FMRP directly binds to Slack channel subunits. (a,b) Slack antibodies co-immunoprecipitated FMRP from WT (a, lane 2) but not from Fmr1^−/y mice synaptosomal samples (a, lane 5). Immunoblots were performed using FMRP (a) or Slack (b) antibodies (n=4). Input synaptosomal samples from WT or Fmr1^−/y mice were isolated prior to co-immunoprecipitation (a, lanes 1, 4, b, lanes 1, 4). (c) FMRP was co-immunoprecipitated using anti-FLAG antibodies from Slack-FLAG HEK cells (c, lane 4) but not from untransfected HEK cells (c, lane 3) or Slo1-FLAG cells (c, lane 8, n=3). (d) The untransfected HEK and Slack-FLAG HEK immunopreciptates were also separately immunoblotted using anti-FLAG antibody detecting Slack-FLAG channels in transfected cells. (e) Slack-FMRP complexes contained known FMRP mRNA targets. Map1b and Arc mRNA were detected by isolating RNA after co-IP with anti-Slack antibodies followed by RT-PCR in WT samples (e, lanes 3, 4) but not Fmr1^−/y samples (e, lanes 9, 10; n=3).

FMRP alters gating of full-length Slack-B channels, but not Slack-BΔ804, in inside-out patches. (a) Current recordings before (control), 20 seconds after application of 100nM FMRP(1–298) to the cytoplasmic face of the patch, and after washout of FMRP(1–298). Top traces show representative 10 second examples of recording at −40mV and bottom traces show expanded time views of the same patch. The increase in channel activity (top trace) and the suppression of substates, S, (bottom trace) are evident after FMRP reactivity. Dashed lines indicate closed state and number of channels. (b) All-points amplitude histograms plotted on linear (top) and logarithmic scale (bottom), of control, FMRP(1–298) treated, and recovery (after washout). (c) Application of heat-inactivated FMRP(1–298) had no effect on Slack-B channel activity; n=5. (d) Top traces show representative 10 second recording at −80 mV and bottom traces show expanded time views of Slack-BΔ804 channel activity before (control) and after application of FMRP(1–298). There are no changes in NP_o or in the occurrence of substates, S. (e) All-points amplitude histograms show no significant changes in Slack-BΔ804 channel activity in response to FMRP(1–298).

I_K(Na) is reduced in Fmr1^−/y MNTB neurons. (a) Images of Slack-B immunoreactivity in MNTB of WT and Fmr1^−/y mice (scale bar=40μm) (b) Immunoblots for Slack-B in synaptosomal samples of WT and Fmr1^−/y mice. As a loading control, the same samples were probed with antibodies against GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) (c) Quantification of Slack immunoreactivity. MNTB sections (n=63) were quantified using the mean optical density of pixels above threshold (OD IHC±s.e.m.). There was a 15% increase in Slack immunoreactivity in MNTB sections from Fmr1^−/y mice compared to WT (p=0.02, n=3). Synaptosomes from WT and Fmr1^−/y mice contained similar amounts of Slack (OD IM±s.e.m.; n=3). (d) Representative traces of I_K(Na) in lateral MNTB neurons from WT and Fmr1^−/y mice (20mM internal Na⁺). Outward currents evoked by voltage steps between −80 to +60mV in 20mV increments from a holding potential of −40mV. 1μM TTX and 20μM ZD-7288 were included in the bath to block Na⁺ and hyperpolarization-activated cation currents (I_H), respectively. (e) I–V plots of K_Na as a proportion of total K⁺ currents. The K_Na component of current from Fmr1^−/y mice (n=5) was significantly reduced relative to WT mice (n=4, p=0.02; two-sample t-test).