Sustained antidepressant effect of ketamine through NMDAR trapping in the LHb.

Ketamine, an N-methyl-D-aspartate receptor (NMDAR) antagonist1, has revolutionized the treatment of depression because of its potent, rapid and sustained antidepressant effects2-4. Although the elimination half-life of ketamine is only 13 min in mice5, its antidepressant activities can last for at least 24 h6-9. This large discrepancy poses an interesting basic biological question and has strong clinical implications. Here we demonstrate that after a single systemic injection, ketamine continues to suppress burst firing and block NMDARs in the lateral habenula (LHb) for up to 24 h. This long inhibition of NMDARs is not due to endocytosis but depends on the use-dependent trapping of ketamine in NMDARs. The rate of untrapping is regulated by neural activity. Harnessing the dynamic equilibrium of ketamine-NMDAR interactions by activating the LHb and opening local NMDARs at different plasma ketamine concentrations, we were able to either shorten or prolong the antidepressant effects of ketamine in vivo. These results provide new insights into the causal mechanisms of the sustained antidepressant effects of ketamine. The ability to modulate the duration of ketamine action based on the biophysical properties of ketamine-NMDAR interactions opens up new opportunities for the therapeutic use of ketamine.

Goblet cell LRRC26 regulates BK channel activation and protects against colitis in mice.

Goblet cells (GCs) are specialized cells of the intestinal epithelium contributing critically to mucosal homeostasis. One of the functions of GCs is to produce and secrete MUC2, the mucin that forms the scaffold of the intestinal mucus layer coating the epithelium and separates the luminal pathogens and commensal microbiota from the host tissues. Although a variety of ion channels and transporters are thought to impact on MUC2 secretion, the specific cellular mechanisms that regulate GC function remain incompletely understood. Previously, we demonstrated that leucine-rich repeat-containing protein 26 (LRRC26), a known regulatory subunit of the Ca2+-and voltage-activated K+ channel (BK channel), localizes specifically to secretory cells within the intestinal tract. Here, utilizing a mouse model in which MUC2 is fluorescently tagged, thereby allowing visualization of single GCs in intact colonic crypts, we show that murine colonic GCs have functional LRRC26-associated BK channels. In the absence of LRRC26, BK channels are present in GCs, but are not activated at physiological conditions. In contrast, all tested MUC2- cells completely lacked BK channels. Moreover, LRRC26-associated BK channels underlie the BK channel contribution to the resting transepithelial current across mouse distal colonic mucosa. Genetic ablation of either LRRC26 or BK pore-forming α-subunit in mice results in a dramatically enhanced susceptibility to colitis induced by dextran sodium sulfate. These results demonstrate that normal potassium flux through LRRC26-associated BK channels in GCs has protective effects against colitis in mice.

The functionally relevant site for paxilline inhibition of BK channels.

The tremorgenic fungal alkaloid paxilline (PAX) is a commonly used specific inhibitor of the large-conductance, voltage- and Ca2+-dependent BK-type K+ channel. PAX inhibits BK channels by selective interaction with closed states. BK inhibition by PAX is best characterized by the idea that PAX gains access to the channel through the central cavity of the BK channel, and that only a single PAX molecule can interact with the BK channel at a time. The notion that PAX reaches its binding site via the central cavity and involves only a single PAX molecule would be consistent with binding on the axis of the permeation pathway, similar to classical open channel block and inconsistent with the observation that PAX selectively inhibits closed channels. To explore the potential sites of interaction of PAX with the BK channel, we undertook a computational analysis of the interaction of PAX with the BK channel pore gate domain guided by recently available liganded (open) and metal-free (closed) Aplysia BK channel structures. The analysis unambiguously identified a preferred position of PAX occupancy that accounts for all previously described features of PAX inhibition, including state dependence, G311 sensitivity, stoichiometry, and central cavity accessibility. This PAX-binding pose in closed BK channels is supported by additional functional results.

Regulation of BK Channels by Beta and Gamma Subunits.

