Differential regulation of BK channels by fragile X mental retardation protein

Abstract

Fragile X mental retardation protein (FMRP) is an RNA-binding protein prominently expressed in neurons. Missense mutations or complete loss of FMRP can potentially lead to fragile X syndrome, a common form of inherited intellectual disability. In addition to RNA regulation, FMRP was also proposed to modulate neuronal function by direct interaction with the large conductance Ca2+- and voltage-activated potassium channel (BK) β4 regulatory subunits (BKβ4). However, the molecular mechanisms underlying FMRP regulation of BK channels were not studied in detail. We have used electrophysiology and super-resolution stochastic optical reconstruction microscopy (STORM) to characterize the effects of FMRP on pore-forming BKα subunits, as well as the association with regulatory subunits BKβ4. Our data indicate that, in the absence of coexpressed β4, FMRP alters the steady-state properties of BKα channels by decreasing channel activation and deactivation rates. Analysis using the Horrigan-Aldrich model revealed alterations in the parameters associated with channel opening (L0) and voltage sensor activation (J0). Interestingly, FMRP also altered the biophysical properties of BKαβ4 channels favoring channel opening, although not as dramatically as BKα. STORM experiments revealed clustered multi-protein complexes, consistent with FMRP interacting not only to BKαβ4 but also to BKα. Lastly, we found that a partial loss-of-function mutation in FMRP (R138Q) counteracts many of its functional effects on BKα and BKαβ4 channels. In summary, our data show that FMRP modulates the function of both BKα and BKαβ4 channels.

FMRP sustains presynaptic function via control of activity-dependent bulk endocytosis

“…The R138Q mutation does not affect the role of FMRP in translation regulation (16,24). Therefore, its ability to rescue defective ADBE in KO neurons supports the hypothesis that presynaptic defects observed in the absence of FMRP are translation-independent (15).…”

“…We exploited the ability of WT FMRP to rescue TMR-dextran uptake to perform molecular replacement studies using two loss of function mutants: FMRP I304N -that does not bind ribosomes (De Boulle et al, 1993) and FMRP R138Q -that does not bind BK channels (Kshatri et al, 2020;Myrick et al, 2015) ( Figure 4C). When these experiments were performed, FMRP WT restored TMR-dextran uptake in Fmr1 KO cultures as previously observed ( Figure 4D).…”

“…However, it also has protein synthesis-independent presynaptic functions, such as mediating ion channel gating, activation, and density (reviewed in Ferron, 2016). For example, FMRP interacts with Slack potassium channels (Brown et al, 2010), N-type calcium channels (Ferron et al, 2014) and large conductance voltage and calcium-gated big potassium (BK) channels (Deng et al, 2013;Deng and Klyachko, 2016;Kshatri et al, 2020). The absence of FMRP at CA3-CA1 hippocampal synapses leads to reduced BK channel activity and excessive AP broadening (Deng et al, 2013;Deng and Klyachko, 2016;Wang et al, 2014), resulting in increased presynaptic calcium influx during high frequency stimulation (Deng et al, 2011;Deng et al, 2013).…”

FMRP differentially regulates BK channels

“…For example, FMRP is thought to increase the open probability of type II BK channels by binding to their auxiliary β 4 subunits, thereby modifying neuronal action potentials and neurotransmission (1,2). In this issue of JGP, Kshatri et al demonstrate that FMRP also regulates type I BK channels lacking β subunits, indicating that the effects of FMRP on BK currents are more complex than originally thought (3).…”

“…FMRP also clustered with BKαβ 4 channels, but its effects on this type of BK channel were relatively modest, only slowing the rate of deactivation by a factor of ∼2. Kshatri et al found that, rather than abrogating the effects of β 4 by sequestering it, FMRP potentiates β 4 function: while β 4 enhances voltage sensor activation in BKα channels, FMRP promotes pore opening to further enhance channel activity (3).…”