[MAGUKs: beyond ionic channel anchoring]. / Les MAGUK: au-delà de l'accrochage des canaux ioniques.

A family of anchoring proteins named MAGUK (for membrane associated guanylate kinase) has emerged as a key element in the organization of protein complexes in specialized membrane regions. These proteins are characterized by the presence of multipe protein-protein interaction domains including PDZ and SH3 domains. The MAGUK family comprises the post-synaptic density 95 (PSD-95) protein and closely related molecules such as chapsyn-110, synapse-associated protein 102 (SAP-102), and SAP-97. These are located either on the pre- and/or post-synaptic sides of synapses or at cell-cell adhesion sites of epithelial cells. MAGUK proteins interact with glutamate receptors and various ionic channels. For instance, an interaction has been reported between the first two PDZ domains of MAGUK proteins and several channels via a consensus sequence Thr/Ser-X-Val/Leu usually located at their carboxy terminus. The role of these anchoring proteins in channel function is not fully understood. MAGUK proteins enhance the current density by increasing the number of functional channels to the sarcolemma. They can also facilitate signaling between channels and several enzymes or G protein-dependent signaling pathways. In the heart also, MAGUK proteins are abundantly expressed and they interact with various channels including Shaker Kv1.5 and connexins.

Different isoforms of synapse-associated protein, SAP97, are expressed in the heart and have distinct effects on the voltage-gated K+ channel Kv1.5.

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.

Expression, regulation and role of the MAGUK protein SAP-97 in human atrial myocardium.

OBJECTIVE: In various cell types, membrane-associated guanylate kinases proteins called MAGUK play a major role in the spatial localization and clustering of ion channels. Here, we studied the expression and role of these anchoring proteins in human right atrial myocardium by means of various molecular, biochemical and physiological methods. METHODS AND RESULTS: SAP-97, PSD-95, Chapsyn and SAP-102 messengers were detected by reverse transcriptase-polymerase chain reaction (RT-PCR) on mRNA extracted from both whole myocardium and isolated myocytes. Western blot revealed that the MAGUK protein SAP-97 and, to a lesser extent, PSD-95, is abundantly expressed in human atrial myocardium, while Chapsyn are almost undetectable. Confocal microscopic visualization of cryosection of atrial myocardium stained with the anti-PSD-95 family antibody showed positive staining at the plasma membrane level and cell extremity. Calpain-I cleaved both SAP-97 and PSD-95 proteins, resulting in an accumulation of short bands, including an 80-kDa band that was also detected in the cytosolic protein fraction. Immunoprecipitation of SAP-97 co-precipitated hKv1.5 channels, and vice versa. Co-expression of cloned SAP-97 and hKv1.5 channels in Chinese hamster ovarian (CHO) cells increased the K(+) current (157.00+/-19.45 pA/pF vs. 344.50+/-58.58 pA/pF at +50 mV). CONCLUSIONS: The protein SAP-97 is abundantly expressed in human atrial myocardium in association with hKv1.5 channels, and probably contributes to regulating the functional expression of the latter.

Dedifferentiation of atrial myocytes during atrial fibrillation: role of fibroblast proliferation in vitro.

OBJECTIVES: Severe myocyte alterations, characterized by enlarged myocytes and myolysis, is observed in fibrillating and dilated atria and contributes to atrial fibrillation. The aim of this study was to determine the nature of this cellular remodeling process and factors involved in its regulation. METHODS: In vivo, contractile proteins were studied in 24 human right atrial specimens by means of immunohistochemical techniques. In an attempt to reproduce in vitro the myocyte remodeling and to study its regulation, human atrial myocytes were cultured (n=27) and analyzed immunocytochemically; intracellular Ca(2+) transients (Ca(i)-tr) in response to electrical stimulation were monitored using Fura-2/AM. RESULTS: In diseased specimens, sarcomeres, seen at the periphery of myolytic myocytes, stained positively with antibodies against sarcomeric proteins of the Z-band (alpha-actinin and titin epitope T12) but not with antibodies against titin epitope T11 (I-band) or desmin (intermediate filament). beta-myosin heavy chain (MHC) and smooth muscle alpha-actin, two proteins of the fetal program, were re-expressed. In culture, diseased myocytes also showed myolysis and glycogen accumulation; their sarcomeres stained positively with anti-alpha-actinin, anti-T12, anti-beta-MHC and anti-smooth muscle alpha-actin but not with anti-titin T11 or anti-desmin antibodies. At confluence, myocytes regained a normal sarcomeric apparatus and were excitable, as shown by electrical Ca(i)-tr triggering. This redifferentiation process was inhibited by fibroblast proliferation. CONCLUSION: In diseased atria, myolytic myocytes are in a dedifferentiated state resembling that of immature muscle cells. In vitro, fibroblast proliferation prevents the reversibility of this cellular alteration.

Mechanisms of action of antiarrhythmic agent bertosamil on hKv1.5 channels and outward potassium current in human atrial myocytes.

We analyzed the mechanism of action of the antiarrhythmic agent bertosamil on hKv1.5 channels expressed in Chinese hamster ovary cells (I(hKv1.5)) and on the outward current (I(o)) of human atrial myocytes (HAMs) by using the whole cell patch-clamp technique to record current. External application of 10 microM bertosamil inhibited I(hKv1.5), accelerated its time-dependent decay, and slowed its deactivation. When bertosamil was applied at rest or intracellularly (50 microM), it accelerated the rate of I(hKv1.5) inactivation without change of the peak amplitude. At the steady-state effect of intracellular bertosamil, external drug application only inhibited I(hKv1.5). When cesium was the charge carrier, bertosamil inhibited I(hKv1.5) but had no effect on its time course. Intracellular tetraethylammonium inhibited I(hKv1.5), suppressed its inactivation, and prevented bertosamil effects. Bertosamil-treated I(hKv1.5) became highly sensitive to the rate of membrane stimulation and to cumulative inactivation phenomenon. In HAMs, bertosamil also increased the rate and extent of I(o) inactivation and slowed its recovery from inactivation, whereas after drug application I(o) became highly sensitive to cumulative inactivation phenomenon. In conclusion, bertosamil 1) causes a use-dependent inhibition of the current upon external drug application, and 2) accelerates the rate of current inactivation when applied at rest or intracellularly. These effects result from both an open-channel block and acceleration of the rate of channel inactivation and contribute to the modulation by bertosamil of I(o) in HAM.