Different Isoforms of Synapse-associated Protein, SAP97, Are Expressed in the Heart and Have Distinct Effects on the Voltage-gated K+ Channel Kv1.5

Abstract

The SAP97 isoforms differ by alternatively spliced insertion domains that regulate protein localization and oligomerization. We used reverse transcription-PCR to identify SAP97 isoforms of human and rat myocardium. In Chinese hamster ovary cells, cloned protein expression was studied using Western blot, confocal imaging of green fluorescent protein-tagged proteins, and patch clamp technique. The two main cardiac SAP97 isoforms contained both I3 and I1B inserts and differed by the I1A insert. Both isoforms co-precipitated with hKv1.5 channels. Only the isoform lacking I1A increased the current (by 215 ؎ 22%), whatever the level of channel expression. To examine the involvement of the proline-rich I1A insert in the effect of SAP97, a W623F mutation in the Src homology 3 domain was created, and that restored the effect of the SAP97 on current. SAP97 isoform containing an I1A and I2 domain instead of the I3 domain stimulated the current, whereas SAP97 after deletion of the Src homology 3 and guanylate kinase-like domains did not. In cells co-expressing I3(؉I1A) or I3(؊I1A), green fluorescent protein-tagged Kv1.5 channels were organized in plaque-like structures at the plasma membrane level, whereas intracellular aggregates of channels predominated with the I2 isoform. The two cardiac SAP97 isoforms have different effects on the hKv1.5 current, depending on their capacity to form channel clusters.Localization of ionic channels in distinct plasma membrane domains is critical for cell function. For instance, in the myocardium, a number of ionic channels are concentrated in the intercalated disk, where they contribute to the normal propagation of the electrical influx between myocytes. The functional properties of ionic channels are generally regulated by various auxiliary proteins that compose, together with the ␣-subunits of the channels, large protein networks.The mechanisms that regulate this highly specialized organization and localization of ionic channels are unclear. Important clues have come from the identification of a family of anchoring proteins named MAGUK (membrane-associated guanylate kinases) that appear to play a critical role in the formation and subcellular localization of channel complexes (1).The MAGUK protein SAP97 (the mammalian homologue of Dlg in Drosophila) is abundantly expressed in both human and rat ventricular myocardium and is associated with potassium Kir2.2 and Kv1.5 channels (2-4). Like other MAGUK proteins, SAP97 bears multiple sites of protein-protein interactions, namely three PDZ (postsynaptic, disc large, zonula occludens) domains, an Src homology 3 (SH3) 1 region, and a guanylate kinase-like domain (GUK). The PDZ domains are the best characterized and bind to the carboxyl-terminal peptide motif (S/T)X(V/L) in a number of proteins, including voltage-gated and inwardly rectifying K ϩ channels (1). It has been reported that the SH3 domain interacts with PXXPR-like sequences in several proteins, whereas the partners of GUK domain are members of the GKAP/SAPAP1/DAP1 family or brain-enr...

Dynamic of Ion Channel Expression at the Plasma Membrane of Cardiomyocytes

“…Indeed, Kv1.5 current is not enhanced by the cardiac SAP97 isoform containing the I1A domain, which prevents protein oligomerization (145,199). Oligomerization of SAP97 is regulated by inter-and intramolecular interactions (302), which determines the conformation of SAP97: compact "closed" state (preventing binding of ligands to the SH3 and GUK domains) or "open" state, allowing access to the protein binding sites (456).…”

“…In the heart, two SAP97 isoforms are expressed, both containing the alternatively spliced insertions I3 and I1B, but differing in the presence or absence of I1A (145). SAP97 contains different combinations of alternatively spliced insertions that regulate the subcellular localization of the protein and its capacity to homo-or heterodimerize (247,270).…”

SAP97 and Dystrophin Macromolecular Complexes Determine Two Pools of Cardiac Sodium Channels Na v 1.5 in Cardiomyocytes

“…9,10 SAP97 regulates the targeting and localization of cardiac potassium channels, such as Kir2.x 11 and K v 1.5 in myocytes. 10 El-Haou et al 12 recently demonstrated that SAP97 interacts with K v 4.2 and K v 4.3 channels via their PDZ-domain binding motif. They showed that suppression of SAP97 expression in rat myocytes decreases the K v 4.x-mediated current, whereas its overexpression increases it.…”

Discs Large 1 (Dlg1) Scaffolding Protein Participates with Clathrin and Adaptator Protein Complex 1 (AP-1) in Forming Weibel-Palade Bodies of Endothelial Cells

“…As already shown in other cell types derived from the cardiovascular system, i.e. smooth muscle cells and cardiomyocytes, the larger and the shorter isoforms of Dlg1 differ, in endothelial cells, by an N-terminal insertion of a small proline-rich sequence named I1A that is due to the presence of a site of alternative splicing in the mRNA (data not shown) (35,36). In the larger form, an SH3 binding site is thus present that may bind the SH3 domain of a number of protein tyrosine kinases or of Dlg1 itself in an intra-or an extramolecular way that favors a "closed state" or the homomultimerization of the protein, respectively (37).…”