The carboxyl‐terminal region of ahnak provides a link between cardiac L‐type Ca²⁺channels and the actinbased 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.

Impaired functional communication between the L-type calcium channel and mitochondria contributes to metabolic inhibition in the mdx heart

“…Consistent with this argument and with previous reports, we find that immobilizing the beta subunit of I Ca-L by exposing wt myocytes to a peptide derived against the alpha-interacting domain (AID) of I Ca-L slows inactivation of the current (AID-TAT; Fig. 1 A, Inset) (6,9,10). Because the auxiliary beta subunit regulates inactivation of I Ca-L current (14,15), we propose that the alterations in I Ca-L inactivation in the mdx myocyte result from disruption of the cytoskeleton due to the absence of dystrophin.…”

Expression of the Giant Protein AHNAK (Desmoyokin) in Muscle and Lining Epithelial Cells

“…Cytoplasmic staining was also observed in fibroblast or certain epithelial cells, such as those in the colon ( Figure 5F). Exclusive membrane location of AHNAK has also been recently reported in rat cardiomyocytes by Hohaus et al (2002), using two region-specific AHNAK antibodies raised against either amino-terminal or carboxylterminal epitopes. These findings contrast with the predominant nuclear localization of AHNAK observed in cultured cells lines of nonepithelial origin (Shtivelman and Bishop 1993).…”

“…In the cardiomyocyte, AHNAK modulates membrane-bound L-type calcium channel activity (Haase et al 1999). Intriguingly, organization of the cortical actin network has been implicated in modulation of voltage-gated ion channel activity (Maguire et al 1998), and in vitro interaction between the carboxyl-terminal region of AHNAK and actin has recently been described (Hohaus et al 2002). Peptides from the central domain of AHNAK also bind and activate PLC ␥ in the presence of arachidonic acid (Sekiya et al 1999).…”

AHNAK-mediated Activation of Phospholipase C-γ1 through Protein Kinase C

“…The division of AHNAK into N (amino acid residues 1-257), C1 (amino acid residues 4640 -5386), and C2 (amino acid residues 5258 -5643) domains was based on a previously published report (4). The cDNA fragments were amplified by PCR and inserted into the pcDNA3-HA vector by digestion with EcoRI and XhoI.…”

“…The carboxyl-terminal region of AHNAK proteins was reported to play an important role in cellular localization and in interaction with L-type Ca 2ϩ channels in cardiac cells (4) and with the calcium-binding S100B protein in rat embryo fibroblast cells (5). In low calcium concentrations, AHNAK proteins are mainly localized in the nucleus, but the increase in intracellular calcium levels leads the protein to translocate to plasma membrane (3).…”