Regulation of neuronal excitability by interaction of fragile X mental retardation protein with slack potassium channels

Abstract

Loss of the RNA-binding protein fragile X mental retardation protein (FMRP) represents the most common form of inherited intellectual disability. Studies with heterologous expression systems indicate that FMRP interacts directly with Slack Na(+)-activated K(+) channels (K(Na)), producing an enhancement of channel activity. We have now used Aplysia bag cell (BC) neurons, which regulate reproductive behaviors, to examine the effects of Slack and FMRP on excitability. FMRP and Slack immunoreactivity were colocalized at the periphery of isolated BC neurons, and the two proteins could be reciprocally coimmunoprecipitated. Intracellular injection of FMRP lacking its mRNA binding domain rapidly induced a biphasic outward current, with an early transient tetrodotoxin-sensitive component followed by a slowly activating sustained component. The properties of this current matched that of the native Slack potassium current, which was identified using an siRNA approach. Addition of FMRP to inside-out patches containing native Aplysia Slack channels increased channel opening and, in current-clamp recordings, produced narrowing of action potentials. Suppression of Slack expression did not alter the ability of BC neurons to undergo a characteristic prolonged discharge in response to synaptic stimulation, but prevented recovery from a prolonged inhibitory period that normally follows the discharge. Recovery from the inhibited period was also inhibited by the protein synthesis inhibitor anisomycin. Our studies indicate that, in BC neurons, Slack channels are required for prolonged changes in neuronal excitability that require new protein synthesis, and raise the possibility that channel-FMRP interactions may link changes in neuronal firing to changes in protein translation.

Figure 1.

Slack channels and FMRP interact and are colocalized in BC neurons. A, Protein was extracted from BC clusters and used to immunoprecipitate FMRP with a mouse anti-FMRP antibody. Samples were immunoblotted with a rabbit anti-Slack antibody, and an immunoreactive band was detected at 90 kDa. There was no immunoreactive band detected in control immunoprecipitations using an anti-mouse IgG or when immunoblotting with secondary anti-rabbit antibody alone. Additionally, no immunoreactive band was detected when the rabbit anti-Slack antibody was preincubated with purified rat Slack before immunoblotting. Input samples were positive for the 90 kDa immunoreactive band. B, The reverse coimmunoprecipitation experiment showed that Slack immunoprecipitates with FMRP (immunoreactive band at 75 kDa). Control experiments were negative as described in A. C, Aplysia BC neurons are immunoreactive with both Slack and FMRP antibodies. Confocal immunofluorescence staining for FMRP (left top) (green); Slack (middle top) (red); Slack and FMRP colocalize in BC neurons (left bottom). Differential interference contrast (DIC) imaging is shown (middle bottom). Scale bar, 30 μm.

Figure 2.

Detection of sodium-activated potassium current in Aplysia BC neurons. A, Injection of Na⁺ increased outward currents while injection of K⁺ had no effect. Calibration: 5 nA, 50 ms. Traces show superimposed currents evoked from a holding potential of −60 mV to command potentials between −60 and +70 mV in 10 mV increments. B, Bar graphs summarizing the effects of injection of Na⁺ (n = 6), or K⁺ (n = 3) on peak currents are measured at +20 mV. Data are expressed as mean ± SEM. *Significant difference from control (p < 0.05; n = 6; paired t test). C, Current–voltage relationships for the difference current measured by subtracting peak currents before and after Na⁺ injection. D, Single-channel inside-out patches recordings from BC neurons shows increased open probability of K_Na channels with increasing Na⁺ concentration (holding potential, +30 mV). Calibration: 5 pA, 100 ms. E, All-points amplitude histograms for 10 s of recording in 0, 120, or 200 mm Na⁺. F, K_Na current–voltage (I--V plot) relationship in symmetric 470 mm KCl. Unitary conductance is ∼167 pS (n = 3).

Figure 3.

