ANKYRIN(G) IS ASSOCIATED WITH THE POSTSYNAPTIC MEMBRANE AND THE SARCOPLASMIC-RETICULUM IN THE SKELETAL-MUSCLE FIBER

Ankyrins are a multi-gene family of peripheral proteins that link ion channels and cell adhesion molecules to the spectrin-based skeleton in specialized membrane domains, In the mammalian skeletal myofiber, ank yrins were immunolocalized in several membrane domains, namely the cos tameres, the postsynaptic membrane and the triads, Ank1 and Ank3 trans cripts were previously detected in skeletal muscle by northern blot an alysis. However, the ankyrin isoforms associated with these domains we re not identified, with the exception of an unconventional Ank1 gene p roduct that was recently localized at discrete sites of the sarcoplasm ic reticulum. Here we study the expression and subcellular distributio n of the Ank3 gene products, the ankyrins(G), in the rat skeletal musc le fiber. Northern blot analysis of rat skeletal muscle mRNAs using do main-specific Ank3 cDNA probes revealed two transcripts of 8.0 kb and 5.6 kb containing the spectrin-binding and C-terminal, but not the ser ine-rich, domains. Reverse transcriptase PCR analysis of rat skeletal muscle total RNA confirmed the presence of Ank3 transcripts that lacke d the serine-rich and tail domains, a major insert of 7813 bp at the j unction of the spectrin-binding and C-terminal domains that was previo usly identified in brain Ank3 transcripts. Immunoblot analysis of tota l skeletal muscle homogenates using ankyrin(G)-specific antibodies rev ealed one major 100 kDa ankyrin(G) polypeptide, Immunofluorescence lab eling of rat diaphragm cryosections showed that ankyrin(s)(G) are sele ctively associated with (1) the depths of the postsynaptic membrane fo lds, where the voltage-dependent sodium channel and N-CAM accumulate, and (2) the sarcoplasmic reticulum, as confirmed by codistribution wit h the sarcoplasmic reticulum Ca2+-ATPase (SERCA 1), At variance with a nkyrin(s)(G), ankyrin(s)(R) (ank1 gene products) accumulate at the sar colemma and at sarcoplasmic structures, in register with A-bands. Both ankyrin isoforms codistributed over Z-lines and at the postsynaptic m embrane. These data extend the notion that ankyrins are differentially localized within myofibers, and point to a role of the ankyrin(G), fa mily in the organization of the sarcoplasmic reticulum and the postsyn aptic membrane.