Potassium Ion Channel Interactions Protein-kchip1 Gene Function Study

It is well known Ca²⁺ plays an important role in regulating a variety of neuronal processes, such as neurotransmission, cytoskeletal dynamics, gene expression and signal transduction. The actions of Ca²⁺ are often mediated by Ca²⁺- binding proteins, which may act as either Ca²⁺ buffers or Ca²⁺ sensors. Neuronal Calcium Sensor (NCS) is a large family of Ca²⁺-binding proteins, and is very important in modulating the Ca²⁺ mediated signals transduction.More than 20 NCS proteins have been identified from various species. These proteins are highly conserved through out the evolution, sharing high homolog within their C-terminal. One important structural feature of NCS is containing four EF-Hand calcium binding motifs in each protein. NCS proteins can be classified into three subclasses. Classâ… , includes visinin, recoverin and S-modulin. Classâ…¡, consists of hippocalcin, frequenin, and NCS-1. The four newly identified KChIP proteins constitute the third class. KChIPs share more than 65% identity at the amino acid level with a distinct N-terminus.KChIPs can interact with A-type potassium channels (Kv4 channels), acting as theβsubunit of fast transient A-type potassium channels. KChIP1 can slow the inactivation period of Kv4.2, while speed this period of Kv4.1. Targeting deletion of KChIP2 gene in mouse genome leads to a complete loss of calcium-dependent transient outward potassium current in cardiac myocytes and confers susceptibility to ventricular tachycardia, which further supports a role of KChIPs in modulating potassium channels. KChIP3 can interact with Kv4.2,Kv4.3and Kv4.4, regulating the kinetic properties of Kv4 channels. KChIP4 also can interact with Kv4.2and Kv4.3. Mutation in the Ca²⁺ binding motif of KChIP4 results in a complete loss of potassium current.Nevertheless, KChIPs may function in vivo with many other proteins. In our previous study, we used the medium fragment of APC( Adenomatous polyposis coli) as the bait to screen the human fetal brain cDNA library by Yeast Two Hybrid and identified KChIP1, suggesting its role in APC related neuronal regulation. KChIP3 is referred to calsenilin for its interaction with Alzheimer's disease related presenilin-2, although the functional significance of these interactions remains to be elucidated. KChIP3 is also called DREAM, a downstream regulatory element antagonist modulator. DREAM (KChIP3)-deficient mice exhibit reduced responses in models of acute pain through a transcriptional mechanism. Upon single transfection, KChIP4 was diffusely distributed in cytoplasm as well as in nuclei. Double transfection with PS2 dramatically changed the distribution of KChIP4 into a reticular pattern, overlapping with that of PS2 in the perinuclear area and endoplasmic reticulum membranes, supporting KChIP4 interaction with presenilin-2.Preliminary study has demonstrated that KChIPs is an important protein with multifunction, however, the function of KChIP proteins in brain where they are predominantly expressed remains unclear. To answer this question, the function of KChIP1 in brain was carried out in this study. The study has 3 parts.In the first part, by in situ hybridization, immunohistochemical assay and immunostaining, we found KChIP1 is predominantly expressed in a subpopulation of parvalbumin-positive GABAergic neurons in mouse brain, suggesting its functional relationship with GABAergic inhibitory neurons.In the second part, we deleted the KChIP1 gene in mouse genome by a targeting construct. The fact that no embryonic lethality or significant difference in brain and weight was observed in these mice compared with their wild-type littermates hinted that KChIP1 may be not requisite for embryo growth and development. However, the mice bearing KChIP1 deletion showed impaired motor coordination, suggesting that KChIP1 may play important roles in controlling motor function. Moreover, the mutant mice showed increased susceptibility to anti-GABAergic convulsive drug-PTZ-induced seizure, further supporting its roles in synaptic transmission of GABAergic inhibitory neurons.In the third part, in the mutant mouse brain, we identified a novel Purkinje neuron-specific KChIP1 splicing variant, KChIP1c. KChIP1c has a distinct N-terminus from KChIP1a and KChIP1b that is encoded by an alternative exon. Like KChIP1a and KChIP1b, KChIP1c interacts with Kv4.3 and promotes the plasma membrane localization of Kv4.3 in transfected cells. In Xenopus oocytes, KChIP1c enhanced Kv4.3 currents amplitude that is significantly larger than those caused by KChIP1a and KChIP1b. In contrast, KChIP1c did not affect the inactivation of Kv4.3 in its closed state. The results suggest that KChIP1c may function in modulating Kv4.3 currents with a mechanism different from KChIP1a and KChIP1b.In summary, our results suggested: KChIP1 can change cells' excitability through interaction with potassium channel protein Kv4.2 and Kv4.3, therefore regulating balance; KChIP1 can also modulate inhibitory synaptic transmission through increasing presynaptic GABA release, which may play an important role in the anti-epileptic attack and the maintenance of the normal nervous status; Different KChIP1s, differently distributed, may function differently in regulating A-type potassium channels. These results may provide useful basis in diagnosis and treatment of KChIP1 related degenerative diseases.