A novel BK channel-targeted peptide suppresses sound evoked activity in the mouse inferior colliculus.
ABSTRACT: Large conductance calcium-activated (BK) channels are broadly expressed in neurons and muscle where they modulate cellular activity. Decades of research support an interest in pharmaceutical applications for modulating BK channel function. Here we report a novel BK channel-targeted peptide with functional activity in vitro and in vivo. This 9-amino acid peptide, LS3, has a unique action, suppressing channel gating rather than blocking the pore of heterologously expressed human BK channels. With an IC50 in the high picomolar range, the apparent affinity is higher than known high affinity BK channel toxins. LS3 suppresses locomotor activity via a BK channel-specific mechanism in wild-type or BK channel-humanized Caenorhabditis elegans. Topical application on the dural surface of the auditory midbrain in mouse suppresses sound evoked neural activity, similar to a well-characterized pore blocker of the BK channel. Moreover, this novel ion channel-targeted peptide rapidly crosses the BBB after systemic delivery to modulate auditory processing. Thus, a potent BK channel peptide modulator is open to neurological applications, such as preventing audiogenic seizures that originate in the auditory midbrain.
Project description:Non-mammalian vertebrates rely on electrical resonance for frequency tuning in auditory hair cells. A key component of the resonance exhibited by these cells is an outward calcium-activated potassium current that flows through large-conductance calcium-activated potassium (BK) channels. Previous work in midshipman fish (Porichthys notatus) has shown that BK expression correlates with seasonal changes in hearing sensitivity and that pharmacologically blocking these channels replicates the natural decreases in sensitivity during the winter non-reproductive season. To test the hypothesis that reducing BK channel function is sufficient to change auditory thresholds in fish, morpholino oligonucleotides (MOs) were used in larval zebrafish (Danio rerio) to alter expression of slo1a and slo1b, duplicate genes coding for the pore-forming ?-subunits of BK channels. Following MO injection, microphonic potentials were recorded from the inner ear of larvae. Quantitative real-time PCR was then used to determine the MO effect on slo1a and slo1b expression in these same fish. Knockdown of either slo1a or slo1b resulted in disrupted gene expression and increased auditory thresholds across the same range of frequencies of natural auditory plasticity observed in midshipman. We conclude that interference with the normal expression of individual slo1 genes is sufficient to increase auditory thresholds in zebrafish larvae and that changes in BK channel expression are a direct mechanism for regulation of peripheral hearing sensitivity among fishes.
Project description:BACKGROUND: BK channels are usually activated by membrane depolarization and cytoplasmic Ca(2+). Especially,the activity of BK channel (?+?4) can be modulated by martentoxin, a 37 residues peptide, with Ca(2+)-dependent manner. gBK channel (glioma BK channel) and BK channel (?+?1) possessed higher Ca(2+) sensitivity than other known BK channel subtypes. METHODOLOGY AND PRINCIPAL FINDINGS: The present study investigated the modulatory characteristics of martentoxin on these two BK channel subtypes by electrophysiological recordings, cell proliferation and Ca(2+) imaging. In the presence of cytoplasmic Ca(2+), martentoxin could enhance the activities of both gBK and BK channel (?+?1) subtypes in dose-dependent manner with EC(50) of 46.7 nM and 495 nM respectively, while not shift the steady-state activation of these channels. The enhancement ratio of martentoxin on gBK and BK channel (?+?1) was unrelated to the quantitative change of cytoplasmic Ca(2+) concentrations though the interaction between martentoxin and BK channel (?+?1) was accelerated under higher cytoplasmic Ca(2+). The selective BK pore blocker iberiotoxin could fully abolish the enhancement of these two BK subtypes induced by martentoxin, suggesting that the auxiliary ? subunit might contribute to the docking for martentoxin. However, in the absence of cytoplasmic Ca(2+), the activity of gBK channel would be surprisingly inhibited by martentoxin while BK channel (?+?1) couldn't be affected by the toxin. CONCLUSIONS AND SIGNIFICANCE: Thus, the results shown here provide the novel evidence that martentoxin could increase the two Ca(2+)-hypersensitive BK channel subtypes activities in a new manner and indicate that ? subunit of these BK channels plays a vital role in this enhancement by martentoxin.
