A primate-specific, brain isoform of KCNH2 affects cortical physiology, cognition, neuronal repolarization and risk of schizophrenia

Abstract

Organized neuronal firing is crucial for cortical processing and is disrupted in schizophrenia. Using rapid amplification of 5' complementary DNA ends in human brain, we identified a primate-specific isoform (3.1) of the ether-a-go-go-related K(+) channel KCNH2 that modulates neuronal firing. KCNH2-3.1 messenger RNA levels are comparable to full-length KCNH2 (1A) levels in brain but three orders of magnitude lower in heart. In hippocampus from individuals with schizophrenia, KCNH2-3.1 expression is 2.5-fold greater than KCNH2-1A expression. A meta-analysis of five clinical data sets (367 families, 1,158 unrelated cases and 1,704 controls) shows association of single nucleotide polymorphisms in KCNH2 with schizophrenia. Risk-associated alleles predict lower intelligence quotient scores and speed of cognitive processing, altered memory-linked functional magnetic resonance imaging signals and increased KCNH2-3.1 mRNA levels in postmortem hippocampus. KCNH2-3.1 lacks a domain that is crucial for slow channel deactivation. Overexpression of KCNH2-3.1 in primary cortical neurons induces a rapidly deactivating K(+) current and a high-frequency, nonadapting firing pattern. These results identify a previously undescribed KCNH2 channel isoform involved in cortical physiology, cognition and psychosis, providing a potential new therapeutic drug target.

Figure 1. Genetic association of 7q36.1 with risk for schizophrenia

(Top) Inverse log of the p-value for single SNPs from association results for the CBDB/NIMH Sibling Study (CBDB-blue), NIMHGI (NIMHGI-black), for the German case-control study (German-green) and for the pooled 5 sample meta-analysis (Pooled-red). Only markers with p-values less than 0.1 are shown. A physical map of the region is given and depicts known genes within the region. (Bottom) The LD structure of the genotyped markers is shown for 370 unrelated healthy Caucasian controls and depicted as r. Graphic created using the R package snp.plotter.

Figure 2. Association of risk SNPs with cognitive measures, brain structure volumes, and regional brain activity during memory-based tasks

(A) M30 genotype versus hippocampal gray matter volumes in healthy control subjects. Heatmap depicts linear decrease in regional gray matter volume from subjects homozygous for the risk associated allele, A, (n = 16) to heterozygote carriers (n = 61) to non carriers (n= 64). Only voxels corresponding to a p<0.05 FDR corrected threshold are shown. (B) Percent blood oxygen level-dependent (BOLD) signal change in healthy controls during the encoding conditions of a declarative memory task in the left posterior hippocampus (MNI coordinates of peak cluster: −34 −25 −15 mm) showing a significant linear increase of activation in homozygote carriers (n= 14) relative to heterozygote carriers (n=37), and to non carriers (n=28) of the risk associated allele at M30. Plot depicts voxel mean ±1 SEM. Heatmap colors correspond to degree of increase in BOLD signal with each copy of M30 risk allele (T). Only voxels surviving p< 0.05 FWE correction are shown) statistical t-maps and mean (±1 SEM) (D) Thresholded (p< 0.05 FWE corrected) statistical t-maps and mean ((±1 SEM) percent BOLD signal change during the executive working memory task in the right dorsolateral prefrontal cortex (DLPFC) (MNI coordinates of peak cluster: 26 30 42 mm) showing a significant linear increase of activation in homozygote carriers (n=24) relative to heterozygote carriers (n=71), and to non carriers (n=81) of the risk associated allele at M30.

Figure 3. Regional gene expression and association with risk genotype

(A) Differences in mRNA expression within the hippocampus of 29 schizophrenia patients/59 healthy control subjects, and within the DLPFC of a largely overlapping set of 31 schizophrenia patients/69 healthy control subjects. Expression values are normalized fold differences versus the mean of healthy controls. P-values represent main effect of diagnosis in the final regression model. (B) Association of M30 risk-genotype (T carriers) with isoforms 3.1 expression within the hippocampus (data from A) with respect to diagnosis. All error bars represent one standard error of the mean.

