Intermediate filaments, composed of desmin and of keratins, play important roles in linking contractile elements to each other and to the sarcolemma in striated muscle. We examined the contractile properties and morphology of fast-twitch skeletal muscle from mice lacking keratin 19. Tibialis anterior muscles of keratin-19-null mice showed a small but significant decrease in mean fiber diameter and in the specific force of tetanic contraction, as well as increased plasma creatine kinase levels. Costameres at the sarcolemma of keratin-19-null muscle, visualized with antibodies against spectrin or dystrophin, were disrupted and the sarcolemma was separated from adjacent myofibrils by a large gap in which mitochondria accumulated. The costameric dystrophin-dystroglycan complex, which co-purified with γ-actin, keratin 8 and keratin 19 from striated muscles of wild-type mice, co-purified with γ-actin but not keratin 8 in the mutant. Our results suggest that keratin 19 in fast-twitch skeletal muscle helps organize costameres and links them to the contractile apparatus, and that the absence of keratin 19 disrupts these structures, resulting in loss of contractile force, altered distribution of mitochondria and mild myopathy. This is the first demonstration of a mammalian phenotype associated with a genetic perturbation of keratin 19.

We used degenerate primers for the amino-and carboxyl-terminal ends of the rod domains of intermediate filament proteins in reverse transcriptase-PCR experiments to identify and clone cytokeratins 8 and 19 (K8 and K19) from cardiac muscle of the adult rat. Northern blots showed that K8 has a 2.2-kb transcript and K19 has a 1.9-kb transcript in both adult cardiac and skeletal muscles. Immunolocalization of the cytokeratins in adult cardiac muscle with isoform-specific antibodies for K8 and K19 showed labeling at Z-lines within the muscle fibers and at Z-line and M-line domains at costameres at the sarcolemmal membrane. Dystrophin and K19 could be co-immunoprecipitated and co-purified from extracts of cardiac muscle, suggesting a link between the cytokeratins and the dystrophin-based cytoskeleton at the sarcolemma. Furthermore, transfection experiments indicate that K8 and K19 may associate with dystrophin through a specific interaction with its actin-binding domain. Consistent with this observation, the cytokeratins are disrupted at the sarcolemmal membrane of skeletal muscle of the mdx mouse that lacks dystrophin. Together these results indicate that at least two cytokeratins are expressed in adult striated muscle, where they may contribute to the organization of both the myoplasm and sarcolemma.

Cytokeratins 8 and 19 concentrate at costameres of striated muscle and copurify with the dystrophin-glycoprotein complex, perhaps through the interaction of the cytokeratins with the actin-binding domain of dystrophin. We overexpressed dystrophin's actin-binding domain (Dys-ABD), K8 and K19, as well as closely related proteins, in COS-7 cells to assess the basis and specificity of their interaction. Dys-ABD alone associated with actin microfilaments. Expressed with K8 and K19, which form filaments, Dys-ABD associated preferentially with the cytokeratins. This interaction was specific, as the homologous ABD of ␤I-spectrin failed to interact with K8/K19 filaments, and Dys-ABD did not associate with desmin or K8/K18 filaments. Studies in COS-7 cells and in vitro showed that Dys-ABD binds directly and specifically to K19. Expressed in muscle fibers in vivo, K19 accumulated in the myoplasm in structures that contained dystrophin and spectrin and disrupted the organization of the sarcolemma. K8 incorporated into sarcomeres, with no effect on the sarcolemma. Our results show that dystrophin interacts through its ABD with K19 specifically and are consistent with the idea that cytokeratins associate with dystrophin at the sarcolemma of striated muscle. INTRODUCTIONMutations in the gene for dystrophin cause Duchenne or Becker Muscular Dystrophy, but we still know little about dystrophin's functions in normal muscle or how its absence leads to the loss of myofibers. Dystrophin is the major cytoskeletal component of a large, transmembrane complex (Ahn and Kunkel, 1993;Matsumura and Campbell, 1994;Ozawa et al., 1998) that plays a role in signaling from the plasma membrane of skeletal myofibers (sarcolemma) to the cytoplasm (Rando, 2001;Lapidos et al., 2004) and in transmitting the force of contraction across the sarcolemma to extracellular structures (Campbell, 1995;Bloch and Gonzalez-Serratos, 2003). Here, we focus on the latter role, and particularly, on the nature of the connections made between the contractile apparatus and the sarcolemma, at sites termed "costameres" (Bloch and Gonzalez-Serratos, 2003;Ervasti, 2003).Costameres are riblike structures that surround each myofiber at the sarcolemma and that are enriched in a number of integral and peripheral membrane proteins (Bloch and Gonzalez-Serratos, 2003;Ervasti, 2003), including dystrophin (Masuda et al., 1992;Minetti et al., 1992;Porter et al., 1992;Straub et al., 1992;Williams and Bloch, 1999a;Rybakova et al., 2000). Costameres and the connections between the contractile apparatus and the sarcolemma that they anchor are present at the plasma membrane overlying both the Z and M lines of superficial myofibrils of fast-twitch muscles (Porter et al., 1992;Williams et al., 2000). They also can be oriented at the plasma membrane parallel to the longitudinal axis of the myofibers, creating a rectilinear, structural lattice at the sarcolemma. Dystrophin and its associated proteins are enriched at all these sites (Porter et al., 1992;Williams and Bloch, 1999a;Williams et al...

