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. 2019 Jan 2;39(1):28-43.
doi: 10.1523/JNEUROSCI.1593-18.2018. Epub 2018 Nov 2.

Voltage-Independent SK-Channel Dysfunction Causes Neuronal Hyperexcitability in the Hippocampus of Fmr1 Knock-Out Mice

Pan-Yue Deng  1   2 , Dan Carlin  3 , Young Mi Oh  3 , Leila K Myrick  4   5   6 , Stephen T Warren  4   5   6 , Valeria Cavalli  3 , Vitaly A Klyachko  7   2
Affiliations

Voltage-Independent SK-Channel Dysfunction Causes Neuronal Hyperexcitability in the Hippocampus of Fmr1 Knock-Out Mice

Pan-Yue Deng et al. J Neurosci. .

Abstract

Neuronal hyperexcitability is one of the major characteristics of fragile X syndrome (FXS), yet the molecular mechanisms of this critical dysfunction remain poorly understood. Here we report a major role of voltage-independent potassium (K+)-channel dysfunction in hyperexcitability of CA3 pyramidal neurons in Fmr1 knock-out (KO) mice. We observed a reduction of voltage-independent small conductance calcium (Ca2+)-activated K+ (SK) currents in both male and female mice, leading to decreased action potential (AP) threshold and reduced medium afterhyperpolarization. These SK-channel-dependent deficits led to markedly increased AP firing and abnormal input-output signal transmission of CA3 pyramidal neurons. The SK-current defect was mediated, at least in part, by loss of FMRP interaction with the SK channels (specifically the SK2 isoform), without changes in channel expression. Intracellular application of selective SK-channel openers or a genetic reintroduction of an N-terminal FMRP fragment lacking the ability to associate with polyribosomes normalized all observed excitability defects in CA3 pyramidal neurons of Fmr1 KO mice. These results suggest that dysfunction of voltage-independent SK channels is the primary cause of CA3 neuronal hyperexcitability in Fmr1 KO mice and support the critical translation-independent role for the fragile X mental retardation protein as a regulator of neural excitability. Our findings may thus provide a new avenue to ameliorate hippocampal excitability defects in FXS.SIGNIFICANCE STATEMENT Despite two decades of research, no effective treatment is currently available for fragile X syndrome (FXS). Neuronal hyperexcitability is widely considered one of the hallmarks of FXS. Excitability research in the FXS field has thus far focused primarily on voltage-gated ion channels, while contributions from voltage-independent channels have been largely overlooked. Here we report that voltage-independent small conductance calcium-activated potassium (SK)-channel dysfunction causes hippocampal neuron hyperexcitability in the FXS mouse model. Our results support the idea that translation-independent function of fragile X mental retardation protein has a major role in regulating ion-channel activity, specifically the SK channels, in hyperexcitability defects in FXS. Our findings may thus open a new direction to ameliorate hippocampal excitability defects in FXS.

Keywords: FMRP; SK channels; action potential; excitability; presynaptic function; threshold.

