Positions of the cytoplasmic end of BK α S0 helix relative to S1–S6 and of β1 TM1 and TM2 relative to S0–S6

The large-conductance, voltage- and Ca²⁺-gated K⁺ (BK) channel consists of four α subunits, which form a voltage- and Ca²⁺-gated channel, and up to four modulatory β subunits. The β1 subunit is expressed in smooth muscle, where it slows BK channel kinetics and shifts the conductance–voltage (G-V) curve to the left at [Ca²⁺] > 2 µM. In addition to the six transmembrane (TM) helices, S1–S6, conserved in all voltage-dependent K⁺ channels, BK α has a unique seventh TM helix, S0, which may contribute to the unusual rightward shift in the G-V curve of BK α in the absence of β1 and to a leftward shift in its presence. Such a role is supported by the close proximity of S0 to S3 and S4 in the voltage-sensing domain. Furthermore, on the extracellular side of the membrane, one of the two TM helices of β1, TM2, is adjacent to S0. We have now analyzed induced disulfide bond formation between substituted Cys residues on the cytoplasmic side of the membrane. There, in contrast, S0 is closest to the S2–S3 loop, from which position it is displaced on the addition of β1. The cytoplasmic ends of β1 TM1 and TM2 are adjacent and are located between the S2–S3 loop of one α subunit and S1 of a neighboring α subunit and are not adjacent to S0; i.e., S0 and TM2 have different trajectories through the membrane. In the absence of β1, 70% of disulfide bonding of W43C (S0) and L175C (S2–S3) has no effect on V₅₀ for activation, implying that the cytoplasmic end of S0 and the S2–S3 loop move in concert, if at all, during activation. Otherwise, linking them together in one state would obstruct the transition to the other state, which would certainly change V₅₀.     

Location of modulatory β subunits in BK potassium channels

Large-conductance voltage- and calcium-activated potassium (BK) channels contain four pore-forming α subunits and four modulatory β subunits. From the extents of disulfide cross-linking in channels on the cell surface between cysteine (Cys) substituted for residues in the first turns in the membrane of the S0 transmembrane (TM) helix, unique to BK α, and of the voltage-sensing domain TM helices S1–S4, we infer that S0 is next to S3 and S4, but not to S1 and S2. Furthermore, of the two β1 TM helices, TM2 is next to S0, and TM1 is next to TM2. Coexpression of α with two substituted Cys’s, one in S0 and one in S2, and β1 also with two substituted Cys’s, one in TM1 and one in TM2, resulted in two αs cross-linked by one β. Thus, each β lies between and can interact with the voltage-sensing domains of two adjacent α subunits.     

The single transmembrane segment determines the modulatory function of the BK channel auxiliary γ subunit

The large-conductance, calcium-activated potassium (BK) channels consist of the pore-forming, voltage- and Ca²⁺-sensing α subunits (BKα) and the tissue-specific auxiliary β and γ subunits. The BK channel γ1 subunit is a leucine-rich repeat (LRR)–containing membrane protein that potently facilitates BK channel activation in many tissues and cell types through a vast shift in the voltage dependence of channel activation by ∼140 mV in the hyperpolarizing direction. In this study, we found that the single transmembrane (TM) segment together with its flanking charged residues is sufficient to fully modulate BK channels upon its transplantation into the structurally unrelated β1 subunit. We identified Phe273 and its neighboring residues in the middle of the TM segment and a minimum of three intracellular juxtamembrane Arg residues as important for the γ1 subunit’s modulatory function and observed functional coupling between residues of these two locations. We concluded that the TM segment is a key molecular determinant for channel association and modulation and that the intracellular positively charged cluster is involved mainly in channel association, likely through its TM-anchoring effect. Our findings provide insights into the structure–function relationship of the γ1 subunit in understanding its potent modulatory effects on BK channels.     