Slack contributes to outward K⁺ current in BC neurons. A, Slack staining was reduced in BC neurons injected with siRNA-Slack. Scale bar, 25 μm. B, C, Immunoblots demonstrating that Slack protein level is decreased in Slack siRNA-treated BC neurons. Data are expressed as mean ± SEM (n = 3; *p < 0.05, paired t test). Immunoblots against rabbit anti-actin antibodies are provided as a control for equivalence of protein samples. No change in Slack protein levels was observed in scrambled siRNA-treated BC neurons (n = 3; NS, paired t test). D, Averaged traces of outward current in control and Slack siRNA-treated neurons. Calibration: 5 nA, 50 ms. Subtracted currents for treatment with either Slack siRNA (E) (calibration: 0.5 nA, 50 ms), scrambled siRNA (F) (calibration: 0.5 nA, 50 ms), Shab siRNA (G) (calibration: 0.4 nA, 50 ms), and Slack siRNA pretreated with 100 μm TTX (H) (calibration: 0.5 nA, 50 ms). All currents were recorded by depolarizing the membrane from a holding potential of −60 mV to a test potential of +70 mV.

Figure 4.

Injection of recombinant FMRP(1–298) into BC neurons increased Slack-like currents. A, Superimposed currents before and after injection of FMRP(1–298) (left, calibration: 10 nA, 250 ms) and difference currents (right, calibration: 2 nA, 250 ms). Holding potential was maintained at −60 mV, the outward current was evoked by pulses to +70 mV. B, Injection of heat-inactivated FMRP(1–298) had no effect on outward current in nASW. Calibration: 2 nA, 100 ms. C, Pretreatment of BC neurons with 100 μm TTX before injection of FMRP(1–298) eliminated the fast component of FMRP-induced outward current. Calibration: 0.5 nA, 100 ms. D, Difference current recorded on switching the external medium from Na⁺-containing nASW to zero Na⁺ ASW. Calibration: 1 nA, 100 ms. E, Injection of FMRP(1–298) had no effect when neurons were recorded in zero Na⁺ ASW external medium. Calibration: 2 nA, 100 ms. F, Current–voltage relationship of subtracted Slack-like current in nASW. G, Group data for steady-state difference currents produced by injecting either FMRP(1–298) in nASW, inactivated FMRP(1–298) in nASW, or FMRP(1–298) in zero Na⁺ ASW (n = 4 in each group; test potential, +70 mV).

Figure 5.

Addition of FMRP(1–298) increases K_Na channel open probability, hyperpolarizes membrane potential, and decreases input resistance in BC neurons. A, Representative traces before and after addition of FMRP(1–298) to an excised inside-out patch containing Na⁺-activated K⁺ channels (80 mm Na⁺ at cytoplasmic face; holding potential, +30 mV; calibration: 2 pA, 0.2 s; C, closed state; O, open state). B, All-points amplitude histograms before and after addition of FMRP(1–298) to an excised patch as in A (C, closed state; S, subconducance state; O, open state). C, Group data for effect of FMRP(1–298) or heat-inactivated FMRP(1–298) on channel open probability (n = 4). Data are expressed as the mean ± SEM (*p < 0.05; n = 4; paired t test). D, Injection of FMRP(1–298) hyperpolarizes membrane potential and decreases input resistance in BC neurons. Representative traces of the effect of injection of FMRP(1–298) or heat-inactivated FMRP(1–298) on action potentials evoked by injecting 0.6 nA current pulses. Calibration: 10 mV, 50 ms. E, Summary of changes in membrane potential, input resistance following injection of FMRP(1–298). Data are expressed as mean ± SEM (n = 4, **p < 0.005, unpaired t test; n = 4, *p < 0.05, paired t test).

Figure 6.

Recovery from the inhibited state of BC neurons requires new protein synthesis. A, Panels shows a control discharge evoked by a first stimulus (top) and failure to trigger discharge within 1 h of the termination of the first discharge (center). The bottom panel shows a full-length discharge triggered the next day. Calibration: 50 μV and 2, 0.5, and 2 min for top to bottom panels. The arrows indicate times of stimulation. B, Treatment with Slack siRNA prevents recovery from the inhibited state. Top trace, Discharge after 5 d of Slack siRNA treatment. Center trace, Stimulation within 1 h of the termination of the first discharge. Bottom trace, Shortened discharge triggered 24 h after the first discharge in the Slack siRNA-treated neurons. Calibration: 50 μV and 2, 0.5, and 2 min for top to bottom panels, respectively. C, Discharge durations on two consecutive days after no treatment (Control) or treatment with Slack or scrambled siRNA (mean ± SEM; n = 6; **p = 0.0021, paired t test, two-tailed). D, Effects of anisomycin on mean durations of first discharges triggered on 2 consecutive days (n = 15; ***p = 0.0001, paired t tests, two-tailed). The bar graph at right shows duration on day 2 with no prior stimulation on day 1 (n = 10).