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.
are ongoing at two more locations); for an 871 atom (protein, 50 water molecules, and the ion) pore section, quantum calculations optimized (energy minimized) the section, and the energy and ion hydration at each ion position were determined. The protein structures the water column. It appears that hydration may explain the reason for the strong conservation of the threonine at the bottom of the selectivity filter, which interacts with water in a characteristic way, producing a ''basket'' of four water molecules H-bonded to the threonines. One remaining problem is determining how the ion exchanges its own hydration shell to be cosolvated by the four threonine hydroxyl groups, breaking the water ''basket''. Once that is accomplished, the ion can proceed through the selectivity filter. At the entrance to the pore at the bottom of the water column (i.e., the gate), hydration is strongly rearranged when an ion arrives. A small increase in diameter from the closed state, approximately 5 Å , is adequate to admit the ion and hold it by a local increase in the density of the water hydrating it; a wide open gate would often lose the ion back to solution, greatly diminishing the current. The entering ion remains held as the ion above it moves; following this, the gate ion can move up to the center of the pore cavity. Optimizations were done using HF/6-31G*, while energy calculations used B3LYP/6-31G** on the optimized structures.
Z L 0.26 0.29 0.23 0.27 J 0 0.05 0.17 0.12 0.15 Z J 0.61 0.58 0.59 0.55 D 13 13 16 11 V HC, mV 124 77 91 87 V HO, mV 18 -35 -28 -23
H þ currents when expressed in HEK-293 cells. The data indicate that H þ transfer is unlikely to be mediated by H þ shuttling through pairs of nearly acidic side chains, but are consistent with the hypothesis that 'aqueous' or water-wire proton transfer is the primary mechanism of H þ conduction in Hv1. Different mutant combinations produce similar defects in pH-dependent gating, suggesting that interactions among networked side chains confer emergent biophysical properties in Hv1. Experimental data were used to build a refined activatedstate Hv1 VS model structure (Hv1 J) that was subjected to molecular dynamics simulation. Although Hv1 J is similar overall to Hv1 B (Ramsey, et al., 2010), differences in the positions of specific side chains suggest a channel-opening conformational rearrangement that follows S4 movement, as predicted from gating currents (De La Rosa, et al., 2016).
location-dependent, and overlapping, making dissection of this cacophonous phenomenon difficult. Pinpointing the physiological role of solo ion channels is further impeded by a lack in existing technologies capable of determining when and where ion channel types are activated in native tissue. We have generated an endogenous channel activity probe (ECAP) that optically tracks voltage activation of Kv2 type K þ channels. Guided by a conformation-selective, peptide toxin, the fluorescently tagged ECAP binds to resting voltage sensing domain of Kv2 and dissociates from activated voltage sensors. We demonstrate that loss of fluorescent ECAP signal from CA1 neurons in rat hippocampal slices coincides with voltage activation of endogenous Kv2 channels. Confirmatory tests show that the ECAP colocalizes with Kv2-GFP channels in neurons and in cultured cells with or without the presence of the channel's auxiliary subunit AMIGO-1, but not with other channel subtypes. ECAP signal dynamics were both frequency and voltage-stimuli dependent, setting methodical precedence for current and future explorations tracking channel activation or protein conformational changes during physiological signaling.
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