A BK (Slo1) channel journey from molecule to physiology

Calcium and voltage-activated potassium (BK) channels are key actors in cell physiology, both in neuronal and non-neuronal cells and tissues. Through negative feedback between intracellular Ca²⁺ and membrane voltage, BK channels provide a damping mechanism for excitatory signals. Molecular modulation of these channels by alternative splicing, auxiliary subunits and post-translational modifications showed that these channels are subjected to many mechanisms that add diversity to the BK channel α subunit gene. This complexity of interactions modulates BK channel gating, modifying the energetic barrier of voltage sensor domain activation and channel opening. Regions for voltage as well as Ca²⁺ sensitivity have been identified, and the crystal structure generated by the 2 RCK domains contained in the C-terminal of the channel has been described. The linkage of these channels to many intracellular metabolites and pathways, as well as their modulation by extracellular natural agents, has been found to be relevant in many physiological processes. This review includes the hallmarks of BK channel biophysics and its physiological impact on specific cells and tissues, highlighting its relationship with auxiliary subunit expression.     

Activation of the BK (SLO1) potassium channel by mallotoxin

Pharmacologic approaches to activate K+ channels represent an emerging strategy to regulate membrane excitability. Here we report the identification and characterization of a lipid soluble toxin, mallotoxin (rottlerin), which potently activates the large conductance voltage and Ca2+-activated K+ channel (BK) expressed in a heterologous expression system and human vascular smooth muscle cells, shifting the conductance/voltage relationship by >100 mV. Probing the mechanism of action, we discover that the BK channel can be activated in the absence of divalent cations (Ca2+, Mg2+), suggesting that the mallotoxin mechanism of action involves the voltage-dependent gating of the channel. Mallotoxin-activated channels remain incrementally sensitive to Ca2+ and beta subunits. In comparison to other small hydrophobic poisons, anesthetic agents, and protein toxins that inhibit ion channel activity, mallotoxin potently activates channel activity. In certain respects, mallotoxin acts as a BK channel beta1 subunit mimetic, preserving BK channel Ca2+ sensitivity yet adjusting the set-point for BK channel activation to a more hyperpolarized membrane potential.     

Elimination of the BK(Ca) channel's high-affinity Ca(2+) sensitivity

We report here a combination of site-directed mutations that eliminate the high-affinity Ca(2+) response of the large-conductance Ca(2+)-activated K(+) channel (BK(Ca)), leaving only a low-affinity response blocked by high concentrations of Mg(2+). Mutations at two sites are required, the "Ca(2+) bowl," which has been implicated previously in Ca(2+) binding, and M513, at the end of the channel's seventh hydrophobic segment. Energetic analyses of mutations at these positions, alone and in combination, argue that the BK(Ca) channel contains three types of Ca(2+) binding sites, one of low affinity that is Mg(2+) sensitive (as has been suggested previously) and two of higher affinity that have similar binding characteristics and contribute approximately equally to the power of Ca(2+) to influence channel opening. Estimates of the binding characteristics of the BK(Ca) channel's high-affinity Ca(2+)-binding sites are provided.     

Cytosolic phospholipase A2 is required for optimal ATP activation of BK channels in GH(3) cells

To test the hypothesis that ATP activation of BK channels in GH(3) cells involves cytosolic phospholipase A(2) (cPLA(2)) as a potential protein target for phosphorylation, we first inhibited the activity of cPLA(2) by both pharmacologic and molecular biologic approaches. Both approaches resulted in a decrease rather than an increase in BK channel activity by ATP, suggesting that in the absence of cPLA(2), phosphorylation of other regulatory elements, possibly the BK channel protein itself, results in inactivation rather than activation of the channel. The absence of changes in activity in the presence of the non-substrate ATP analog 5'-adenylyl-beta,gamma-imidodiphosphate verified that ATP hydrolysis was required for channel activation by ATP. Experiments with an activator and inhibitor of protein kinase C (PKC) support the hypothesis that PKC can be involved in the activation of BK channels by ATP; and in the absence of PKC, other kinases appear to phosphorylate additional elements in the regulatory pathway that reduce channel activity. Our data point to cPLA(2)-alpha (but not cPLA(2)-gamma) as one target protein for phosphorylation that is intimately associated with the BK channel protein.     

