Subcellular localization and transcription regulatory potency of KCNIP/Calsenilin/DREAM/KChIP proteins in cultured primary cortical neurons do not provide support for their role in CRE‐dependent gene expression

Abstract

KCNIP3/KChIP3 (voltage‐dependent K+ channel interacting protein 3), alias Calsenilin and downstream regulatory element antagonist modulator (DREAM), is a multifunctional protein that modulates A‐type potassium channels, affects processing of amyloid precursor protein and regulates transcription. KCNIP3 has been described to negatively influence the activity of CREB (cAMP/Ca2+‐response element binding protein), an essential factor in neuronal activity‐dependent gene expression regulation. However, reports on intracellular localization of KCNIP3 in neurons are diverse and necessitate additional analyses of distribution of KCNIPs in cells to clarify the potential of KCNIP3 to fulfill its functions in different cell compartments. Here, we examined localization of the entire family of highly similar KCNIP proteins in neuronal cells and show that over‐expressed isoforms of KCNIP1/KChIP1, KCNIP2/KChIP2, KCNIP3/KChIP3, and KCNIP4/KChIP4 had varied, yet partially overlapping subcellular localization. In addition, although some of the over‐expressed KCNIP isoforms localized to the nucleus, endogenous KCNIPs were not detected in nuclei of rat primary cortical neurons. Moreover, we analyzed the role of KCNIP proteins in cAMP/Ca2+‐response element (CRE)‐dependent transcription by luciferase reporter assay and electrophoretic mobility shift assay and report that our results do not support the role for KCNIPs, including DREAM/Calsenilin/KChIP3, in modulation of CREB‐mediated transcription in neurons.

Kcnip1 a Ca2+-dependent transcriptional repressor regulates the size of the neural plate in Xenopus

“…3A, middle and right panels). These results are in accordance with the subcellular distribution of the Kcnip1 counterpart in mammalian cell cultures [38]. In addition, the nuclear localization of the GFP-tagged Kcnip1 is in agreement with a role for Kcnip1 as transcriptional regulator in X. laevis embryos.…”

Modulatory mechanisms and multiple functions of somatodendritic A-type K+ channel auxiliary subunits

“…Furthermore, S-palmitoylation is a reversible process, suggesting that trafficking of Kv4 channels may be regulated by the cell. Indeed, studies by Pruunsild and Timmusk (2012) using over-expression of tagged KChIP variants in neurons indicate that, while N-myristoylated and transmembrane KChIPs consistently show respectively cytoplasmic punctuate and perinuclear localization, many of the supposed S-palmitoylated KChIPs were diffusedly distributed throughout the neuron (Pruunsild and Timmusk, 2012). …”

“…Based on sequence similarity, it is likely that the transmembrane-encoding N-terminal exons of KChIP2x, KChIP3x, and KChIP4a have been retained from the ancestral KChIP gene, and they all show similar functional effects, including ER retention, suppression of Kv4 functional expression, and modulation of channel gating properties (Jerng and Pfaffinger, 2008). KChIP4e, which is also predicted to have a transmembrane domain, likewise has a perinuclear subcellular localization, even though a fusion protein between the KChIP4e variable N-terminus with eGFP was not found to associate with the membrane fraction (Pruunsild and Timmusk, 2012). In summary, while N-myristoylation and S-palmitoylation can specifically promote trafficking of KChIPs to the post-ER or the Golgi bodies, the transmembrane segment found in KChIP2x, KChIP3x, and KChIP4a affect both channel gating as well as trafficking.…”

“…The variable N-terminal domains of KChIP2x, KChIP3x, and KChIP4a feature a stretch of hydrophobic amino acids that folds into a transmembrane segment, facilitates membrane partition, and promotes ER retention ( Figure 5A ; Jerng and Pfaffinger, 2008; Pruunsild and Timmusk, 2012). In intact cells, biotinylation studies of N-terminal cysteine mutants show that the KChIP4a N-terminus is extracellular and the transmembrane segment begins with leucine at residue 3.…”

“…For example, even though KChIP4e has a putative transmembrane N-terminus and shows perinuclear localization, KChIP4e increases surface expression of Kv4.2 channels, does not slow inactivation kinetics, and accelerates recovery from inactivation (Jerng and Pfaffinger, 2008; Pruunsild and Timmusk, 2012). Also, while KChIP2x and Kv3x slow inactivation similar to KChIP4a, they accelerated recovery like typical KChIPs (Jerng and Pfaffinger, 2008).…”