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. 2011 Jun;3(6):309-19.
doi: 10.1002/emmm.201100135. Epub 2011 Mar 24.

A CAG repeat polymorphism of KCNN3 predicts SK3 channel function and cognitive performance in schizophrenia

Sabrina Grube  1 , Martin F Gerchen, Bartosz Adamcio, Luis A Pardo, Sabine Martin, Dörthe Malzahn, Sergi Papiol, Martin Begemann, Katja Ribbe, Heidi Friedrichs, Konstantin A Radyushkin, Michael Müller, Fritz Benseler, Joachim Riggert, Peter Falkai, Heike Bickeböller, Klaus-Armin Nave, Nils Brose, Walter Stühmer, Hannelore Ehrenreich
Affiliations

A CAG repeat polymorphism of KCNN3 predicts SK3 channel function and cognitive performance in schizophrenia

Sabrina Grube et al. EMBO Mol Med. 2011 Jun.

Abstract

KCNN3, encoding the small conductance calcium-activated potassium channel SK3, harbours a polymorphic CAG repeat in the amino-terminal coding region with yet unproven function. Hypothesizing that KCNN3 genotypes do not influence susceptibility to schizophrenia but modify its phenotype, we explored their contribution to specific schizophrenic symptoms. Using the Göttingen Research Association for Schizophrenia (GRAS) data collection of schizophrenic patients (n = 1074), we performed a phenotype-based genetic association study (PGAS) of KCNN3. We show that long CAG repeats in the schizophrenic sample are specifically associated with better performance in higher cognitive tasks, comprising the capacity to discriminate, select and execute (p < 0.0001). Long repeats reduce SK3 channel function, as we demonstrate by patch-clamping of transfected HEK293 cells. In contrast, modelling the opposite in mice, i.e. KCNN3 overexpression/channel hyperfunction, leads to selective deficits in higher brain functions comparable to those influenced by SK3 conductance in humans. To conclude, KCNN3 genotypes modify cognitive performance, shown here in a large sample of schizophrenic patients. Reduction of SK3 function may constitute a pharmacological target to improve cognition in schizophrenia and other conditions with cognitive impairment.

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Figures

Figure 1
Figure 1. SK3 CAG repeat lengths sum is associated with higher cognitive function in schizophrenia but does not constitute a genetic risk factor for the disease

  1. A. KCNN3 is located at 1q21.3 and spans 162.8 kbp. The nine exons (boxes) encode two different splicing variants (1, 2); the coding region is shaded in grey. The black line in exon1 indicates the position of the polymorphic CAG repeat.

  2. B. The region around the CAG repeat is highly conserved among species.

  3. C,D. Neither the distribution of the individual sum of repeat lengths of both alleles (C) nor that of the individual difference between repeat lengths of both alleles (D) is different between schizophrenic patients (n = 1060) and healthy controls (n = 1135). Hence, these readouts of the SK3 CAG repeat polymorphism do not support a genetic risk for developing schizophrenia.

  4. E,F. In contrast, the PGAS approach allows identification of a role for the SK3 CAG repeat polymorphism in higher cognitive function.

  5. E. Intercorrelation network of cognitive target variables (dark ovals) and cognitive control variables (light ovals) in the GRAS population of schizophrenic patients. Line thickness indicates the degree of correlation between two respective tests after standardization by Blom transformation and adjustment for covariates sex, age, antipsychotic medication and negative symptoms.

  6. F. Scatter plot of the covariate-adjusted composite score calculated as mean of all standardized (Blom transformed) cognitive target variables. Adjusted was for covariates sex, age, antipsychotic medication and negative symptoms. Linear regression analysis reveals a significant effect (p < 0.0001) of allelic repeat lengths sum on the composite score.

