Genetic upregulation of BK channel activity normalizes multiple synaptic and circuit defects in a mouse model of fragile X syndrome

Abstract

Single-channel recordings in CA3 pyramidal neurons revealed that large-conductance calcium-activated K(+) (BK) channel open probability was reduced by loss of fragile X mental retardation protein (FMRP) and that FMRP acts on BK channels by modulating the channel's gating kinetics. Fmr1/BKβ4 double knockout mice were generated to genetically upregulate BK channel activity in the absence of FMRP. Deletion of the BKβ4 subunit alleviated reduced BK channel open probability via increasing BK channel open frequency, but not through prolonging its open duration. Genetic upregulation of BK channel activity via deletion of BKβ4 normalized action potential duration, excessive glutamate release and short-term synaptic plasticity during naturalistic stimulus trains in excitatory hippocampal neurons in the absence of FMRP. Genetic upregulation of BK channel activity via deletion of BKβ4 was sufficient to normalize excessive epileptiform activity in an in vitro model of seizure activity in the hippocampal circuit in the absence of FMRP. Loss of fragile X mental retardation protein (FMRP) causes fragile X syndrome (FXS), yet the mechanisms underlying the pathophysiology of FXS are incompletely understood. Recent studies identified important new functions of FMRP in regulating neural excitability and synaptic transmission via both translation-dependent mechanisms and direct interactions of FMRP with a number of ion channels in the axons and presynaptic terminals. Among these presynaptic FMRP functions, FMRP interaction with large-conductance calcium-activated K(+) (BK) channels, specifically their auxiliary β4 subunit, regulates action potential waveform and glutamate release in hippocampal and cortical pyramidal neurons. Given the multitude of ion channels and mechanisms that mediate presynaptic FMRP actions, it remains unclear, however, to what extent FMRP-BK channel interactions contribute to synaptic and circuit defects in FXS. To examine this question, we generated Fmr1/β4 double knockout (dKO) mice to genetically upregulate BK channel activity in the absence of FMRP and determine its ability to normalize multilevel defects caused by FMRP loss. Single-channel analyses revealed that FMRP loss reduced BK channel open probability, and this defect was compensated in dKO mice. Furthermore, dKO mice exhibited normalized action potential duration, glutamate release and short-term dynamics during naturalistic stimulus trains in hippocampal pyramidal neurons. BK channel upregulation was also sufficient to correct excessive seizure susceptibility in an in vitro model of seizure activity in hippocampal slices. Our studies thus suggest that upregulation of BK channel activity normalizes multi-level deficits caused by FMRP loss.

Figure 1. BKβ4 deletion alleviates the decreased BK channel open probability in the absence of FMRP

A, traces of BK single channel recordings at −80 mV, 10 μm free Ca²⁺, 33°C, from CA3 PCs of acute hippocampal slices. C, closed state; O, open state; ★, subconductance state. B–D, all‐point amplitude histograms (B) were fitted by two Gaussian fits, representing the open (peak around −20 pA) and closed (peak around 0 pA) states. BK unitary current was determined by the amplitude difference between two peaks of Gaussian fits, which then was used to estimate BK single channel conductance (C). Open probabilities were calculated by the area under the Gaussian fits [open area/(open area + closed area)], and summarized in D. E–G, representative cumulative probability of open dwell time (E), closed dwell time (F) and inter‐open intervals (G). Insets represent the mean open dwell time constant (E), the mean closed dwell time constant (F), and the mean open frequency (G). **P < 0.01 Fmr1 KO vs. WT; ^## P < 0.01 dKO vs. KO; ⁺ P < 0.01, ⁺⁺ P < 0.01 dKO vs. WT; ns, not significant. Error bars represent SEM.

Figure 2. BKβ4 deletion normalizes excessive AP broadening defects in excitatory CA3 neurons caused by loss of FMRP

A, upper panel, sample traces of the 1st and last APs of the 25‐AP train at 60 Hz. Long line indicates the baseline of the first APs, short lines AP duration measured at −10 mV level. A, lower panel, summarized data of actual AP widths for baseline and burst. B, time course of normalized AP width during 25‐AP train at 60 Hz. AP duration was normalized to its own basal values. C, grouped data of actual AP widths of baseline and burst for different background mice. D, time course of normalized AP width during 25‐AP train at 60 Hz among different background mice. *P < 0.05, **P < 0.01 vs. WT; ^## P < 0.01 vs. Fmr1 KO; ^&& P < 0.01 vs. β4 KO; ns, not significant. Error bars represent SEM.