Ca2+- and voltage-gated K+ channels of large conductance (BK channels) are expressed in a diverse variety of both excitable and inexcitable cells, with functional properties presumably uniquely calibrated for the cells in which they are found. Although some diversity in BK channel function, localization, and regulation apparently arises from cell-specific alternative splice variants of the single pore-forming α subunit ( KCa1.1, Kcnma1, Slo1) gene, two families of regulatory subunits, ß and γ, define BK channels that span a diverse range of functional properties. We are just beginning to unravel the cell-specific, physiological roles served by BK channels of different subunit composition.

The LRRC family of BK channel regulatory subunits: potential roles in health and disease.

Large conductance K+ channels, termed BK channels, regulate a variety of cellular and physiological functions. Although universally activated by changes in voltage or [Ca2+ ]i , the threshold for BK channel activation varies among loci of expression, often arising from cell-specific regulatory subunits including a family of leucine rich repeat-containing (LRRC) γ subunits (LRRC26, LRRC52, LRRC55 and LRRC38). The 'founding' member of this family, LRRC26, was originally identified as a tumour suppressor in various cancers. An LRRC26 knockout (KO) mouse model recently revealed that LRRC26 is also highly expressed in secretory epithelial cells and partners with BK channels in the salivary gland and colonic goblet cells to promote sustained K+ fluxes likely essential for normal secretory function. To accomplish this, LRRC26 negatively shifts the range of BK channel activation such that channels contribute to K+ flux near typical epithelial cell resting conditions. In colon, the absence of LRRC26 increases vulnerability to colitis. LRRC26-containing BK channels are also likely important regulators of epithelial function in other loci, including airways, female reproductive tract and mammary gland. Based on an LRRC52 KO mouse model, LRRC52 regulation of large conductance K+ channels plays a role both in sperm function and in cochlear inner hair cells. Although our understanding of LRRC-containing BK channels remains rudimentary, KO mouse models may help define other organs in which LRRC-containing channels support normal function. A key topic for future work concerns identification of endogenous mechanisms, whether post-translational or via gene regulation, that may impact LRRC-dependent pathologies.

LRRC52 regulates BK channel function and localization in mouse cochlear inner hair cells.

The perception of sound relies on sensory hair cells in the cochlea that convert the mechanical energy of sound into release of glutamate onto postsynaptic auditory nerve fibers. The hair cell receptor potential regulates the strength of synaptic transmission and is shaped by a variety of voltage-dependent conductances. Among these conductances, the Ca2+- and voltage-activated large conductance Ca2+-activated K+ channel (BK) current is prominent, and in mammalian inner hair cells (IHCs) displays unusual properties. First, BK currents activate at unprecedentedly negative membrane potentials (-60 mV) even in the absence of intracellular Ca2+ elevations. Second, BK channels are positioned in clusters away from the voltage-dependent Ca2+ channels that mediate glutamate release from IHCs. Here, we test the contributions of two recently identified leucine-rich-repeat-containing (LRRC) regulatory γ subunits, LRRC26 and LRRC52, to BK channel function and localization in mouse IHCs. Whereas BK currents and channel localization were unaltered in IHCs from Lrrc26 knockout (KO) mice, BK current activation was shifted more than +200 mV in IHCs from Lrrc52 KO mice. Furthermore, the absence of LRRC52 disrupted BK channel localization in the IHCs. Given that heterologous coexpression of LRRC52 with BK α subunits shifts BK current gating about -90 mV, to account for the profound change in BK activation range caused by removal of LRRC52, we suggest that additional factors may help define the IHC BK gating range. LRRC52, through stabilization of a macromolecular complex, may help retain some other components essential both for activation of BK currents at negative membrane potentials and for appropriate BK channel positioning.

Regulatory γ1 subunits defy symmetry in functional modulation of BK channels.

Structural symmetry is a hallmark of homomeric ion channels. Nonobligatory regulatory proteins can also critically define the precise functional role of such channels. For instance, the pore-forming subunit of the large conductance voltage and calcium-activated potassium (BK, Slo1, or KCa1.1) channels encoded by a single KCa1.1 gene assembles in a fourfold symmetric fashion. Functional diversity arises from two families of regulatory subunits, ß and γ, which help define the range of voltages over which BK channels in a given cell are activated, thereby defining physiological roles. A BK channel can contain zero to four ß subunits per channel, with each ß subunit incrementally influencing channel gating behavior, consistent with symmetry expectations. In contrast, a γ1 subunit (or single type of γ1 subunit complex) produces a functionally all-or-none effect, but the underlying stoichiometry of γ1 assembly and function remains unknown. Here we utilize two distinct and independent methods, a Forster resonance energy transfer-based optical approach and a functional reporter in single-channel recordings, to reveal that a BK channel can contain up to four γ1 subunits, but a single γ1 subunit suffices to induce the full gating shift. This requires that the asymmetric association of a single regulatory protein can act in a highly concerted fashion to allosterically influence conformational equilibria in an otherwise symmetric K+ channel.