Project description:Large-conductance, Ca(2+)- and voltage-sensitive K(+) (BK) channels regulate neuronal functions such as spike frequency adaptation and transmitter release. BK channels are composed of four Slo1 subunits, which contain the voltage-sensing and pore-gate domains in the membrane and Ca(2+) binding sites in the cytoplasmic domain, and accessory ? subunits. Four types of BK channel ? subunits (?1-?4) show differential tissue distribution and unique functional modulation, resulting in diverse phenotypes of BK channels. Previous studies show that both the ?1 and ?2 subunits increase Ca(2+) sensitivity, but different mechanisms may underline these modulations. However, the structural domains in Slo1 that are critical for Ca(2+)-dependent activation and targeted by these ? subunits are not known. Here, we report that the N termini of both the transmembrane (including S0) and cytoplasmic domains of Slo1 are critical for ?2 modulation based on the study of differential effects of the ?2 subunit on two orthologs, mouse Slo1 and Drosophila Slo1. The N terminus of the cytoplasmic domain of Slo1, including the AC region (?A-?C) of the RCK1 (regulator of K(+) conductance) domain and the peptide linking it to S6, both of which have been shown previously to mediate the coupling between Ca(2+) binding and channel opening, is specifically required for the ?2 but not for the ?1 modulation. These results suggest that the ?2 subunit modulates the coupling between Ca(2+) binding and channel opening, and, although sharing structural homology, the BK channel ? subunits interact with structural domains in the Slo1 subunit differently to enhance channel activity.
Project description:To probe structure and gating-associated conformational changes in BK-type potassium (BK) channels, we examined consequences of Cd(2+) coordination with cysteines introduced at two positions in the BK inner pore. At V319C, the equivalent of valine in the conserved Kv proline-valine-proline (PVP) motif, Cd(2+) forms intrasubunit coordination with a native glutamate E321, which would place the side chains of V319C and E321 much closer together than observed in voltage-dependent K(+) (Kv) channel structures, requiring that the proline between V319C and E321 introduces a kink in the BK S6 inner helix sharper than that observed in Kv channel structures. At inner pore position A316C, Cd(2+) binds with modest state dependence, suggesting the absence of an ion permeation gate at the cytosolic side of BK channel. These results highlight fundamental structural differences between BK and Kv channels in their inner pore region, which likely underlie differences in voltage-dependent gating between these channels.
Project description:Large-conductance Ca2+- and voltage-dependent K+ (BK) channels display diverse biological functions while their pore-forming ? subunit is coded by a single Slo1 gene. The variety of BK channels is correlated with the effects of BK? coexpression with auxiliary ? (?1-?4) subunits, as well as newly defined ? subunits. Charybdotoxin (ChTX) blocks BK channel through physically occluding the K+-conduction pore. Human brain enriched ?4 subunit (h?4) alters the conductance-voltage curve, slows activation and deactivation time courses of BK channels. Its extracellular loop (h?4-loop) specifically impedes ChTX to bind BK channel pore. However, the structure of ?4 subunit's extracellular loop and the molecular mechanism for gating kinetics, toxin sensitivity of BK channels regulated by ?4 are still unclear. To address them, here, we first identified four disulfide bonds in h?4-loop by mass spectroscopy and NMR techniques. Then we determined its three-dimensional solution structure, performed NMR titration and electrophysiological analysis, and found that residue Asn123 of ?4 subunit regulated the gating and pharmacological characteristics of BK channel. Finally, by constructing structure models of BK?/?4 and thermodynamic double-mutant cycle analysis, we proposed that BK? subunit might interact with ?4 subunit through the conserved residue Glu264(BK?) coupling with residue Asn123(?4).
Project description:BK channels are regulated by two distinct physiological signals, transmembrane potential and intracellular Ca(2+), each acting through independent modular sensor domains. However, despite a presumably central role in the coupling of sensor activation to channel gating, the pore-lining S6 transmembrane segment has not been systematically studied. Here, cysteine substitution and modification studies of the BK S6 point to substantial differences between BK and Kv channels in the structure and function of the S6-lined inner pore. Gating shifts caused by introduction of cysteines define a pattern and direction of free energy changes in BK S6 distinct from Shaker. Modification of BK S6 residues identifies pore-facing residues that occur at different linear positions along aligned BK and Kv S6 segments. Periodicity analysis suggests that one factor contributing to these differences may be a disruption of the BK S6 ?-helix from the unique diglycine motif at the position of the Kv hinge glycine. State-dependent MTS accessibility reveals that, even in closed states, modification can occur. Furthermore, the inner pore of BK channels is much larger than that of K(+) channels with solved crystal structures. The results suggest caution in the use of Kv channel structures as templates for BK homology models, at least in the pore-gate domain.