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Figures

Figure 1.
Figure 1.
Increased excitability of CA3 PCs in Fmr1 KO mice. A, Sample traces of spontaneous APs recorded at different membrane potentials in WT and Fmr1 KO CA3 PCs. B, Analysis of recordings in A showing an increased number of APs at all tested membrane potentials in Fmr1 KO neurons. C, Spontaneous AP frequency distribution. APs were recorded at −51 mV. A bin size of 0.2 Hz was used to calculate AP firing frequency distribution from a 20-s-long trace per cell. The number of APs within a bin was normalized to the total number of APs. Note the peak frequency of AP firing shifts rightward in Fmr1 KO neurons. D, Cumulative probability of AP frequency showed increased AP firing frequency in Fmr1 KO neurons. Data from C (WT, n = 30; KO, n = 32, p < 0.0001, Kolmogorov–Smirnov test). Inset, Averaged AP firing frequency at −51 mV. E, Threshold estimates for spontaneous APs. Left, Sample AP traces show a hyperpolarizing shift of threshold in Fmr1 KO neurons (arrows). Right, Bar graph shows mean thresholds. F, RMP, input resistance, and membrane capacitance in CA3 PCs of WT and Fmr1 KO mice. Left, RMP: WT, n = 62; KO, n = 85, p = 0.8880; middle, input resistance: WT, n = 25; KO, n = 26, p = 0.88406; right, membrane capacitance: WT, n = 67; KO n = 65, p = 0.463. G, AP parameters (left to right): AP upstroke maximal rising rate (p = 0.653); AP rising time (10–90%), p = 0.147; AP falling time (90–10%), p < 0.0001; AP duration at −10 mV level (p < 0.0001); AP amplitude (p = 0.628); and fast AHP (fAHP) amplitude (p = 0.0014). WT, n = 33; KO, n = 35; **p < 0.01. ns, Not significant. Bar graph data are means ± SEM.
Figure 2.
Figure 2.
Increased CA3 neuron excitability in Fmr1 KO mice is a cell-autonomous defect, but not caused by changes in mGluR5 signaling or INaP. A, Spontaneous AP traces recorded with or without five blockers: DNQX, APV, MPEP, gabazine, and CGP55845. B, Number of APs in control, with five blockers, or riluzole. Note that APs were recorded at −46 mV in the presence of riluzole, because riluzole markedly increased threshold and no AP firing was detected at −51 mV. C, Determination of AP threshold by a ramp current injection (lower trace, ramp rate 0.1 pA/ms). Only the first AP was used to estimate threshold (boxed area). D, Enlargement of box in C. Arrows denote the threshold in both genotypes. E, Analysis of data in C showing a decreased AP voltage threshold in Fmr1 KO neurons. F, AP threshold with and without the five blockers. G, Same as in F, but for riluzole. *p < 0.05, **p < 0.01. Data are means ± SEM.
Figure 3.
Figure 3.
SK-channel defects decrease AP threshold in CA3 PCs of Fmr1 KO mice. A, Effect of Kv1 inhibitor dendrotoxin on AP threshold. B, Same as in A, but for the BK-channel blocker iberiotoxin. C, Same as in A, but for the H-channel blocker ZD7288. D, Same as in A, but for the M-channel blocker XE991. E, SK-channel blocker apamin or UCL1684 abolished the difference in threshold between genotypes. F, SK-channel opener 1-EBIO or NS309 abolished the difference in threshold between genotypes. G, Sample AP traces recorded with or without intracellular administration of 1-EBIO or NS309. H, 1-EBIO or NS309 abolished the difference in AP firing between genotypes. *p < 0.05, **p < 0.01. ns, Not significant. Data are means ± SEM.
Figure 4.
Figure 4.
Decreased SK current in CA3 PCs of Fmr1 KO mice. A, Sample trace of SK current evoked by a depolarizing voltage ramp protocol (5 mV/s). a, Before apamin; b, during apamin; c, apamin-sensitive current. B, SK current evoked by ramp protocol. C, Sample trace of SK current evoked by voltage steps (depolarized from −60 to 0 mV; 10 step durations; Δt = 1, 3, 6, 10, 30, 60, 100, 300, 600, 1000 ms). D, Top, SK currents from C plotted as a function of step duration. Bottom, SK-channel activation curve in Fmr1 KO neurons shifts rightward (fitted by sigmoid function). The half maximum activation step duration (D1/2) was larger in KO neurons (inset). E, AP threshold in the presence of thapsigargin (Thaps), BAPTA, or CdCl2 in WT and Fmr1 KO CA3 PCs. F, Same as in C and D (top), but in the presence of CdCl2. *p < 0.05, **p < 0.01. Data are means ± SEM.
Figure 5.
Figure 5.
