Cell Surface Targeting and Clustering Interactions between Heterologously Expressed PSD-95 and the Shal Voltage-gated Potassium Channel, Kv4.2

Abstract

Kv4.2 is a voltage-gated potassium channel that is critical in controlling the excitability of myocytes and neurons. Processes that influence trafficking and surface distribution patterns of Kv4.2 will affect its ability to contribute to cellular functions. The scaffolding/clustering protein PSD-95 regulates trafficking and distribution of several receptors and Shaker family Kv channels. We therefore investigated whether the C-terminal valine-serine-alanine-leucine (VSAL) of Kv4.2 is a novel binding motif for PSD-95. By using co-immunoprecipitation assays, we determined that full-length Kv4.2 and PSD-95 interact when co-expressed in mammalian cell lines. Mutation analysis in this heterologous expression system showed that the VSAL motif of Kv4.2 is necessary for PSD-95 binding. PSD-95 increased the surface expression of Kv4.2 protein and caused it to cluster, as shown by deconvolution microscopy and biotinylation assays. Deleting the C-terminal VSAL motif of Kv4.2 eliminated these effects, as did substituting a palmitoylation-deficient PSD-95 mutant. In addition to these effects of PSD-95 on Kv4.2 distribution, the channel itself promoted redistribution of PSD-95 to the cell surface in the heterologous expression system. This work represents the first evidence that a member of the Shal subfamily of Kv channels can bind to PSD-95, with functional consequences.The influence that voltage-gated ion channels exert on the integrative physiology of excitable cells is determined by the inherent biophysical properties of the channels and their cell surface distribution. Kv4.2 is a fast transient (A-type) voltagegated potassium (Kv) 1 channel of the Shal subfamily that is found in heart and neurons (1). In myocytes, Kv4.2 is a major component of the I to current, which is crucial for recovery from the QT interval and for repolarization (2). In neurons of the central nervous system, Kv4.2 is expressed primarily somatodendritically (3, 4), where its concentration at postsynaptic sites may affect the back propagation of action potentials (5) and may therefore modulate long term potentiation at synapses (6). In contrast, Kv1.4, a Kv channel of the Shaker subfamily, is primarily expressed on axons (3, 4). Its positioning along the axon and at presynaptic sites (7-9) is thought to regulate the efficiency of action potential propagation (10) and to control neurotransmitter release (11). It is therefore crucial to understand the basis for the specialized distributions of Kv channels in order to fully understand their roles in neuronal excitability. The mechanisms that specify ion channel distributions are not well known and likely differ between those that determine localization in sub-domains of cells (e.g. axon, soma, and dendrite) versus lateral organization at the membrane, such as within channel clusters. Post-synaptic density 95 (PSD-95) is a membrane-associated guanylate kinase that helps localize and cluster numerous proteins. Kv1.4 and other Kv1 members are among the best known examples of in vitro ion channel clusteri...

An Alternatively Spliced Isoform of PSD-93/Chapsyn 110 Binds to the Inwardly Rectifying Potassium Channel, Kir2.1

“…This is demonstrated by the fact that whole-cell Kir2.1 currents recorded in the presence of PSD-93␦ were indistinguishable from those recorded in its absence. Similar results have been obtained for Kv1.4 and Kv4.2 (33,43) and suggest that pathways involved in the insertion or removal of K ϩ channels into or out of the plasma membrane may be more tightly regulated and synchronized than previously thought.…”

“…A role for MAGUKs in the regulation of channel and receptor surface expression is supported by the finding that deletion of the C-terminal PDZ binding motif of NR2B subunits increases the rate of NMDAR internalization in neurons (34), and by a recent study that shows a reduction of surface NR2A and NR2B expression in spinal dorsal horn neurons of PSD-93 knockout mice (39). The rate of internalization of Kv1.4 and Kv4.2 channels is also reduced when coexpressed with PSD-95 (33,43), and at least in the case of Kv1.4, surface stabilization seems to intimately linked with channel clustering, since PSD-95 mutants that bind but do not cluster Kv1.4 allow rapid channel endocytosis (33). Interestingly, whereas PSD-93␦ suppresses the rate of Kir2.1 internalization, it does not seem to affect the overall level of channel expression at the cell surface.…”

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

“…In contrast, the I2 insert fails to address SAP97 to the membrane, leading to SAP97 accumulation in the cytosol or nucleus (10,29). The subsarcolemmal localization of this SAP97 isoform containing the I2 insert may depend more on protein partners such as protein 4.1 (11) or Kv channels, as described for the membrane redistribution of PSD95 by Kv4.2 Shal channels (30). One consequence of the poor capacity of SAP97 lacking the I3 domain to be correctly addressed to the membrane could be also the formation of aggresomes of misfolded proteins (15,31).…”

Structure and Function of Kv4-Family Transient Potassium Channels

“…Another synapse-associated protein found to interact with Kv4.2 is the scaffolding protein PSD-95 (216). Using a surface biotinylation assay, Wong et al (216) showed that coexpression of Kv4.2 with PSD-95 in CHO cells enhanced the surface expression of Kv4.2 ϳ2-fold.…”