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 b...

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Veröffentlicht in:The Journal of biological chemistry 2003-11, Vol.278 (47), p.47046-47052
Hauptverfasser: Godreau, David, Vranckx, Roger, Maguy, Ange, Goyenvalle, Catherine, Hatem, Stéphane N.
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container_end_page 47052
container_issue 47
container_start_page 47046
container_title The Journal of biological chemistry
container_volume 278
creator Godreau, David
Vranckx, Roger
Maguy, Ange
Goyenvalle, Catherine
Hatem, Stéphane N.
description 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.
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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. 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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. 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subjects Adaptor Proteins, Signal Transducing
Animals
Cloning, Molecular
Discs Large Homolog 1 Protein
Electrophysiology
Heart Atria - chemistry
Humans
Kv1.5 Potassium Channel
Membrane Proteins
Myocardium - chemistry
Myocardium - metabolism
Nerve Tissue Proteins - analysis
Nerve Tissue Proteins - biosynthesis
Nerve Tissue Proteins - physiology
Potassium Channels, Voltage-Gated - chemistry
Potassium Channels, Voltage-Gated - metabolism
Protein Binding
Protein Isoforms - analysis
Protein Isoforms - biosynthesis
Protein Isoforms - metabolism
Protein Isoforms - physiology
Rats
Rats, Wistar
Transfection
title Different Isoforms of Synapse-associated Protein, SAP97, Are Expressed in the Heart and Have Distinct Effects on the Voltage-gated K+ Channel Kv1.5
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