Conopeptide Vt3.1 Preferentially Inhibits BK Potassium Channels Containing β4 Subunits via Electrostatic Interactions

BK channel β subunits (β1–β4) modulate the function of channels formed by slo1 subunits to produce tissue-specific phenotypes. The molecular mechanism of how the homologous β subunits differentially alter BK channel functions and the role of different BK channel functions in various physiologic processes remain unclear. By studying channels expressed in Xenopus laevis oocytes, we show a novel disulfide-cross-linked dimer conopeptide, Vt3.1 that preferentially inhibits BK channels containing the β4 subunit, which is most abundantly expressed in brain and important for neuronal functions. Vt3.1 inhibits the currents by a maximum of 71%, shifts the G-V relation by 45 mV approximately half-saturation concentrations, and alters both open and closed time of single channel activities, indicating that the toxin alters voltage dependence of the channel. Vt3.1 contains basic residues and inhibits voltage-dependent activation by electrostatic interactions with acidic residues in the extracellular loops of the slo1 and β4 subunits. These results suggest a large interaction surface between the slo1 subunit of BK channels and the β4 subunit, providing structural insight into the molecular interactions between slo1 and β4 subunits. The results also suggest that Vt3.1 is an excellent tool for studying β subunit modulation of BK channels and for understanding the physiological roles of BK channels in neurophysiology.     

Lipids Modulate the Increase of BK Channel Calcium Sensitivity by the β1 Subunit

Co-expression of the auxiliary β1 subunit with the pore forming α subunit of BK dramatically alters apparent calcium sensitivity. Investigation of the mechanism underlying the increase in calcium sensitivity of BK in smooth muscle has concentrated on the energetic effect of β1′s interaction with α. We take a novel approach, exploring whether β1 modification of calcium sensitivity reflects altered interaction between the channel protein and surrounding lipids. We reconstituted hSlo BK α and BK α+β1 channels into two sets of bilayers. One set contained POPE with POPS, POPG, POPA and POPC, where the length of acyl chains is constant, but surface charge differs. The second set is a series of neutral bilayers formed from DOPE with phosphatidylcholines (PCs) of varying acyl chain lengths: C (14∶1), C (18∶1), C (22∶1) and C (24∶1), and with brain sphingomyelin (SPM), in which surface charge is constant, but bilayer thickness varies. The increase in calcium sensitivity caused by the β1 subunit was preserved in negatively charged lipid bilayers but not in neutral bilayers, indicating that modification of apparent Ca²⁺ sensitivity by β1 is modulated by membrane lipids, requiring negatively charged lipids in the membrane. Moreover, the presence of β1 reduces BK activity in thin bilayers of PC 14∶1 and thick bilayers containing SPM, but has no significant effect on activity of BK in PC 18∶1, PC 22∶1 and PC 24∶1 bilayers. These data suggest that auxiliary β1 subunits fine-tune channel gating not only through direct subunit-subunit interactions but also by modulating lipid-protein interactions.     

Functional dichotomy in the 16S rRNA (m1A1408) methyltransferase family and control of catalytic activity via a novel tryptophan mediated loop reorganization

Methylation of the bacterial small ribosomal subunit (16S) rRNA on the N1 position of A1408 confers exceptionally high-level resistance to a broad spectrum of aminoglycoside antibiotics. Here, we present a detailed structural and functional analysis of the Catenulisporales acidiphilia 16S rRNA (m¹A1408) methyltransferase (‘CacKam’). The apo CacKam structure closely resembles other m¹A1408 methyltransferases within its conserved SAM-binding fold but the region linking core β strands 6 and 7 (the ‘β6/7 linker’) has a unique, extended structure that partially occludes the putative 16S rRNA binding surface, and sequesters the conserved and functionally critical W203 outside of the CacKam active site. Substitution of conserved residues in the SAM binding pocket reveals a functional dichotomy in the 16S rRNA (m¹A1408) methyltransferase family, with two apparently distinct molecular mechanisms coupling cosubstrate/ substrate binding to catalytic activity. Our results additionally suggest that CacKam exploits the W203-mediated remodeling of the β6/7 linker as a novel mechanism to control 30S substrate recognition and enzymatic turnover.     

