title: The carboxyl-terminal region of ahnak provides a link between cardiac L-type Ca2+ channels and the actin-based cytoskeleton.
abstract: Ahnak is a ubiquitously expressed giant protein of 5643 amino acids implicated in cell differentiation and signal transduction. In a recent study, we demonstrated the association of ahnak with the regulatory beta2 subunit of the cardiac L-type Ca2+ channel. Here we identify the most carboxyl-terminal ahnak region (aa 5262-5643) to interact with recombinant beta2a as well as with beta2 and beta1a isoforms of native muscle Ca2+ channels using a panel of GST fusion proteins. Equilibrium sedimentation analysis revealed Kd values of 55 +/- 11 nM and 328 +/- 24 nM for carboxyl-terminal (aa 195-606) and amino-terminal (aa 1-200) truncates of the beta2a subunit, respectively. The same carboxyl-terminal ahnak region (aa 5262-5643) bound to G-actin and cosedimented with F-actin. Confocal microscopy of human left ventricular tissue localized the carboxyl-terminal ahnak portion to the sarcolemma including the T-tubular system and the intercalated disks of cardiomyocytes. These results suggest that ahnak provides a structural basis for the subsarcolemmal cytoarchitecture and confers the regulatory role of the actin-based cytoskeleton to the L-type Ca2+ channel.
authors.affiliation: Max Delbrück Center for Molecular Medicine, 13092 Berlin, Germany.
authors.forename: Annette
authors.initials: A
authors.lastname: Hohaus
authors.forename: Veronika
authors.initials: V
authors.lastname: Person
authors.forename: Joachim
authors.initials: J
authors.lastname: Behlke
authors.forename: Jutta
authors.initials: J
authors.lastname: Schaper
authors.forename: Ingo
authors.initials: I
authors.lastname: Morano
authors.forename: Hannelore
authors.initials: H
authors.lastname: Haase
mesh_terms: D008841:Actin Cytoskeleton / Q000378:metabolism*; D000199:Actins / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D020746:Calcium Channels, L-Type / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008954:Models, Biological; D018482:Muscle, Skeletal; D009206:Myocardium / Q000737:chemistry / Q000378:metabolism*; D009363:Neoplasm Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012508:Sarcolemma / Q000737:chemistry; D017384:Sequence Deletion; D013552:Swine
chemical_list: C075176:AHNAK protein, human; D000199:Actins; D020746:Calcium Channels, L-Type; D008565:Membrane Proteins; D009363:Neoplasm Proteins; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins