J Physiol 587.13 (2009) pp 3123–3124 3123

PERSPECT IVES

Triadin, not essential, but useful

Paul D. AllenDepartment of Anesthesia, Perioperativeand Pain Medicine, Brigham and Women’sHospital, 75 Francis Street, Boston MA 02115,USA

Email: allen@zeus.bwh.harvard.edu

As presented in the review article byMarty et al. (2009), triadins are a familyof proteins that are derived from tissuespecific alternatively spliced products of asingle gene. All triadins share a commonamino terminus and differ primarily inthe C-terminal regions. What are triadinssupposed to do?

The predominant isoforms of triadin areco-localized with RyR and calsequestrin,using immunohistochemistry and canbe immuno-precipitated from heartand skeletal muscle with Anti-RyR andAnti-Casq antibodies (Jones et al. 1995;Zhang et al. 1997; Groh et al. 1999). Becauseof its ability to be immunoprecipitated withcalsequestrin, triadin has been suggestedby many studies to function as a proteinthat binds to calsequestrin to bring it tothe mouth of the calcium release channeland that binds to the RyRs themselves tomodulate their gating. In skeletal muscle keystructural aspects that are involved in bothassociations and triadin’s RyR1 bindingsite have been defined and the motifs intriadin were shown to be the same in thepredominant isoforms expressed both inskeletal muscle (Trisk95) and in heart(CT1) (Kobayashi & Jones, 1999). A secondcalsequestrin binding protein, junctin,was found to be expressed in both skeletalmuscle and heart in addition to triadin andtogether with triadin, calsequestrin and theRyR makes up a quartenerary complex thatwas thought to be essential for proper ECcoupling. Additional isoforms of triadin(Trisk 51, Trisk49 and Trisk32) have beenidentified in skeletal muscle by one group(Marty et al. 2000; Vassilopoulos et al.2005). Despite evidence of the expressionof three mRNA isoforms in the heart, thepredominant isoform in heart is CT1, andthere are also traces of CT3 (Kobayashi &Jones, 1999).

It is important to note that there is somecontroversy associated with the expressionof the additional isoforms in skeletal musclerelated to the antibodies used for their

detection. Trisk 95, 51 and 32 have beenidentified in rat and rabbit skeletal musclewith an N-terminal antibody raised to asequence common to all triadin isoforms.Trisk 49, which shares this epitope with theother three isoforms, could not be detectedwith the N-terminal antibody but has onlybeen detected with an isoform specificantibody raised to a sequence in a non-common region (Marty et al. 2000;Vassilopoulos et al. 2005). In earlierpublications using antibodies that arecommon to all triadins in the N-terminaland luminal regions, only Trisk 95 anda band at ∼66 kDa were detected (Joneset al. 1995; Zhang et al. 1997; Shen et al.2007). Based on comparison of figures,the smaller isoform appears to have thesame mobility as Trisk 51. However, the32/44 kDa pair was only detected in heartwith these antibodies and no smallerisoform was found in skeletal muscle. Eventhough the differences in isoforms couldbe species and/or developmentally specific,the fact that both groups used adults andsignificant species overlap doesn’t resolvethe controversy.

Early studies of triadin function werebased on the effects of acute over-expression of triadin in myotubes andheart (Zhang et al. 2001; Kirchhefer et al.2003; Tijskens et al. 2003; Kirchhefer et al.2004; Rezgui et al. 2005). In this modelEC coupling was almost abolished inresponse to depolarization in the absence ofextracellular Ca2+ while the direct responseof RyRs to caffeine and DHPR currentswere maintained. This suggested that triadinwas essential for skeletal-type EC coupling.The consequences of mutating the putativeRyR1 binding site(s) (D4878A, D4907Aor E4908A) that were identified earlieras binding to the KEKE motif of triadinare associated with discordant results (Leeet al. 2006; Goonasekera et al. 2007).In the study of Lee et al., mutation ofall three amino acids partially disruptedco-immunoprecipitation of triadin by RyR1in a RyR1-null cell line and variably(25–100%) inhibited caffeine induced Ca2+

release in myotubes, but not in HEKcells, where no triadin is expressed. Whensingle mutations were studied individuallyD4907A had the largest effect. The results ofthe study of Goonasekera et al. expressingthe mutated RyRs in RyR1−null primarymyotubes agreed that D4907A had thelargest effect and that caffeine induced

Ca2+ release was depressed in myotubesbut not HEK cells. However they foundthat either the triple mutant or doublemutants containing the D4907A mutationabolished any co-immunoprecipitation oftriadin with RyR1. They also showed thatexpression of selected mutants abolishedEC coupling in response to electricalstimulation and amplified the reduction ofCa2+ transients caused by Cd2+/La3+ orryanodine pretreatment in voltage clampexperiments. Despite inconsistencies, bothconcluded that triadin binding to the secondintracellular loop of RyR1 was importantfor EC coupling. However, the fact thatEC coupling was suppressed while directactivation of RyR1 was not could be dueto several factors including (1) the needfor triadin/RyR1 interactions to support ECcoupling in the presence of triadin, (2)that the mutations cause conformationalchanges in RyR1 that directly block passageof the orthograde signal from the DHPRpreventing channel opening irrespectiveof triadin, and (3) perhaps that withoutbinding to RyR1 there is a relative over-expression of triadin.