Both Transmembrane Domains of BK β1 Subunits Are Essential to Confer the Normal Phenotype of β1-Containing BK Channels

Voltage/Ca²⁺ _i-gated, large conductance K⁺ (BK) channels result from tetrameric association of α (slo1) subunits. In most tissues, BK protein complexes include regulatory β subunits that contain two transmembrane domains (TM1, TM2), an extracellular loop, and two short intracellular termini. Four BK β types have been identified, each presenting a rather selective tissue-specific expression profile. Thus, BK β modifies current phenotype to suit physiology in a tissue-specific manner. The smooth muscle-abundant BK β1 drastically increases the channel''s apparent Ca²⁺ _i sensitivity. The resulting phenotype is critical for BK channel activity to increase in response to Ca²⁺ levels reached near the channel during depolarization-induced Ca²⁺ influx and myocyte contraction. The eventual BK channel activation generates outward K⁺ currents that drive the membrane potential in the negative direction and eventually counteract depolarization-induced Ca²⁺ influx. The BK β1 regions responsible for the characteristic phenotype of β1-containing BK channels remain to be identified. We used patch-clamp electrophysiology on channels resulting from the combination of smooth muscle slo1 (cbv1) subunits with smooth muscle-abundant β1, neuron-abundant β4, or chimeras constructed by swapping β1 and β4 regions, and determined the contribution of specific β1 regions to the BK phenotype. At Ca²⁺ levels found near the channel during myocyte contraction (10 µM), channel complexes that included chimeras having both TMs from β1 and the remaining regions (“background”) from β4 showed a phenotype (V_half, τ_act, τ_deact) identical to that of complexes containing wt β1. This phenotype could not be evoked by complexes that included chimeras combining either β1 TM1 or β1 TM2 with a β4 background. Likewise, β “halves” (each including β1 TM1 or β1 TM2) resulting from interrupting the continuity of the EC loop failed to render the normal phenotype, indicating that physical connection between β1 TMs via the EC loop is also necessary for proper channel function.     

Large Conductance Voltage- and Ca2+-gated Potassium (BK) Channel β4 Subunit Influences Sensitivity and Tolerance to Alcohol by Altering Its Response to Kinases

Tolerance is a well described component of alcohol abuse and addiction. The large conductance voltage- and Ca²⁺-gated potassium channel (BK) has been very useful for studying molecular tolerance. The influence of association with the β4 subunit can be observed at the level of individual channels, action potentials in brain slices, and finally, drinking behavior in the mouse. Previously, we showed that 50 mm alcohol increases both α and αβ4 BK channel open probability, but only α BK develops acute tolerance to this effect. Currently, we explore the possibility that the influence of the β4 subunit on tolerance may result from a striking effect of β4 on kinase modulation of the BK channel. We examine the influence of the β4 subunit on PKA, CaMKII, and phosphatase modulation of channel activity, and on molecular tolerance to alcohol. We record from human BK channels heterologously expressed in HEK 293 cells composed of its core subunit, α alone (Insertless), or co-expressed with the β4 BK auxiliary subunit, as well as, acutely dissociated nucleus accumbens neurons using the cell-attached patch clamp configuration. Our results indicate that BK channels are strongly modulated by activation of specific kinases (PKA and CaMKII) and phosphatases. The presence of the β4 subunit greatly influences this modulation, allowing a variety of outcomes for BK channel activity in response to acute alcohol.     

The smooth muscle-type β1 subunit potentiates activation by DiBAC4(3) in recombinant BK channels

Large-conductance Ca²⁺-activated (BK) channels, expressed in a variety of tissues, play a fundamental role in regulating and maintaining arterial tone. We recently demonstrated that the slow voltage indicator DiBAC₄(3) does not depend, as initially proposed, on the β₁ or β₄ subunits to activate native arterial smooth muscle BK channels. Using recombinant mslo BK channels, we now show that the β₁ subunit is not essential to this activation but exerts a large potentiating effect. DiBAC₄(3) promotes concentration-dependent activation of BK channels and slows deactivation kinetics, changes that are independent of Ca²⁺. K_d values for BK channel activation by DiBAC₄(3) in 0 mM Ca²⁺ are approximately 20 μM (α) and 5 μM (α+β₁), and G-V curves shift up to −40mV and −110 mV, respectively. β₁ to β₂ mutations R11A and C18E do not interfere with the potentiating effect of the subunit. Our findings should help refine the role of the β₁ subunit in cardiovascular pharmacology.     

Molecular basis for differential modulation of BK channel voltage-dependent gating by auxiliary γ subunits

Large conductance Ca²⁺- and voltage-activated potassium (BK) channels are comprised of pore-forming α subunits and various regulatory auxiliary subunits. The BK channel auxiliary γ (BKγ) subunits are a newly identified class of proteins containing an extracellular leucine-rich repeat domain (LRRD), a single transmembrane (TM) segment, and a short cytoplasmic C-terminal tail (C-tail). Although each of the four BKγ proteins shifts the voltage dependence of BK channel activation in a hyperpolarizing direction, they show markedly different efficacies, mediating shifts over a range of 15–145 mV. Analyses of chimeric BKγ subunits created by swapping individual structural elements, and of BKγ deletion and substitution mutants, revealed that differential modulation of BK gating by the four BKγ subunits depends on a small region consisting of the TM segment and the adjacent intracellular cluster of positively charged amino acids. The γ1 and γ2 TM segments contributed approximately −100 mV, and the γ1 and γ3 C-tails contributed approximately −40 mV, to shifting the voltage dependence of BK channel activation, whereas the γ3 and γ4 TM segments and the γ2 and γ4 C-tails contributed much less. The large extracellular LRRDs were mainly functionally interchangeable, although the γ1 LRRD was slightly less effective at enhancing (or slightly more effective at attenuating) the shift in BK channel voltage-dependent gating toward hyperpolarizing potentials than those of the other BKγ subunits. Analysis of mutated BKγ subunits revealed that juxta-membrane clusters of positively charged amino acids determine the functions of the γ1 and γ3 C-tails. Therefore, the modulatory functions of BKγ subunits are coarse- and fine-tuned, respectively, through variations in their TM segments and in the adjacent intracellular positively charged regions. Our results suggest that BK channel modulation by auxiliary γ subunits depends on intra- and/or juxta-membrane mechanisms.     

Activation of Calcium- and Voltage-gated Potassium Channels of Large Conductance by Leukotriene B4

Calcium/voltage-gated, large conductance potassium (BK) channels control numerous physiological processes, including myogenic tone. BK channel regulation by direct interaction between lipid and channel protein sites has received increasing attention. Leukotrienes (LTA4, LTB4, LTC4, LTD4, and LTE4) are inflammatory lipid mediators. We performed patch clamp studies in Xenopus oocytes that co-expressed BK channel-forming (cbv1) and accessory β1 subunits cloned from rat cerebral artery myocytes. Leukotrienes were applied at 0.1 nm–10 μm to either leaflet of cell-free membranes at a wide range of [Ca²⁺]_i and voltages. Only LTB4 reversibly increased BK steady-state activity (EC₅₀ = 1 nm; E_max reached at 10 nm), with physiological [Ca²⁺]_i and voltages favoring this activation. Homomeric cbv1 or cbv1-β2 channels were LTB4-resistant. Computational modeling predicted that LTB4 docked onto the cholane steroid-sensing site in the BK β1 transmembrane domain 2 (TM2). Co-application of LTB4 and cholane steroid did not further increase LTB4-induced activation. LTB4 failed to activate β1 subunit-containing channels when β1 carried T169A, A176S, or K179I within the docking site. Co-application of LTB4 with LTA4, LTC4, LTD4, or LTE4 suppressed LTB4-induced activation. Inactive leukotrienes docked onto a portion of the site, probably preventing tight docking of LTB4. In summary, we document the ability of two endogenous lipids from different chemical families to share their site of action on a channel accessory subunit. Thus, cross-talk between leukotrienes and cholane steroids might converge on regulation of smooth muscle contractility via BK β1. Moreover, the identification of LTB4 as a highly potent ligand for BK channels is critical for the future development of β1-specific BK channel activators.     

Functional Roles of Clusters of Hydrophobic and Polar Residues in the Epithelial Na+ Channel Knuckle Domain

The extracellular regions of epithelial Na⁺ channel subunits are highly ordered structures composed of domains formed by α helices and β strands. Deletion of the peripheral knuckle domain of the α subunit in the αβγ trimer results in channel activation, reflecting an increase in channel open probability due to a loss of the inhibitory effect of external Na⁺ (Na⁺ self-inhibition). In contrast, deletion of either the β or γ subunit knuckle domain within the αβγ trimer dramatically reduces epithelial Na⁺ channel function and surface expression, and impairs subunit maturation. We systematically mutated individual α subunit knuckle domain residues and assessed functional properties of these mutants. Cysteine substitutions at 14 of 28 residues significantly suppressed Na⁺ self-inhibition. The side chains of a cluster of these residues are non-polar and are predicted to be directed toward the palm domain, whereas a group of polar residues are predicted to orient their side chains toward the space between the knuckle and finger domains. Among the mutants causing the greatest suppression of Na⁺ self-inhibition were αP521C, αI529C, and αS534C. The introduction of Cys residues at homologous sites within either the β or γ subunit knuckle domain resulted in little or no change in Na⁺ self-inhibition. Our results suggest that multiple residues in the α subunit knuckle domain contribute to the mechanism of Na⁺ self-inhibition by interacting with palm and finger domain residues via two separate and chemically distinct motifs.     

Positions of β2 and β3 subunits in the large-conductance calcium- and voltage-activated BK potassium channel

Large-conductance voltage- and Ca²⁺-gated K⁺ channels are negative-feedback regulators of excitability in many cell types. They are complexes of α subunits and of one of four types of modulatory β subunits. These have intracellular N- and C-terminal tails and two transmembrane (TM) helices, TM1 and TM2, connected by an ∼100-residue extracellular loop. Based on endogenous disulfide formation between engineered cysteines (Cys), we found that in β2 and β3, as in β1 and β4, TM1 is closest to αS1 and αS2 and TM2 is closest to αS0. Mouse β3 (mβ3) has seven Cys in its loop, one of which is free, and this Cys readily forms disulfides with Cys substituted in the extracellular flanks of each of αS0–αS6. We identified by elimination mβ3-loop Cys152 as the only free Cys. We inferred the disulfide-bonding pattern of the other six Cys. Using directed proteolysis and fragment sizing, we determined this pattern first among the four loop Cys in β1. These are conserved in β2–β4, which have four additional Cys (eight in total), except that mβ3 has one fewer. In β1, disulfides form between Cys at aligned positions 1 and 8 and between Cys at aligned positions 5 and 6. In mβ3, the free Cys is at position 7; position 2 lacks a Cys present in all other β2–β4; and the disulfide pattern is 1–8, 3–4, and 5–6. Presumably, Cys 2 cross-links to Cys 7 in all other β2–β4. Cross-linking of mβ3 Cys152 to Cys substituted in the flanks of αS0–S5 attenuated the protection against iberiotoxin (IbTX); cross-linking of Cys152 to K296C in the αS6 flank and close to the pore enhanced protection against IbTX. In no case was N-type inactivation by the N-terminal tail of mβ3 perturbed. Although the mβ3 loop can move, its position with Cys152 near αK296, in which it blocks IbTX binding, is likely favored.     

New Hyperekplexia Mutations Provide Insight into Glycine Receptor Assembly,Trafficking, and Activation Mechanisms

Hyperekplexia is a syndrome of readily provoked startle responses, alongside episodic and generalized hypertonia, that presents within the first month of life. Inhibitory glycine receptors are pentameric ligand-gated ion channels with a definitive and clinically well stratified linkage to hyperekplexia. Most hyperekplexia cases are caused by mutations in the α1 subunit of the human glycine receptor (hGlyR) gene (GLRA1). Here we analyzed 68 new unrelated hyperekplexia probands for GLRA1 mutations and identified 19 mutations, of which 9 were novel. Electrophysiological analysis demonstrated that the dominant mutations p.Q226E, p.V280M, and p.R414H induced spontaneous channel activity, indicating that this is a recurring mechanism in hGlyR pathophysiology. p.Q226E, at the top of TM1, most likely induced tonic activation via an enhanced electrostatic attraction to p.R271 at the top of TM2, suggesting a structural mechanism for channel activation. Receptors incorporating p.P230S (which is heterozygous with p.R65W) desensitized much faster than wild type receptors and represent a new TM1 site capable of modulating desensitization. The recessive mutations p.R72C, p.R218W, p.L291P, p.D388A, and p.E375X precluded cell surface expression unless co-expressed with α1 wild type subunits. The recessive p.E375X mutation resulted in subunit truncation upstream of the TM4 domain. Surprisingly, on the basis of three independent assays, we were able to infer that p.E375X truncated subunits are incorporated into functional hGlyRs together with unmutated α1 or α1 plus β subunits. These aberrant receptors exhibit significantly reduced glycine sensitivity. To our knowledge, this is the first suggestion that subunits lacking TM4 domains might be incorporated into functional pentameric ligand-gated ion channel receptors.     

Modal affinities of endplate acetylcholine receptors caused by loop C mutations

The time course of the endplate current is determined by the rate and equilibrium constants for acetylcholine receptor (AChR) activation. We measured these constants in single-channel currents from AChRs with mutations at the neurotransmitter-binding sites, in loop C. The main findings are: (a) Almost all perturbations of loop C generate heterogeneity in the channel open probability (“modes”). (b) Modes are generated by different affinities for ACh that can be either higher or lower than in the wild-type receptors. (c) The modes are stable, in so far as each receptor maintains its affinity for at least several minutes. (d) Different agonists show different degrees of modal activity. With the loop C mutation αP197A, there are four modes with ACh but only two with partial agonists. (e) The affinity variations arise exclusively from the αδ-binding site. (f) Substituting four γ-subunit residues into the δ subunit (three in loop E and one in the β5–β5′ linker) reduces modal activity. (g) At each neurotransmitter-binding site, affinity is determined by a core of five aromatic residues. Modes are eliminated by an alanine mutation at δW57 but not at the other aromatics. (h) Modes are eliminated by a phenylalanine substitution at all core aromatics except αY93. The results suggest that, at the αδ agonist site, loop C and the complementary subunit surface can each adopt alternative conformations and interact with each other to influence the position of δW57 with respect to the aromatic core and, hence, affinity.     

