase molecule are separated by a fairly long distance of about 50 A. The coupling mechanism of these two functional sites is not yet fully understood and is currently under active investigation. Within the family of cation transport ATPases, the Ca2+-ATPase of sarcoplasmic reticulum (SR) provides an advantageous experimental system due to its abundance in the native membrane, and the availability of cDNA for expression of functional protein.;The sarcoplasmic reticulum Ca2+-ATPase segment extending from the phosphorylation site (Asp351) to the preceding transmembrane helix M4 (which is involved in Ca2+ binding in conjunction with transmembrane helices M5, M6 and M8), shares a marked sequence homology with the corresponding segments of other cation ATPases. We generated twenty six point mutations in this segment and expressed those mutant enzymes in COS-1 cells. We found that non-conservative mutations of residues which are homologous in various cation ATPases result in strong inhibition of catalytic and transport functions. Mutations of non-homologous residues to match the corresponding residues of other cation ATPases are not inhibitory, and in some cases produce higher activity. The inhibitory mutations specifically affect the phosphorylated intermediate turnover, which is associated with the vectorial translocation of bound Ca2+. The same mutations do not affect the kinetics of ATPase activation by Ca2+, which is required for enzyme phosphorylation by ATP. This indicates that activation of the phosphoryl transfer reaction by Ca2+ binding, and vectorial displacement of bound Ca2+ by enzyme phosphorylation, do not occur simply as the forward and reverse directions of the same process, but are linked to distinct structural features of the enzyme. The peptide segment extending from the phosphorylation site in the enzyme extramembranous headpiece, through the M4 helix in the membrane bound region, sustains a prominent role in transmission of the phosphorylation signal for displacement of bound Ca2+. A critical structural role of this segment is also demonstrated by the interference of specific mutations with membrane assembly of the expressed protein.
    • Understanding the Role of Small Ankryin 1 in Calicum Regulation in Excitable Cells

      Labuza, Amanda; Bloch, Robert J. (2020)
      Small Ankryin 1 (sAnk1) is a 17kD transmembrane protein that plays a role in stabilizing the network sarcoplasmic reticulum in skeletal muscle (Ackermann et al., 2011). Recent studies have shown that sAnk1 can bind to and regulate sarco(endo)plasmic reticulum Ca2+-ATPase1 (SERCA1) activity (Desmond et al., 2015). SERCA1 transports Ca2+ against its gradient into the SR after muscle contraction. SERCA is inhibited by sarcolipin (SLN) in fast twitch skeletal muscle and atrial cardiac muscle and by phospholamban (PLN) in slow twitch muscle and ventricular cardiac muscle. Like SLN and PLN, sAnk1 also interacts with SERCA at least in part through its transmembrane domain (Asahi et al., 2003; Hutter et al., 2002; Desmond et al., 2015). The interaction of SERCA with SLN and PLN has been studied individually and together, but the effects of sAnk1 and its regulatory activity have only recently started to be addressed (Desmond et al., 2015, 2017). Here I show that sAnk1 can interact with PLN or SLN independently of SERCA1. sAnk1 forms a three-way complex with SLN and SERCA1 that ablates SLN inhibition (Desmond et al., 2017). sAnk1 can also form a three-way complex with PLN and SERCA1 that abolishes all inhibition. I show that the complexes that sAnk1 forms with SLN or PLN and SERCA1 are distinct, suggesting unique roles for each protein in SERCA regulation. I also examined sAnk1 and SERCA in several CNS tissues, and found that sAnk1 is not expressed in neurons, but that it is expressed in astrocytes, where it has the potential to bind and regulate SERCA2B. Studying the multi-protein complex of SERCA, sAnk1, SLN, and/or PLN can help us better understand physiological SERCA regulation. This knowledge can lead to better treatment for diseases related to misregulation of calcium, including muscular dystrophies and potentially some neuropathies.