Figure 3. BKβ4 deletion ameliorates spontaneous AP defects in excitatory CA3 neurons caused by loss of FMRP

A, spontaneous AP (sAP) duration measured at −10 mV level. Inset, sample sAP traces showing the level of AP duration measured (short cyan line). Red‐boxed area is enlarged in C. B, data from A averaged by frequency bands shown on x‐axis. C, enlargement of the red box in A indicates the fAHP measurements. fAHP is defined by the potential difference between the baseline and the lowest point within 5 ms after AP peak. Baseline was determined as an average of a 0.5‐ms time period in the recording trace immediately before AP initiation (2–2.5 ms before individual AP threshold). D, summarized data of fAHP amplitude. **P < 0.01, ⁺ P < 0.05 vs. WT; ^## P < 0.01 vs. KO; ^& P < 0.05, ^&& P < 0.01 vs. β4 KO; ns, not significant. Error bars represent SEM.

Figure 4. BKβ4 deletion corrects neurotransmission and short‐term dynamics defects at excitatory hippocampal CA3–CA1 synapses in the absence of FMRP

A, normalized EPSC traces recorded in CA1 PCs in response to SC stimulation with a train of 25 stimuli at 60 Hz. Note that EPSC traces were normalized to their own baseline amplitude measured at 0.2 Hz of the same train for better visual comparison (the first EPSCs of the burst in each condition were scaled to the same value, representing 100% shown as a bar). EPSC stimulus artifacts were removed for clarity. B, summarized data of EPSC amplitude during the train. Data were normalized to their own baseline. C, normalized EPSC amplitudes averaged from the last two EPSCs of the burst. D, the normalized EPSC amplitudes at the end of burst for WT and KO mice among different backgrounds. **P < 0.01, ^## P < 0.01, ^&& P < 0.01 vs. β4 KO; ns, not significant. Error bars represent SEM.

Figure 5. BKβ4 deletion corrects abnormal short‐term dynamics during natural stimulus patterns at excitatory hippocampal CA3–CA1 synapses in the absence of FMRP

A, changes in EPSC amplitude during natural stimulus train plotted as a function of time. The top shows the natural stimulus pattern used (Fenton & Muller, 1998), preceded and followed by four control stimuli at 0.2 Hz. B, same data as in A but plotted as a function of stimulus number for better visual comparison. Inset shows EPSCs 95–102 during the natural stimulus train, scaled to their own controls. C, average synaptic gain during natural stimulus trains plotted as a function of frequency bands. *P < 0.05, ^# P < 0.05, **P < 0.01, ^## P < 0.01, ^& P < 0.05, ^&& P < 0.01 vs. β4 KO; ns, not significant. Error bars represent SEM.

Figure 6. BKβ4 deletion normalizes elevated epileptiform activity in the hippocampal slices caused by FMRP loss in vitro

Epileptiform activity in slices was induced by bath application of 4‐AP (100 μm). Field recording electrodes were put at CA3 PC layer; APs recordings were made by whole‐cell configuration of CA3 PCs 50–100 μm apart from field recording electrodes. A–D, simultaneous field recordings of epileptiform activity from CA3 PC layer and whole‐cell recording of APs from CA3 PCs. Right panels are enlarged plots of the boxed area in the left traces. E, pooled data showing the epileptiform activity latency, i.e. time interval from 4‐AP application to the occurrence of first event. F, frequencies of epileptiform activity measured upon even stabilization. G, duration of epileptiform events. H, number of APs per epileptiform event. Only the APs with a peak potential above 0 mV were counted. **P < 0.01 vs. WT; ^## P < 0.01 vs. KO; ^&& P < 0.01 vs. β4 KO; ns, not significant. Error bars represent SEM.