Knockout of the LRRC26 subunit reveals a primary role of LRRC26-containing BK channels in secretory epithelial cells.

Leucine-rich-repeat-containing protein 26 (LRRC26) is the regulatory γ1 subunit of Ca2+- and voltage-dependent BK-type K+ channels. BK channels that contain LRRC26 subunits are active near normal resting potentials even without Ca2+, suggesting they play unique physiological roles, likely limited to very specific cell types and cellular functions. By using Lrrc26 KO mice with a ß-gal reporter, Lrrc26 promoter activity is found in secretory epithelial cells, especially acinar epithelial cells in lacrimal and salivary glands, and also goblet and Paneth cells in intestine and colon, although absent from neurons. We establish the presence of LRRC26 protein in eight secretory tissues or tissues with significant secretory epithelium and show that LRRC26 protein coassembles with the pore-forming BK α-subunit in at least three tissues: lacrimal gland, parotid gland, and colon. In lacrimal, parotid, and submandibular gland acinar cells, LRRC26 KO shifts BK gating to be like α-subunit-only BK channels. Finally, LRRC26 KO mimics the effect of SLO1/BK KO in reducing [K+] in saliva. LRRC26-containing BK channels are competent to contribute to resting K+ efflux at normal cell membrane potentials with resting cytosolic Ca2+ concentrations and likely play a critical physiological role in supporting normal secretory function in all secretory epithelial cells.

Roles of Na+, Ca2+, and K+ channels in the generation of repetitive firing and rhythmic bursting in adrenal chromaffin cells.

Adrenal chromaffin cells (CCs) are the main source of circulating catecholamines (CAs) that regulate the body response to stress. Release of CAs is controlled neurogenically by the activity of preganglionic sympathetic neurons through trains of action potentials (APs). APs in CCs are generated by robust depolarization following the activation of nicotinic and muscarinic receptors that are highly expressed in CCs. Bovine, rat, mouse, and human CCs also express a composite array of Na+, K+, and Ca2+ channels that regulate the resting potential, shape the APs, and set the frequency of AP trains. AP trains of increasing frequency induce enhanced release of CAs. If the primary role of CCs is simply to relay preganglionic nerve commands to CA secretion, why should they express such a diverse set of ion channels? An answer to this comes from recent observations that, like in neurons, CCs undergo complex firing patterns of APs suggesting the existence of an intrinsic CC excitability (non-neurogenically controlled). Recent work has shown that CCs undergo occasional or persistent burst firing elicited by altered physiological conditions or deletion of pore-regulating auxiliary subunits. In this review, we aim to give a rationale to the role of the many ion channel types regulating CC excitability. We will first describe their functional properties and then analyze how they contribute to pacemaking, AP shape, and burst waveforms. We will also furnish clear indications on missing ion conductances that may be involved in pacemaking and highlight the contribution of the crucial channels involved in burst firing.

Stereospecific binding of a disordered peptide segment mediates BK channel inactivation.

A number of functionally important actions of proteins are mediated by short, intrinsically disordered peptide segments, but the molecular interactions that allow disordered domains to mediate their effects remain a topic of active investigation. Many K+ channel proteins, after initial channel opening, show a time-dependent reduction in current flux, termed 'inactivation', which involves movement of mobile cytosolic peptide segments (approximately 20-30 residues) into a position that physically occludes ion permeation. Peptide segments that produce inactivation show little amino-acid identity and tolerate appreciable mutational substitutions without disrupting the inactivation process. Solution nuclear magnetic resonance of several isolated inactivation domains reveals substantial conformational heterogeneity with only minimal tendency to ordered structures. Channel inactivation mechanisms may therefore help us to decipher how intrinsically disordered regions mediate functional effects. Whereas many aspects of inactivation of voltage-dependent K+ channels (Kv) can be described by a simple one-step occlusion mechanism, inactivation of the voltage-dependent large-conductance Ca2+-gated K+ (BK) channel mediated by peptide segments of auxiliary ß-subunits involves two distinguishable kinetic steps. Here we show that two-step inactivation mediated by an intrinsically disordered BK ß-subunit peptide involves a stereospecific binding interaction that precedes blockade. In contrast, blocking mediated by a Shaker Kv inactivation peptide is consistent with direct, simple occlusion by a hydrophobic segment without substantial steric requirement. The results indicate that two distinct types of molecular interaction between disordered peptide segments and their binding sites produce qualitatively similar functions.