Phosphorylation of a constitutive serine inhibits BK channel variants containing the alternate exon “SRKR”

BK Ca²⁺-activated K⁺ currents exhibit diverse properties across tissues. The functional variation in voltage- and Ca²⁺-dependent gating underlying this diversity arises from multiple mechanisms, including alternate splicing of Kcnma1, the gene encoding the pore-forming (α) subunit of the BK channel, phosphorylation of α subunits, and inclusion of β subunits in channel complexes. To address the interplay of these mechanisms in the regulation of BK currents, two native splice variants, BK₀ and BK_SRKR, were cloned from a tissue that exhibits dynamic daily expression of BK channel, the central circadian pacemaker in the suprachiasmatic nucleus (SCN) of mouse hypothalamus. The BK₀ and BK_SRKR variants differed by the inclusion of a four–amino acid alternate exon at splice site 1 (SRKR), which showed increased expression during the day. The functional properties of the variants were investigated in HEK293 cells using standard voltage-clamp protocols. Compared with BK₀, BK_SRKR currents had a significantly right-shifted conductance–voltage (G-V) relationship across a range of Ca²⁺ concentrations, slower activation, and faster deactivation. These effects were dependent on the phosphorylation state of S642, a serine residue within the constitutive exon immediately preceding the SRKR insert. Coexpression of the neuronal β4 subunit slowed gating kinetics and shifted the G-V relationship in a Ca²⁺-dependent manner, enhancing the functional differences between the variants. Next, using native action potential (AP) command waveforms recorded from SCN to elicit BK currents, we found that these splice variant differences persist under dynamic activation conditions in physiological ionic concentrations. AP-induced currents from BK_SRKR channels were significantly reduced compared with BK₀, an effect that was maintained with coexpression of the β4 subunit but abolished by the mutation of S642. These results demonstrate a novel mechanism for reducing BK current activation under reconstituted physiological conditions, and further suggest that S642 is selectively phosphorylated in the presence of SRKR.     

Knockout of the BK β2 subunit abolishes inactivation of BK currents in mouse adrenal chromaffin cells and results in slow-wave burst activity

Rat and mouse adrenal medullary chromaffin cells (CCs) express an inactivating BK current. This inactivation is thought to arise from the assembly of up to four β2 auxiliary subunits (encoded by the kcnmb2 gene) with a tetramer of pore-forming Slo1 α subunits. Although the physiological consequences of inactivation remain unclear, differences in depolarization-evoked firing among CCs have been proposed to arise from the ability of β2 subunits to shift the range of BK channel activation. To investigate the role of BK channels containing β2 subunits, we generated mice in which the gene encoding β2 was deleted (β2 knockout [KO]). Comparison of proteins from wild-type (WT) and β2 KO mice allowed unambiguous demonstration of the presence of β2 subunit in various tissues and its coassembly with the Slo1 α subunit. We compared current properties and cell firing properties of WT and β2 KO CCs in slices and found that β2 KO abolished inactivation, slowed action potential (AP) repolarization, and, during constant current injection, decreased AP firing. These results support the idea that the β2-mediated shift of the BK channel activation range affects repetitive firing and AP properties. Unexpectedly, CCs from β2 KO mice show an increased tendency toward spontaneous burst firing, suggesting that the particular properties of BK channels in the absence of β2 subunits may predispose to burst firing.     

Two distinct effects of PIP2 underlie auxiliary subunit-dependent modulation of Slo1 BK channels

Phosphatidylinositol 4,5-bisphosphate (PIP₂) plays a critical role in modulating the function of numerous ion channels, including large-conductance Ca²⁺- and voltage-dependent K⁺ (BK, Slo1) channels. Slo1 BK channel complexes include four pore-forming Slo1 (α) subunits as well as various regulatory auxiliary subunits (β and γ) that are expressed in different tissues. We examined the molecular and biophysical mechanisms underlying the effects of brain-derived PIP₂ on human Slo1 BK channel complexes with different subunit compositions that were heterologously expressed in human embryonic kidney cells. PIP₂ inhibited macroscopic currents through Slo1 channels without auxiliary subunits and through Slo1 + γ1 complexes. In contrast, PIP₂ markedly increased macroscopic currents through Slo1 + β1 and Slo1 + β4 channel complexes and failed to alter macroscopic currents through Slo1 + β2 and Slo1 + β2 Δ2–19 channel complexes. Results obtained at various membrane potentials and divalent cation concentrations suggest that PIP₂ promotes opening of the ion conduction gate in all channel types, regardless of the specific subunit composition. However, in the absence of β subunits positioned near the voltage-sensor domains (VSDs), as in Slo1 and probably Slo1 + γ1, PIP₂ augments the negative surface charge on the cytoplasmic side of the membrane, thereby shifting the voltage dependence of VSD-mediated activation in the positive direction. When β1 or β4 subunits occupy the space surrounding the VSDs, only the stimulatory effect of PIP₂ is evident. The subunit compositions of native Slo1 BK channels differ in various cell types; thus, PIP₂ may exert distinct tissue- and divalent cation–dependent modulatory influences.     

