A role for frequenin, a Ca 2+ -binding protein, as a regulator of Kv4 K + -currents

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...

Section: Methodsmentioning

confidence: 82%

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

Wong¹,

Newell²,

Jugloff³

et al. 2002

“…9), analogous to the role of CaM in promoting Ca 2ϩ -induced closure of voltage-gated Ca 2ϩ channels (61,62). Ca 2ϩ -induced regulation of ion channels is also performed by various NCS proteins, including KChIPs (42), NCS-1 (44,70), and CaBP1 (71,72). In this study, we provide evidence that Ncs1p promotes Ca external conditions to help maintain viable cytosolic Ca 2ϩ levels and prevent cell death.…”

Section: Discussionmentioning

confidence: 99%

Neuronal Calcium Sensor-1 (Ncs1p) Is Up-regulated by Calcineurin to Promote Ca2+ Tolerance in Fission Yeast

Hamasaki-Katagiri

Ames

2010

) regulates signaling processes in the fission yeast, Schizosaccharomyces pombe (reviewed in Refs. 1-3), as it does in mammalian cells (reviewed in Ref. 4). Fission yeast contains ion channels (5, 6) and transporters (7-9) homologous to those involved in regulating Ca 2ϩ entry and signal transduction in excitable cells. Thus, stimulation of yeast cells by pheromones or other extracellular stimuli lead to an increase in intracellular Ca 2ϩ levels (10, 11) sensed by Ca 2ϩ -binding proteins belonging to the EF-hand superfamily (12). In S. pombe, a total of 23 genes specifies EF-hand-containing Ca 2ϩ -binding proteins, including cam1 (calmodulin) (13), cnb1 (calcineurin B) (14), and the recently characterized ncs1 gene (Ncs1p) (15), belonging to the neuronal calcium sensor (NCS) 2 family (16) expressed mostly in the central nervous system (17, 18).The primary sequence of S. pombe Ncs1p is Ͼ50% identical to neuronal homologs of the NCS branch of the EF-hand superfamily (19,20). Recoverin, the most intensively studied NCS protein, serves as a Ca 2ϩ sensor in retinal rod and cone cells where it controls the desensitization of rhodopsin (21-24). The NCS family also includes neuronal Ca 2ϩ sensors such as neurocalcin (25), hippocalcin (26), and frequenin (27), as well as the budding yeast homolog, Frq1 (28). All members of the NCS family have around 200 amino acid residues, contain N-terminal myristoylation, and possess four EF-hands.Three-dimensional structures are now known for many NCS proteins, including recoverin (29, 30), frequenin (31), Frq1 (32), neurocalcin (33), and guanylate cyclase activator proteins (34, 35). The Ca 2ϩ -bound forms of these proteins all share a common fold with four EF-hands arranged in a tandem array, with an exposed N-terminal myristoyl group. Binding of Ca 2ϩ to recoverin leads to extrusion of its myristoyl group such that many hydrophobic residues become exposed for target recognition (30, 36). The Ca 2ϩ -induced exposure of the myristoyl group, termed the Ca 2ϩ -myristoyl switch, enables recoverin to bind to membrane targets only at high Ca 2ϩ levels (37, 38). NCS proteins interact with and regulate many different physiological targets. Recoverin binds specifically and regulates rhodopsin kinase (23), whereas the guanylate cyclase activator proteins specifically regulate retinal guanylate cyclases (39, 40). NCS-1 and yeast Frq1 activate phosphatidylinositol 4-kinase important for secretion from the Golgi (28). Hippocalcin and VILIP proteins regulate guanylate cyclase and nicotinamide acetylcholine receptors implicated in synaptic plasticity (41). KChIPs (42), hippocalcin (43), and NCS-1 (44) all bind and regulate various ion channels and thus serve as Ca 2ϩ sensors that control membrane excitability. The mechanism of target regulation in each case is not understood, and it remains unclear how the structurally similar NCS proteins are able to specifically bind and regulate so many different target proteins.Yeast genetics is a powerful tool for dissecting NCS protein interactions in signa...

“…In these cell types, NCS-1 modulates synaptic transmission (14,22,34), secretion (21,25), or cellular excitability (18,20) via complex processes that include regulation of key enzymes for membrane transport (15,17,23,24,35) and specific regulation of various ion channels (16, 18 -20, 28). K ϩ channels were the first ion channel target to be characterized in expression systems (26,36). Co-expression studies described an up-regulation of channel expression and activity, specifically recorded with the K V 4 K ϩ channel family (26,36).…”

Section: Discussionmentioning

confidence: 99%

“…K ϩ channels were the first ion channel target to be characterized in expression systems (26,36). Co-expression studies described an up-regulation of channel expression and activity, specifically recorded with the K V 4 K ϩ channel family (26,36). Modifications in the expression and properties of other ion channels have also been reported.…”

Section: Discussionmentioning

confidence: 99%

Down-regulation of Voltage-gated Ca2+ Channels by Neuronal Calcium Sensor-1 Is β Subunit-specific

Rousset¹,

Cens²,

Gavarini³

et al. 2003

Neuronal Ca 2؉ sensor protein-1 (NCS-1) is a member of the Ca 2؉ binding protein family, with three functional Ca 2؉ binding EF-hands and an N-terminal myristoylation site. NCS-1 is expressed in brain and heart during embryonic and postnatal development. In neurons, NCS-1 facilitates neurotransmitter release, but both inhibition and facilitation of the Ca 2؉ current amplitude have been reported. In heart, NCS-1 co-immunoprecipitates with K ؉ channels and modulates their activity, but the potential effects of NCS-1 on cardiac Ca 2؉ channels have not been investigated. To directly assess the effect of NCS-1 on the various types of Ca 2؉ channels we have co-expressed NCS-1 in Xenopus oocytes, with Ca V 1.2, Ca V 2.1, and Ca V 2.2 Ca 2؉ channels, using various subunit combinations. The major effect of NCS-1 was to decrease Ca 2؉ current amplitude, recorded with the three different types of ␣ 1 subunit. When expressed with Ca V 2.1, the depression of Ca 2؉ current amplitude induced by NCS-1 was dependent upon the identity of the ␤ subunit expressed, with no block recorded without ␤ subunit or with the ␤ 3 subunit. Current-voltage and inactivation curves were also slightly modified and displayed a different specificity toward the ␤ subunits. Taken together, these data suggest that NCS-1 is able to modulate cardiac and neuronal voltage-gated Ca 2؉ channels in a ␤ subunit specific manner.