Cadmium-cysteine coordination in the BK inner pore region and its structural and functional implications.

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.

SLO3 auxiliary subunit LRRC52 controls gating of sperm KSPER currents and is critical for normal fertility.

Following entry into the female reproductive tract, mammalian sperm undergo a maturation process termed capacitation that results in competence to fertilize ova. Associated with capacitation is an increase in membrane conductance to both Ca(2+) and K(+), leading to an elevation in cytosolic Ca(2+) critical for activation of hyperactivated swimming motility. In mice, the Ca(2+) conductance (alkalization-activated Ca(2+)-permeable sperm channel, CATSPER) arises from an ensemble of CATSPER subunits, whereas the K(+) conductance (sperm pH-regulated K(+) current, KSPER) arises from a pore-forming ion channel subunit encoded by the slo3 gene (SLO3) subunit. In the mouse, both CATSPER and KSPER are activated by cytosolic alkalization and a concerted activation of CATSPER and KSPER is likely a common facet of capacitation-associated increases in Ca(2+) and K(+) conductance among various mammalian species. The properties of heterologously expressed mouse SLO3 channels differ from native mouse KSPER current. Recently, a potential KSPER auxiliary subunit, leucine-rich-repeat-containing protein 52 (LRRC52), was identified in mouse sperm and shown to shift gating of SLO3 to be more equivalent to native KSPER. Here, we show that genetic KO of LRRC52 results in mice with severely impaired fertility. Activation of KSPER current in sperm lacking LRRC52 requires more positive voltages and higher pH than for WT KSPER. These results establish a critical role of LRRC52 in KSPER channels and demonstrate that loss of a non-pore-forming auxiliary subunit results in severe fertility impairment. Furthermore, through analysis of several genotypes that influence KSPER current properties we show that in vitro fertilization competence correlates with the net KSPER conductance available for activation under physiological conditions.

Functional regulation of BK potassium channels by γ1 auxiliary subunits.

Many K(+) channels are oligomeric complexes with intrinsic structural symmetry arising from the homo-tetrameric core of their pore-forming subunits. Allosteric regulation of tetramerically symmetric proteins, whether by intrinsic sensing domains or associated auxiliary subunits, often mirrors the fourfold structural symmetry. Here, through patch-clamp recordings of channel population ensembles and also single channels, we examine regulation of the Ca(2+)- and voltage-activated large conductance Ca(2+)-activated K(+) (BK) channel by associated γ1-subunits. Through expression of differing ratios of γ1:α-subunits, the results reveal an all-or-none functional regulation of BK channels by γ-subunits: channels either exhibit a full gating shift or no shift at all. Furthermore, the γ1-induced shift exhibits a state-dependent labile behavior that recapitulates the fully shifted or unshifted behavior. The γ1-induced shift contrasts markedly to the incremental shifts in BK gating produced by 1-4 ß-subunits and adds a new layer of complexity to the mechanisms by which BK channel functional diversity is generated.

Barium ions selectively activate BK channels via the Ca2+-bowl site.