Based on these studies one mightexpect that the triadin KO mouse wouldvery likely produce an embryonic lethalphenotype in homozygous mice due toan early cardiac death and a birth lethalphenotype if a skeletal muscle specificKO could be accomplished. We foundneither phenotype. Moreover homozygoustriadin KO mice lived, grew and reproducednormally (Shen et al. 2007). Western blotanalysis of sarcoplasmic reticulum (SR)proteins in skeletal muscle showed thatthe absence of triadin expression wasassociated with a small down-regulation ofJunctophilin-1, junctin, and calsequestrinin fast twitch muscles but resulted invery modest contractile dysfunction.Ca2+ imaging studies in null lumbricalismuscles and myotubes showed that theabsence of triadin did not prevent skeletaltype excitation–contraction coupling andonly slightly reduced the Ca2+ transientamplitude in response to electricalstimulation and reduced the amplitudebut not the characteristics of the caffeineresponse. This decrease was attributedto a modest decrease in Ca2+ stores.[3H]Ryanodine binding studies of skeletalmuscle SR vesicles detected no differencesin Ca2+ activation or Ca2+ and Mg2+

inhibition of binding between wild-typeC© 2009 The Author. Journal compilation C© 2009 The Physiological Society DOI: 10.1113/jphysiol.2009.172015

3124 Perspectives J Physiol 587.13

and triadin- null animals but Bmax wassignificantly higher in null vesicles. Thelatter could be the result of the factthat null myotubes and adult fibres hadsignificantly increased myoplasmic restingfree Ca2+ as measured directly withmicroelectrodes. Subtle ultrastructuralchanges, evidenced by the appearance ofrare longitudinally oriented triads and theoccasional presence of calsequestrin in thesacs of the longitudinal SR, were presentin fast but not slow twitch-null muscles.These data support an indirect role fortriadin in regulating myoplasmic Ca2+

homeostasis and a minor role in organizingthe molecular complex of the triad but notin regulating skeletal-type EC coupling.Two studies using shRNA knockdownof triadin in C2C12 cells confirm thatsuppression of triadin expression hassmall effects on the amplitude of the Ca2+

release in response to KCl depolarizationor caffeine (Fodor et al. 2008; Wanget al. 2009). One shRNA study foundthat the mechanism for this change was areduced Ca2+ store, the other did not. Bothdemonstrated that skeletal EC coupling wasintact and one confirmed the increase inresting free Ca2+ previously observed intriadin-null myotubes and muscles (Wanget al. 2009). The frequency of localizedCa2+ release events was increased withsuppression of triadin expression and theamplitude of both sparks and embers waslarger.

Triadin overexpression clearly causesa detriment in Ca2+ homeostasis witha significant decrease in EC couplingefficiency, especially in the presence ofreduced extracellular Ca2+. It has beenrecently shown by the Marty lab that over-expression of triadin in Cos7 cells causes achange in the architecture of the ER and amodification of the microtubules that is verysimilar to overexpression of an anchoringprotein (see accompanying paper). Thismight suggest that one of the roles of triadinmay be that of an anchoring protein. Whilemodification in SR architecture may bethe explanation for the deficits seen whentriadin is overexpressed in skeletal myotubesand may also serve as a possible mechanismfor the absence of EC coupling when RyR1’striadin binding sites are mutated, thisremains to be demonstrated. It is unlikely,however, that this is triadin’s primary rolebecause although there was remodelling ofthe architecture of the SR in triadin-nullanimals, this was a rare event, and wascompletely absent in slow fibres.

What remains to be determined is whattriadin really does. Our recent unpublisheddata in null animals in vivo and null-EDLmuscles in vitro confirm that triadin isnot essential for EC coupling and suggestthat SR stores in triadin−null animalsmay be smaller and are depleted morerapidly than in WT muscles, which maybe related to the fact that the rate ofCa2+ entry after store depletion is slower(Vassilopoulos et al. 2007). Together thesestudies demonstrate that triadin is requiredto maintain normal intracellular Ca2+

homeostasis when the muscle is stressedand suggest that the presence of triadin maybe needed for proper mobilization of theSR Ca2+ sensor Stim1. The mechanism forhow over-expression disrupts EC couplingremains to be determined but may be theresult of a disruption of normal SR andmicrotubule architecture (see Marty et al.2009).

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C© 2009 The Author. Journal compilation C© 2009 The Physiological Society