Abnormal SK currents cause decreased mAHP in CA3 PCs of Fmr1 KO mice. A, Sample traces of mAHP after spontaneous APs (baseline membrane potential at −51 mV). Note that SK-channel blocker apamin markedly decreased mAHP amplitude in both genotypes. B, Decreased mAHP amplitude after single APs in KO neurons. SK-channel blockers apamin or UCL1684 markedly decreased mAHP and abolished differences between genotypes. C, Sample traces of mAHP with or without SK-channel opener 1-EBIO or NS309. D, Either 1-EBIO or NS309 increased mAHP amplitude both in WT and KO neurons and abolished differences between genotypes. Control bars are the same as in B. E, Same as in A, but for mAHP after a burst of 25 APs at 60 Hz (baseline membrane potential at −65 mV). F, Same as in B, but for mAHP after AP bursts. G, Same as in C, but for mAHP after AP bursts. H, Same as in D, but for mAHP after AP bursts. Control bars are the same as in F. *p < 0.05, **p < 0.01. ns, Not significant. Data are means ± SEM.
Figure 6.
Figure 6.
FMRP interacts with SK2, while expression of all SK-channel isoforms is unaltered in the CA3 of Fmr1 KO mice. A, Western blot analysis from the CA3 region showed unaltered expression of all SK-channel isoforms in Fmr1 KO mice. SK-channel intensity was normalized to α-tubulin on the same blot. Samples were then normalized to the average WT value. L and S denote long and short isoforms of SK2 (Allen et al., 2011). SK1: N = 9, p = 0.711; SK2-L: N = 6, p = 0.815; SK2-S: N = 6, p = 0.493; SK3, N = 6, p = 0.988. B, Estimate of total available SK-channel contribution to threshold based on Figure 3E,F. C, In vitro FMRP-SK-channel binding assay. Brain lysates from WT mice were incubated with GST-tagged FMRP298 or GST tag fragments, and the levels of all SK-channel isoforms (SK1, SK2, and SK3) bound to FMRP fragments were analyzed by Western blot with the indicated antibodies. Representative Western blots from n = 4 experiments. Ponceau staining is shown as control for an equal amount of GST-tagged FMRP298 or GST tag fragments used in this assay. D, E, Acute intracellular administration of FMRP298 normalized abnormal AP threshold (D) and mAHP (E) in Fmr1 KO neurons, but heat-inactivated FMRP298 failed to do this. WT data in D are from Figure 2E. *p < 0.05, **p < 0.01. ns, Not significant. Data are means ± SEM.
Figure 7.
Figure 7.
Genetic reintroduction of FMRP234 rescued hyperexcitability in CA3 pyramidal neurons of Fmr1 KO mice. A, Construction of BAC transgenic mouse with truncated FMRP234 (amino acids 1–234). Top, BAC containing the murine Fmr1 was modified to retain only codons for amino acids 1–234, retaining all 5′ and 3′ untranslated regions. Bottom, Diagram of predicted truncated FMRP234. B, BAC DNA was injected into fertilized oocytes and then implanted into pseudopregnant females (top). Three independent founder pups were recovered (bottom, *). C, Western blot of whole-brain cytoplasmic (C) or nuclear (N) lysates from WT, Fmr1 KO, and BAC transgenic mice with FMRP234 on a background of WT, Fmr1 KO, or heterozygote (HET) showing expression of the FMRP234. D, Immunostaining of cortical slices from WT, Fmr1 KO, and BAC transgenic mice on Fmr1 KO background showing reactivity to 2F5-1 FMRP antibody in WT and BAC transgenic (FMRP234) mice. E, Reintroduction of FMRP234 restored the AP threshold defects. F, Reintroduction of FMRP234 restored the mAHP defects after single APs. G, The same as in F, but for mAHP after a burst of 25 APs at 60 Hz (at −65 mV). H, I, Reintroduction of FMRP234 normalized the AP firing at −51 mV. H, Sample AP traces. I, Summarized data. *p < 0.05, **p < 0.01. ns, Not significant. Data are means ± SEM.
Figure 8.
Figure 8.
Abnormal SK-channel activity causes defective signal transmission in CA3 PCs of Fmr1 KO mice. A, Sample trace of EPSCs and APs recorded from CA3 PCs evoked by stimulating MFs. Stimulation protocol (top; 40 Hz × 25 stimuli); EPSCs (middle) representing input from MFs, and APs (bottom) representing CA3 PC output in response to MF input. The stimulation artifacts are shown in AP traces, but were removed from EPSC traces for clarity. Note the multiple APs that were much more frequently observed after each stimulus in Fmr1 KO than in WT neurons (*). B, EPSC amplitudes were indistinguishable in Fmr1 KO and WT neurons, both in absolute value at baseline and relative changes during the trains. C, AP number after each stimulus was markedly increased during the burst in Fmr1 KO neurons. D, E, Effect of intracellular 1-EBIO on MF-CA3 PC transmission. D, Sample AP traces. Note the AP failures both in WT and Fmr1 KO neurons (↑). E, SK-opener 1-EBIO rescued signal transmission of MF-CA3 PCs. F, G, Same as in D and E except for intracellular administration of NS309. *p < 0.05, **p < 0.01. ns, Not significant. Data are means ± SEM.
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