Synaptic localization of KChIP1 in a subset of parvalbulmin-positive GABAergic neurons presynaptically in vivo. a: Adult rat cortex was immunostained with a polyclonal antibody against KChIP1 (green), and b: a monoclonal antibody against synaptophysin (red), and c: a monoclonal antibody against GFAP (blue). Sections were examined under confocal microscopy. d: An overlay image shows colocalization (yellow) of KChIP1 and synaptophysin at the plasma membrane of neuronal cell bodies and processes. No GFAP-labeled cells were KChIP1 positive. Arrows indicate colocalization of KChIP1 and synaptophysin. Bar = 40 μm.

KChIP1 inhibits potassium currents in cultured hippocampal neurons. a: Co-immunoprecipitation of Kv4.3 and KChIP1-EGFP. HEK293 cell lysates co-expressing Flag-tagged Kv4.3 and KChIP1-EGFP were immunoprecipitated with an anti-Flag antibody (left panel; IP: Kv4.3) or an anti-EGFP antibody (right panel; IP: EGFP) followed by immunoblotting with anti-EGFP (left panel; IB: EGFP) or anti-Flag (right panel; IB: Kv4.3), respectively. b: Potentiation of Kv4.3 current density by Kv4.3-EGFP in Xenopus oocytes. Kv4.3 was co-expressed with either KChIP1-EGFP fusion protein, KChIP1, or EGFP in Xenopus oocytes. KChIP1-EGFP and KChIP1 but not EGFP significantly increased Kv4.3 current density (*P < 0.001). c, d: KChIP1-EGFP modulates inactivation of Kv4.3 in Xenopus oocytes. Kv4.3 was co-expressed with either KChIP1-EGFP fusion protein (black line), KChIP1 (red line), or EGFP (green line) in Xenopus oocytes. Expression of KChIP1-EGFP or KChIP1 enhanced inactivation of Kv4.3 from both the open state (c) and the closed state (d) compared with EGFP alone.

KChIP1 decreases potassium but not calcium current densities. a: Inhibition of potassium currents by KChIP1. Representative recordings from control neurons expressing EGFP alone or KChIP1-EGFP. Potassium current density in KChIP1+ neurons was significantly smaller than that seen in control (*P < 0.01, n = 18). Whole-cell potassium currents were evoked by a voltage step to -15 mV from a holding potential of -75 mV. Steady-state amplitudes were normalized to cell capacitance. b Expression of KChIP1 does not affect Ca²⁺ channels. Representative recordings from control (EGFP+) and KChIP1+ (KChIP1-EGFP) neurons. Ba²⁺ current density in KChIP+ neurons (143.17 ± 11.1 pA/pF, n = 11) was similar to that observed in controls (124.3 ± 8.9 pA/pF, n =12; *P > 0.1). Whole cell Ba²⁺ currents were evoked by a voltage step to 0 mV from a holding potential of -80 mV.

KChIP1 KO mice display enhanced anxiety-like behavior. a: In the light/dark test, KChIP1 KO mice (n = 6) spent significantly less time in the light compartment compared to WT controls (n = 5) (*P < 0.05.). b: In the open field test, KChIP1 KO mice had significantly less ambulatory counts compared to WT controls (n = 7). (* P < 0.02). c: KChIP1 KO mice (n = 9) travelled significantly less within the centre area compared to WT controls (n = 7). d: In the elevated plus maze test, KChIP1 KO mice (n = 9) spent significantly less time in the open arms to WT mice (n = 7), however e: KChIP1 KO mice (n = 9) made similar amount of entries into the arms compared to WT controls (n = 7) (* P < 0.05). f: In a rota-rod test for motor performance, KChIP1 KO mice (n = 6) exhibited similar falling latencies as WT control mice (n = 6). g: On a thermal hot plate test, KChIP1 KO mice (n = 6) displayed similar latencies for nociceptive behavior display as WT control mice (n = 6). h: In a tail flick assay, KChIP1 KO mice (n = 6) displayed similar latencies for tail flicks as WT control mice (n = 6).