Small ankyrin 1, or sAnk1, is a small, alternatively spliced product of the erythroid ankyrin gene, ANK1, that is expressed in striated muscle and concentrated in the network sarcoplasmic reticulum (SR) surrounding the Z disks and M lines. We have characterized sAnk1 in muscle homogenates and SR vesicles, and have identified the region that targets it to the network SR. Selective extractions and partitioning into Triton X-114 show that sAnk1 behaves like the SR Ca-ATPase and so is an integral protein of the SR membrane. Mild proteolytic treatment of isolated SR vesicles indicates that sAnk1 is oriented with its hydrophilic, C-terminal sequence exposed to the solution, which is equivalent to the cytoplasmic face of the SR membrane in situ. SDS-PAGE in non-reducing gels suggests that sAnk1 is present as dimers and larger oligomers in the native SR. These results suggest that sAnk1 is oligomeric and oriented with its C-terminus exposed to the cytoplasm, where it may interact with proteins of the contractile apparatus. The N-terminal 29 amino acid hydrophobic sequence of sAnk1, which is predicted to span the SR membrane, is sufficient to target proteins to and anchor them in internal membranes of HEK 293 cells. It also targets reporter proteins to the network SR of skeletal myofibers and is thus the first example of a sequence that targets proteins to a particular compartment of the SR.

(1-3). However, the mechanism by which Ca 2ϩ pool depletion is coupled to activation of "storeoperated" Ca 2ϩ entry channels remains an important but unsolved question (1-5). Recently, several major channels have been shown to be regulated by thiol nitrosylation, a process becoming recognized as an important NO-mediated post-translational modification effecting control over a diverse array of signaling and regulatory proteins (6 -9). Such S-nitrosylationmediated effects are direct and independent of activation of guanylyl cyclase, which is a major target for NO and a frequent mediator of the actions of NO (10, 11). Studies have revealed that nitrosothiol formation underlies the direct modifying action of NO on a number of important plasma membrane and intracellular channels for Ca 2ϩ and other ions including the N-methyl-D-aspartate receptor (12), cyclic nucleotide-gated cation channel (13,14), Ca 2ϩ -activated K ϩ channel (15), L-type Ca 2ϩ channel (16), and most recently, the ryanodine receptor Ca 2ϩ release channel (17). For several of these channels, NO donor-induced S-nitrosylation results in channel activation, and this activation is mimicked by alkylation of the same thiol groups (13-17). Because of the reactivity of thiols toward NO, the sphere of influence of NO can be highly restricted; hence, rather than being diffusion-dependent, NO (or an equivalent of the nitrosonium ion, NO ϩ ) may be donated and exchanged between neighboring protein thiols by local transnitrosation events (6 -9, 13, 14). Here, we have utilized a combination of membrane-permeant NO donors and alkylators to probe the role of S-nitrosylation in the process of Ca 2ϩ entry and its relationship to Ca 2ϩ pool depletion. EXPERIMENTAL PROCEDURESIntracellular Calcium Measurements-The DDT 1 MF-2 hamster smooth muscle and DC-3F Chinese hamster lung fibroblast lines were cultured as described previously (20,21). Cells grown on coverslips for 1 day were loaded with fura-2/acetoxymethylester as described previously (22, 23). Fluorescence measurements (505 nm emission) are shown as 340/380 nm (excitation) ratios obtained from groups of 10 -12 cells. Details of Ca 2ϩ measurements were recently described for DDT 1 MF-2 (24) and DC-3F cells (21). Resting Ca 2ϩ levels were approximately 60 -90 nM in DDT 1 MF-2 cells and 25-50 nM in DC-3F cells; maximal activation by GEA3162 resulted in up to 600 nM Ca 2ϩ