Figure 2
Figure 2. SK3 overexpressing (T/T) mice show selective deficits in higher cognition

  1. A,B. Comparative distribution of SK3 immunoreactivity in hippocampus (sagittal section) is presented for wildtype (A) versus SK3 T/T mice (B). Insets show magnifications of stained granule neurons in dentate gyrus and pyramidal neurons in Ammon's horn (CA2).

  2. C. Densitometrical quantification of SK3 protein expression in hippocampus and heart of WT and SK3 T/T mice (n = 6–7; **Student's t-test, t1,12 = 3.724, p = 0.0029; ***Student's t-test, t1,11 = 4.902, p = 0.0005). SK3 expression is presented as % of ß-actin (ß-Act) and normalized to the WT. Sample Western blots are shown below the bar chart. For quantification, the 74 kDa band was used for WT (endogeneous SK3) and the 70 kDa band for T/T (transgenic SK3 expression).

  3. D. WT and T/T behaviour is unaltered in elevated plus maze. Presented is the percentage of time spent in open arms against the total time spent in both open and closed arms.

  4. E-G. No differences in open field are observed between genotypes regarding time spent in different zones (E) or velocity (F). The latency to reach the wall after initial start from the centre of the open field (G) is significantly higher in SK3 T/T mice compared to WT (*Student's t-test, t1,26 = 2.318, p = 0.029).

  5. H. In hole board, SK3 T/T mice show significantly less exploratory activity than WT (***Student's t-test, t1,26 = 4.110, p = 0.0004).

  6. I. The two genotypes do not differ in motor performance/motor learning on rota-rod.

  7. J. SK3 T/T mice demonstrate longer escape latency in the hidden platform task (*two-way repeated measures ANOVA, F1,26 = 5.43, p = 0.028); in the probe trial (inset), SK3 T/T mice display absence of preference for the target quadrant (*Student's t-test, t1,26 = 2.250, p = 0.033).

  8. K. In contextual and cue memory test of fear conditioning, SK3 T/T mice freeze less (*Student's t-test, t1,26 = 2.212, p = 0.036, ***Student's t-test, t1,26 = 3.779, p = 0.0008). For all behavioural experiments: n = 13–15; data presented as mean ± s.e.m.; two-sided Student's t-tests used; * indicates p < 0.05, **p < 0.01 and ***p < 0.001.

Figure 3
Figure 3. KCNN3 CAG repeat length affects electrophysiological properties of SK3

  1. For electrophysiological measurements, the eGFPhSK3(CAG)n construct has been employed to generate clones with different repeat lengths. Indicated are locations of the CAG repeat (labelled in orange), of the primers (labelled in purple) and of the restriction enzyme sites used for cloning.

  2. Live transfected HEK293 cells show intense green fluorescent signal on the plasma membrane; cell nuclei are stained with Hoechst33342 (blue).

  3. Western blot analysis of eGFP and SK3 demonstrates comparable SK3 fusion protein size and expression level of the different constructs in transfected cells.

  4. The average SK3 current response (expressed as apamin-sensitive current density) for eGFPhSK3(CAG)11 (n = 8; green), eGFPhSK3(CAG)18 (n = 7; blue) and eGFPhSK3(CAG)24 (n = 9; red) is displayed. The result of fitting is superimposed as a solid line. Error bars represent s.e.m.

  5. Normalized apamin-sensitive current density (versus the corresponding individual current density maximum) for clones eGFPhSK3(CAG)11, eGFPhSK3(CAG)18 and eGFPhSK3(CAG)24. Inward rectification starting as early as at +20 mV is evident for eGFPhSK3(CAG)24. Colours as in panel D.

Note: Colour version is available online.
See this image and copyright information in PMC

Comment in

  • Beyond the GWAS in schizophrenia.
    Malhotra AK. Malhotra AK. EMBO Mol Med. 2011 Jun;3(6):303-5. doi: 10.1002/emmm.201100137. Epub 2011 May 3. EMBO Mol Med. 2011. PMID: 21542131 Free PMC article. No abstract available.

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