Potentiation of Neuronal Nicotinic Receptors by 17β-Estradiol: Roles of the Carboxy-Terminal and the Amino-Terminal Extracellular Domains

The endogenous steroid 17β-estradiol (βEST) potentiates activation of neuronal nicotinic receptors containing α4 subunits. Previous work has shown that the final 4 residues of the α4 subunit are required for potentiation. However, receptors containing the α2 subunit are not potentiated although it has these 4 residues, and only one amino acid difference in the C-terminal tail (FLAGMI vs. WLAGMI). Previous work had indicated that the tryptophan residue was involved in binding an analog of βEST, but not in potentiation by βEST. To determine the structural basis for the loss of potentiation we analyzed data from chimeric subunits, which indicated that the major factor underlying the difference between α2 and α4 is the tryptophan/phenylalanine difference, while the N-terminal extracellular domain is a less significant factor. When the tryptophan in α4 was mutated, both phenylalanine and tyrosine conferred lower potentiation while lysine and leucine did not. The reduction reflected a reduced maximal magnitude of potentiation, indicating that the tryptophan is involved in transduction of steroid effects. The regions of the α4 N-terminal extracellular domain involved in potentiation lie near the agonist-binding pocket, rather than close to the membrane or the C-terminal tail, and appear to be involved in transduction rather than binding. These observations indicate that the C-terminal region is involved in both steroid binding (AGMI residues) and transduction (W). The role of the N-terminus appears to be independent of the C-terminal tryptophan and likely reflects an influence on conformational changes caused during channel activation by agonist and potentiation by estradiol.     

The leucine-rich repeat domains of BK channel auxiliary γ subunits regulate their expression,trafficking, and channel-modulation functions

As high-conductance calcium- and voltage-dependent potassium channels, BK channels consist of pore-forming, voltage-, and Ca²⁺-sensing α and auxiliary subunits. The leucine-rich repeat (LRR) domain–containing auxiliary γ subunits potently modulate the voltage dependence of BK channel activation. Despite their dominant size in whole protein masses, the function of the LRR domain in BK channel γ subunits is unknown. We here investigated the function of these LRR domains in BK channel modulation by the auxiliary γ1–3 (LRRC26, LRRC52, and LRRC55) subunits. Using cell surface protein immunoprecipitation, we validated the predicted extracellular localization of the LRR domains. We then refined the structural models of mature proteins on the membrane via molecular dynamic simulations. By replacement of the LRR domain with extracellular regions or domains of non-LRR proteins, we found that the LRR domain is nonessential for the maximal channel-gating modulatory effect but is necessary for the all-or-none phenomenon of BK channel modulation by the γ1 subunit. Mutational and enzymatic blockade of N-glycosylation in the γ1–3 subunits resulted in a reduction or loss of BK channel modulation by γ subunits. Finally, by analyzing their expression in whole cells and on the plasma membrane, we found that blockade of N-glycosylation drastically reduced total expression of the γ2 subunit and the cell surface expression of the γ1 and γ3 subunits. We conclude that the LRR domains play key roles in the regulation of the expression, cell surface trafficking, and channel-modulation functions of the BK channel γ subunits.