Figure 4. Detection and quantification of Isoform 3.1 mRNA and protein

(A) PCR of KCNH2-1A and Isoform 3.1 using isoform specific primer pairs in human heart, hippocampus, and fetal brain tissue extracts. PCR products for KCNH2-1A correspond to 373 and 463 bps, respectively, and for Isoform 3.1 at 388 and 478 bp, respectively. The band ∼1.5 kb in Iso1-Ex4 lanes corresponds to small amounts of unspliced RNA or genomic DNA contaminant present in the samples. (B) Protein expression of KCH2-1A and Isoform 3.1 in human, primate, and mouse frontal cortex. Positive control lanes include HEK cells transfected with full-length KCH2-1A (WT) and Isoform 3.1. Proteins extracted from human brain regions show two distinct bands, one equivalent in size to isoform 3.1 transfected HEK cells. However, the larger protein band observed in human brain did not correspond to the size of KCNH2-1A transfected HEK proteins. Instead, the larger band occurs at ∼160 kDa, which is the reported size of KCNH2-1A from in vivo protein extracts suggesting post-translational modifications. (D) Primary rat neurons transfected with KCNH2-1A or Isoform 3.1 containing vectors.

Figure 5. Characterization of KCNH2 currents in HEK293T cells expressing KCNH2-1A and Isoform 3.1

(A) Schematic diagram of KCNH2-1A and Isoform 3.1 domain structures. Blue box: PAS domain. Green box: conserved amino acid sequences l between isoforms. Numbers correspond to amino acid positions. (B) Currents evoked by voltage steps (4 sec) from V_H of −60 mV to potentials from −100 to +50 mV in 10-mV increments, followed by a voltage pulse to −120 mV (5 sec). Upper panel: voltage protocol. Middle and lower panels: traces (corrected for leak currents) recorded from cells transfected with KCNH2-1A and Isoform 3.1 cDNAs, respectively. (C) Effects of E-4031on tail currents evoked by a test pulse to −120 mV from holding potential of +50 mV, using the same protocol as in B. Traces recorded from the same cells before (black) and after (red) treatment with 10 µM E-4031 are superimposed. Upper and lower panels show KCNH2 currents recorded from cells transfected with KCNH2-1A and Isoform 3.1 cDNAs, respectively. (D) Steady-state activation curves of KCNH2-1A and Isoform 3.1 tail currents, which were induced by the protocol shown in B. The reversal membrane potential (Vrev) under the recording conditions was −96 mV. The V_1/2 for KCNH2-1A and Isoform 3.1 are −16.37 ± 2.55 mV and −6.54 ± 4.04 mV, respectively (p<0.05). In this and subsequent figures, the data are presented as mean ±1 SEM. Numbers in parentheses indicate the number of cells recorded. (E) Tail currents evoked by voltage steps from +60 mV to potentials between −120 mV and −70 mV in 10 mV increments. Traces in upper, middle and lower panels represent tail currents of KCNH2-1A, Isoform 3.1 and co-transfection KCNH2-1A with Isoform3.1, respectively. (F) Semilogarithmic plot of deactivation time constants of KCNH2-1A and Isoform3.1 currents at different repolarizing voltages. Upper plot (between −210 and −100 mV) corresponds to τ2 for HEK cells transfected with KCNH2-1A alone or cotransfected KCNH2-1A and Isoform 3.1, whereas the lower plots correspond to τ1 for all transfection combinations.

Figure 6. Effect of Isoform 3.1 on repolarization-induced tail currents and firing patterns in cortical neurons

(A) KCNH2-mediated tail currents in GFP-(left) and Isoform 3.1-transfected (right) neurons before and after application of E4031. Upper plot diagrams the volatage protocol. Lower plot represents E-4031-sensitive current (i.e. hERG current) generated by subtracting pre and post-inhibitor currents. (B) Semilogarithmic plot of deactivation time constants of E-4031-sensitive currents at different re-polarizing voltages in transfected primary cortical neurons. Between −120 and −100 mV decay of E-4031-sensitive currents was fitted by double-exponential functions (lower curves: τ1, upper curves: τ2), whereas between −90 and −70 mV time course followed single-exponential functions (τ). (C) Effect of Isoform 3.1 over-expression in rat cortical neurons on action potential discharge evoked by long depolarizing pulse (40 pA, 1 sec) before (left) and after (right) application of E-4031. (D) Spike frequencies (number of spikes per second) versus applied depolarizing currents in transfected primary cortical neurons. (E) Effect of Isoform 3.1 on spike frequency adaptation depicted as instantaneous frequency (inverse of interpulse interval) versus the corresponding spikes interval evoked by a 4-pA depolarizing pulse. (F) Effect of E-4031 on repetitive action potential discharge of transfected cortical neurons. Error bars ±1 SEM. *: significantly different, p<0.01.