Activation of Ca(2+)-dependent BK channels is increased via binding of micromolar Ca(2+) to two distinct high-affinity sites per BK α-subunit. One site, termed the Ca(2+) bowl, is embedded within the second RCK domain (RCK2; regulator of conductance for potassium) of each α-subunit, while oxygen-containing residues in the first RCK domain (RCK1) have been linked to a separate Ca(2+) ligation site. Although both sites are activated by Ca(2+) and Sr(2+), Cd(2+) selectively favors activation via the RCK1 site. Divalent cations of larger ionic radius than Sr(2+) are thought to be ineffective at activating BK channels. Here we show that Ba(2+), better known as a blocker of K(+) channels, activates BK channels and that this effect arises exclusively from binding at the Ca(2+)-bowl site. Compared with previous estimates for Ca(2+) bowl-mediated activation by Ca(2+), the affinity of Ba(2+) to the Ca(2+) bowl is reduced about fivefold, and coupling of binding to activation is reduced from ∼3.6 for Ca(2+) to about ∼2.8 for Ba(2+). These results support the idea that ionic radius is an important determinant of selectivity differences among different divalent cations observed for each Ca(2+)-binding site.

Cysteine scanning and modification reveal major differences between BK channels and Kv channels in the inner pore region.

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 α-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.

LRRC52 (leucine-rich-repeat-containing protein 52), a testis-specific auxiliary subunit of the alkalization-activated Slo3 channel.

KSper, a pH-dependent K(+) current in mouse spermatozoa that is critical for fertility, is activated by alkalization in the range of pH 6.4-7.2 at membrane potentials between -50 and 0 mV. Although the KSper pore-forming subunit is encoded by the Slo3 gene, heterologously expressed Slo3 channels are largely closed at potentials negative to 0 mV at physiological pH. Here we identify a Slo3-associating protein, LRRC52 (leucine-rich repeat-containing 52), that shifts Slo3 gating into a range of voltages and pH values similar to that producing KSper current activation. Message for LRRC52, a homolog of the Slo1-modifying LRRC26 protein, is enriched in testis relative to other homologous LRRC subunits and is developmentally regulated in concert with that for Slo3. LRRC52 protein is detected only in testis. It is markedly diminished from Slo3(-/-) testis and completely absent from Slo3(-/-) sperm, indicating that LRRC52 expression is critically dependent on the presence of Slo3. We also examined the ability of other LRRC subunits homologous to LRRC26 and LRRC52 to modify Slo3 currents. Although both LRRC26 and LRRC52 are able to modify Slo3 function, LRRC52 is the stronger modifier of Slo3 function. Effects of other related subunits were weaker or absent. We propose that LRRC52 is a testis-enriched Slo3 auxiliary subunit that helps define the specific alkalization dependence of KSper activation. Together, LRRC52 and LRRC26 define a new family of auxiliary subunits capable of critically modifying the gating behavior of Slo family channels.

Deletion of the Slo3 gene abolishes alkalization-activated K+ current in mouse spermatozoa.

Mouse spermatozoa express a pH-dependent K(+) current (KSper) thought to be composed of subunits encoded by the Slo3 gene. However, the equivalence of KSper and Slo3-dependent current remains uncertain, because heterologous expression of Slo3 results in currents that are less effectively activated by alkalization than are native KSper currents. Here, we show that genetic deletion of Slo3 abolishes all pH-dependent K(+) current at physiological membrane potentials in corpus epididymal sperm. A residual pH-dependent outward current (I(Kres)) is observed in Slo3(-/-) sperm at potentials of >0 mV. Differential inhibition of KSper/Slo3 and I(Kres) by clofilium reveals that the amplitude of I(Kres) is similar in both wild-type (wt) and Slo3(-/-) sperm. The properties of I(Kres) suggest that it likely represents outward monovalent cation flux through CatSper channels. Thus, KSper/Slo3 may account for essentially all mouse sperm K(+) current and is the sole pH-dependent K(+) conductance in these sperm. With physiological ionic gradients, alkalization depolarizes Slo3(-/-) spermatozoa, presumably from CatSper activation, in contrast to Slo3/KSper-mediated hyperpolarization in wt sperm. Slo3(-/-) male mice are infertile, but Slo3(-/-) sperm exhibit some fertility within in vitro fertilization assays. Slo3(-/-) sperm exhibit a higher incidence of morphological abnormalities accentuated by hypotonic challenge and also exhibit deficits in motility in the absence of bicarbonate, revealing a role of KSper under unstimulated conditions. Together, these results show that KSper/Slo3 is the primary spermatozoan K(+) current, that KSper may play a critical role in acquisition of normal morphology and sperm motility when faced with hyperosmotic challenges, and that Slo3 is critical for fertility.