Clustering and Enhanced Activity of an Inwardly Rectifying Potassium Channel, Kir4.1, by an Anchoring Protein, PSD-95/SAP90

Abstract

An inwardly rectifying potassium channel predominantly expressed in glial cells, Kir4.1/K AB -2, has a sequence of Ser-Asn-Val in its carboxyl-terminal end, suggesting a possible interaction with an anchoring protein of the PSD-95 family. We examined the effects of PSD-95 on the distribution and function of Kir4.1 in a mammalian cell line. When Kir4.1 was expressed alone, the channel immunoreactivity was distributed homogeneously. In contrast, when co-expressed with PSD-95, prominent clustering of Kir4.1 in the cell membrane occurred. Kir4.1 was co-immunoprecipitated with PSD-95 in the co-expressed cells. Glutathione S-transferase-fusion protein of COOH terminus of Kir4.1 bound to PSD-95. These interactions disappeared when the SerAsn-Val motif was deleted. The magnitude of whole-cell Kir4.1 current was increased by 2-fold in cells co-expressing Kir4.1 and PSD-95 compared with cells expressing Kir4.1 alone. SAP97, another member of the PSD-95 family, showed similar effects on Kir4.1. Furthermore, we found that Kir4.1 as well as SAP97 distributed not diffusely but clustered in retinal glial cells. Therefore, PSD-95 family proteins may be a physiological regulator of the distribution and function of Kir4.1 in glial cells.Distribution of ion channels in specialized areas of cell membrane is essential for various cell functions: for example, voltage-gated Na ϩ and K ϩ channels are concentrated at nodes of Ranvier for saltatory conduction in myelinated nerves (1). At the neuromuscular junction, the nicotinic acetylcholine receptor channels are concentrated at the postsynaptic membrane to initiate excitation-contraction coupling in skeletal muscle (2). Some molecular mechanisms responsible for the specific distribution of channel proteins have been characterized, e.g. agrin and rapsyn of the dystroglycan complex are involved in accumulation and clustering of nicotinic acetylcholine receptors (2). However, different molecular mechanisms may be responsible for clustering different channels. Recent studies using yeast two-hybrid method have revealed that clustering of voltagegated K ϩ (3) and NMDA 1 channels (4), was mediated by PSD-95 family proteins. PSD-95/SAP90 (5, 6) belongs to the membrane-associated anchoring proteins and is characterized with three PDZ domains in its amino terminus, an SH3 domain, and a carboxyl-terminal guanylate kinase homology domain. So far several members of the PSD-95 family have been cloned (7-9). The PDZ domains of PSD-95 and the Thr/Ser-XVal motifs of the COOH termini of voltage-gated K ϩ and NMDA channels are supposed to interact for aggregation. Although co-localization of NMDA receptors and PSD-95 in cultured hippocampal neurons may indicate that these two proteins interact with each other in vivo (4), a physiological role for this interaction has not been fully elucidated.Inwardly rectifying K ϩ channels comprise a family with more than ten members (10). These K ϩ channels play a pivotal role in determining resting membrane potential, in regulating action potential duration, and in t...

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

“…There are already some reports on the consequences on potassium currents of the interaction between Kv channels and the proteins SAP97 and PSD95 (2,4,(17)(18)(19). However, the mechanisms underlying this effect are poorly understood.…”

Differential Assembly of Inwardly Rectifying K+ Channel Subunits, Kir4.1 and Kir5.1, in Brain Astrocytes

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Direct Interaction between the Actin-binding Protein Filamin-A and the Inwardly Rectifying Potassium Channel, Kir2.1

“…Members of the Kir2 subfamily of inward rectifying potassium channels have previously been shown to interact with the PDZ domains of selected members of the membrane-associated guanylate kinase protein family (31)(32)(33)(34). Interaction with membrane-associated guanylate kinases is believed to facilitate the subcellular targeting of ion channels and the formation of functional ion channel signaling complexes.…”