Over-expression of KChIP1 facilitates GABA-mediated synaptic transmission in cultured hippocampal neurons. a: Expression of KChIP1-EGFP was detected by immunoblot analysis in neurons infected with KChIP1-EGFP (KChIP1) but not in neurons infected with EGFP alone. Detection of actin in the lysates with an anti-actin antibody is shown as a loading control. b: Representative recordings from EGFP+ (Control) and KChIP1-EGFP neurons (KChIP1) are shown. c: The paired pulse ratio (PPR) recorded in control cells was significantly larger than that seen in KChIP1-expressing cells (*P < 0.01). d, E: mIPSCs from autaptic inhibitory neurons. Representative mIPSC recordings are shown from EGFP+ (d) and KChIP1-EGFP+ neurons (e). f: Cumulative probability of amplitude and averaged mIPSCs (inset) from the same two cells as on f and e. g: The frequency of mIPSCs in cells expressing KChIP1 (0.88 ± 0.14 Hz) was more than twice that observed in control cells expressing EGFP alone (0.37 ± 0.13; *P < 0.05). h: There was no difference in the amplitude distribution of mIPSCs in these neurons (KChIP-EGFP: filled dots; EGFP alone: empty dots).

Disruption of mouse KChIP1 gene potentiates potassium currents in cultured Purkinje neurons. a: Lack of KChIP1 mRNA in KChIP1^-/- mouse brain tissue. KChIP1a and KChIP1b mRNAs in wild-type (WT) and KChIP1^-/- (KO) mouse brain were analyzed by RT-PCR. The brain tissues examined include cerebellum (Ce), hippocampus (Hipp), cortex (Cx) and thalamus (Tha). Expression of GAPDH was monitored as a positive control. b: Primary cultures of Purkinje cells from KChIP1^-/- mouse cerebella. A phase contrast image (left panel) and an immunofluorescence image of Purkinje cells stained by with anti-calbindin antibody (right panel) are illustrated. c, d: Potentiation of potassium currents in KChIP1^-/- Purkinje cells. Representative recordings from wild-type (WT) and KChIP1^-/- (KO) Purkinje cells (c). Potassium current density in WT was significantly smaller than that seen in KO neurons (d, *P < 0.05). Whole-cell potassium currents were evoked by a voltage step to -15 mV from a holding potential of -75 mV. Steady-state amplitudes were normalized to cell capacitance.

KChIP1 expression in the adult mouse brain. a, b: In situ hybridization. Adult mouse brain sections were hybridized in situ with an anti-sense RNA probe synthesized using KChIP1 cDNA as the template. Coronal (a) and sagittal (b) views are shown. Hybridization signals are seen in a subset of neurons distributed in the cerebral cortex, hippocampus, and thalamus. High expression of KChIP1 was detected in the medial habenular nucleus (a, MHb), the reticular thalamic nucleus (a and b, Rt), the dorsomedial part of the ventromedial hypothalamic nucleus (a, VMHDM), and the Purkinje cell layer of the cerebellum (b, Ce). The montage was produced from photographs taken with a deconvolution microscope using a 5× objective and Slidebook software (Intelligent Imaging Innovations, Inc.). c: Immunohistochemical detection of KChIP1 in the brain. Adult mouse brain sections were immunostained with a polyclonal anti-KChIP1 antibody. Expression of KChIP1 was analyzed under confocal microscopy. The distribution of KChIP1 protein was similar to that of KChIP1 mRNA detected by in situ hybridization. KChIP1 was detected in a subpopulation of neurons in the cerebral cortex (Cx, yellow arrow); high levels of KChIP1 were observed in the medial habenular nucleus (MHb, yellow arrow), reticular thalamic nucleus (Rt, yellow arrow), and Purkinje cell layer of the cerebellum (Ce, yellow arrow). Bar = 50 μm in Cx and 100 μm in MHb, Rt, and Ce.