E. Kordeli et al., ANKYRIN(G) IS ASSOCIATED WITH THE POSTSYNAPTIC MEMBRANE AND THE SARCOPLASMIC-RETICULUM IN THE SKELETAL-MUSCLE FIBER, Journal of Cell Science, 111, 1998, pp. 2197-2207
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.