Project description:Calcium- and voltage-activated potassium channels (BK) are regulated by a multiplicity of signals. The prevailing view is that different BK gating mechanisms converge to determine channel opening and that these gating mechanisms are allosterically coupled. In most instances the pore forming ? subunit of BK is associated with one of four alternative ? subunits that appear to target specific gating mechanisms to regulate the channel activity. In particular, ?1 stabilizes the active configuration of the BK voltage sensor having a large effect on BK Ca(2+) sensitivity. To determine the extent to which ? subunits regulate the BK voltage sensor, we measured gating currents induced by the pore-forming BK ? subunit alone and with the different ? subunits expressed in Xenopus oocytes (?1, ?2IR, ?3b, and ?4). We found that ?1, ?2, and ?4 stabilize the BK voltage sensor in the active conformation. ?3 has no effect on voltage sensor equilibrium. In addition, ?4 decreases the apparent number of charges per voltage sensor. The decrease in the charge associated with the voltage sensor in ? ?4 channels explains most of their biophysical properties. For channels composed of the ? subunit alone, gating charge increases slowly with pulse duration as expected if a significant fraction of this charge develops with a time course comparable to that of K(+) current activation. In the presence of ?1, ?2, and ?4 this slow component develops in advance of and much more rapidly than ion current activation, suggesting that BK channel opening proceeds in two steps.
Project description:Large conductance voltage- and Ca(2+)-activated potassium channels (BK channels) are important feedback regulators in excitable cells and are potently regulated by protein kinases. The present study reveals a dual role of protein kinase C (PKC) on BK channel regulation. Phosphorylation of S(695) by PKC, located between the two regulators of K(+) conductance (RCK1/2) domains, inhibits BK channel open-state probability. This PKC-dependent inhibition depends on a preceding phosphorylation of S(1151) in the C terminus of the channel alpha-subunit. Phosphorylation of only one alpha-subunit at S(1151) and S(695) within the tetrameric pore is sufficient to inhibit BK channel activity. We further detected that protein phosphatase 1 is associated with the channel, constantly counteracting phosphorylation of S(695). PKC phosphorylation at S(1151) also influences stimulation of BK channel activity by protein kinase G (PKG) and protein kinase A (PKA). Though the S(1151)A mutant channel is activated by PKA only, the phosphorylation of S(1151) by PKC renders the channel responsive to activation by PKG but prevents activation by PKA. Phosphorylation of S(695) by PKC or introducing a phosphomimetic aspartate at this position (S(695)D) renders BK channels insensitive to the stimulatory effect of PKG or PKA. Therefore, our findings suggest a very dynamic regulation of the channel by the local PKC activity. It is shown that this complex regulation is not only effective in recombinant channels but also in native BK channels from tracheal smooth muscle.
Project description:The regulation of surface levels of protein is critical for proper cell function and influences properties including cell adhesion, ion channel contributions to current flux, and the sensitivity of surface receptors to ligands. Here we demonstrate a two-color labeling system in live cells using a single fluorogen activating peptide (FAP) based fusion tag, which enables the rapid and simultaneous quantification of surface and internal proteins. In the nervous system, BK channels can regulate neural excitability and neurotransmitter release, and the surface trafficking of BK channels can be modulated by signaling cascades and assembly with accessory proteins. Using this labeling approach, we examine the dynamics of BK channel surface expression in HEK293 cells. Surface pools of the pore-forming BK? subunit were stable, exhibiting a plasma membrane half-life of >10 h. Long-term activation of adenylyl cyclase by forskolin reduced BK? surface levels by 30%, an effect that could not be attributed to increased bulk endocytosis of plasma membrane proteins. This labeling approach is compatible with microscopic imaging and flow cytometry, providing a solid platform for examining protein trafficking in living cells.
Project description:Large conductance calcium- and voltage-gated potassium (BK) channels are important regulators of physiological homeostasis and their function is potently modulated by protein kinase A (PKA) phosphorylation. PKA regulates the channel through phosphorylation of residues within the intracellular C terminus of the pore-forming alpha-subunits. However, the molecular mechanism(s) by which phosphorylation of the alpha-subunit effects changes in channel activity are unknown. Inhibition of BK channels by PKA depends on phosphorylation of only a single alpha-subunit in the channel tetramer containing an alternatively spliced insert (STREX) suggesting that phosphorylation results in major conformational rearrangements of the C terminus. Here, we define the mechanism of PKA inhibition of BK channels and demonstrate that this regulation is conditional on the palmitoylation status of the channel. We show that the cytosolic C terminus of the STREX BK channel uniquely interacts with the plasma membrane via palmitoylation of evolutionarily conserved cysteine residues in the STREX insert. PKA phosphorylation of the serine residue immediately upstream of the conserved palmitoylated cysteine residues within STREX dissociates the C terminus from the plasma membrane, inhibiting STREX channel activity. Abolition of STREX palmitoylation by site-directed mutagenesis or pharmacological inhibition of palmitoyl transferases prevents PKA-mediated inhibition of BK channels. Thus, palmitoylation gates BK channel regulation by PKA phosphorylation. Palmitoylation and phosphorylation are both dynamically regulated; thus, cross-talk between these 2 major posttranslational signaling cascades provides a mechanism for conditional regulation of BK channels. Interplay of these distinct signaling cascades has important implications for the dynamic regulation of BK channels and physiological homeostasis.