Title

Triadin transmembrane topology and its submolecular interactions with other triadic proteins in skeletal muscle

Date of Award

1995

Availability

Article

Degree Name

Doctor of Philosophy (Ph.D.)

Department

Molecular and Cellular Pharmacology

First Committee Member

Anthony H. Caswell, Committee Chair

Abstract

Native triadin in skeletal muscle is a glycoprotein linked by disulfide bonds of variable subunit number. Its binding to the dihydropyridine receptor (DHPr) and the ryanodine receptor (RyR) in triads indicates triadin might be the mediator for the signal transmission from the T-tubule membrane to the TC of SR during excitation and contraction coupling (E-C coupling). In order to study the transmembrane topology of triadin and localize the interacting domains within triadic proteins, triadin fusion peptides are synthesized using an expression system which includes a phosphorylation site. $\alpha\sb1$ DHPr$\sb{664-799}$ (664-799, numbers correspond to the amino acid sequence) is also synthesized since this portion of DHPr $\alpha\sb1$ subunit is considered to be the major determinant of skeletal muscle type E-C coupling.Triadin membrane topology is studied using the two triadin mAbs, GE4.90 and AE8.91, since both of them were previously shown to have cytoplasmic epitopes. GE4.90 binds to triadin at the C-terminal 34 amino acids and AE8.91 binds to an epitope between residues 110-165, indicating these portions of triadin are cytosolic. Proteolytic fragments of triadin are employed to investigate the glycosylation sites and the nature of the disulfide formation. Evidence shows that the asparagine at residue 625 is glycosylated in vivo and triadin forms a disulfide-linked polymer through mirror image intermolecular disulfide bonds of both cysteines at residues 270 and 671. These in combination with other evidence indicate that one $\alpha$ helix and three $\beta$ sheet segments account for the membrane spanning domains of triadin. This membrane topology gives triadin extensive cytoplasmic and luminal domains.Radio-labelled DHPr$\sb{664-799}$ binds to triadin in triads. Both the DHPr$\sb{664-799}$ and RyR are shown to interact with triadin fusion peptides specifically and extensively. The interaction of RyR and triadin or triadin fusion peptides is shown to be controlled in a complex manner by the known RyR/Ca$\sp{2+}$ release channel modulators.In conclusion, triadin appears to be in a strategic position to receive the muscle excitation signal from the DHPr cytoplasmic II-III loop and pass it to the RyR to allow the Ca$\sp{2+}$ release from the internal store and initiate muscle activation.

Keywords

Biology, Molecular; Biology, Cell

Link to Full Text

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