Multiple cholesterol recognition/interaction amino acid consensus (CRAC) motifs in cytosolic C tail of Slo1 subunit determine cholesterol sensitivity of Ca2+- and voltage-gated K+ (BK) channels

Large conductance, Ca(2+)- and voltage-gated K(+) (BK) channel proteins are ubiquitously expressed in cell membranes and control a wide variety of biological processes. Membrane cholesterol regulates the activity of membrane-associated proteins, including BK channels. Cholesterol modulation of BK channels alters action potential firing, colonic ion transport, smooth muscle contractility, endothelial function, and the channel alcohol response. The structural bases underlying cholesterol-BK channel interaction are unknown. Such interaction is determined by strict chemical requirements for the sterol molecule, suggesting cholesterol recognition by a protein surface. Here, we demonstrate that cholesterol action on BK channel-forming Cbv1 proteins is mediated by their cytosolic C tail domain, where we identified seven cholesterol recognition/interaction amino acid consensus motifs (CRAC4 to 10), a distinct feature of BK proteins. Cholesterol sensitivity is provided by the membrane-adjacent CRAC4, where Val-444, Tyr-450, and Lys-453 are required for cholesterol sensing, with hydrogen bonding and hydrophobic interactions participating in cholesterol location and recognition. However, cumulative truncations or Tyr-to-Phe substitutions in CRAC5 to 10 progressively blunt cholesterol sensitivity, documenting involvement of multiple CRACs in cholesterol-BK channel interaction. In conclusion, our study provides for the first time the structural bases of BK channel cholesterol sensitivity; the presence of membrane-adjacent CRAC4 and the long cytosolic C tail domain with several other CRAC motifs, which are not found in other members of the TM6 superfamily of ion channels, very likely explains the unique cholesterol sensitivity of BK channels.     

大电导钾离子通道(BK)结构与功能的研究进展

大电导钙离子激活钾通道(BK)是细胞膜上唯一接受细胞内Ca2+和膜电位双重调控的离子通道.最新发表的关于BK通道电镜结构及其胞质功能域的晶体结构的文章,第一次展示了BK通道各亚基的组装,并证实通道各功能域在通道门控机制中存在紧密的相互作用.近年来,针对BK通道的功能调节及其门控动力学模拟的研究取得较多进展,有助于更好地理解BK通道发挥生理功能的门控机制,并揭示BK通道相关疾病的病理生理学基础.     

Intracellular Mg(2+) enhances the function of BK-type Ca(2+)-activated K(+) channels.

BK channels modulate neurotransmitter release due to their activation by voltage and Ca(2+). Intracellular Mg(2+) also modulates BK channels in multiple ways with opposite effects on channel function. Previous single-channel studies have shown that Mg(2+) blocks the pore of BK channels in a voltage-dependent manner. We have confirmed this result by studying macroscopic currents of the mslo1 channel. We find that Mg(2+) activates mslo1 BK channels independently of Ca(2+) and voltage by preferentially binding to their open conformation. The mslo3 channel, which lacks Ca(2+) binding sites in the tail, is not activated by Mg(2+). However, coexpression of the mslo1 core and mslo3 tail produces channels with Mg(2+) sensitivity similar to mslo1 channels, indicating that Mg(2+) sites differ from Ca(2+) sites. We discovered that Mg(2+) also binds to Ca(2+) sites and competitively inhibits Ca(2+)-dependent activation. Quantitative computation of these effects reveals that the overall effect of Mg(2+) under physiological conditions is to enhance BK channel function.     

BK channels are linked to inositol 1,4,5-triphosphate receptors via lipid rafts: a novel mechanism for coupling [Ca(2+)](i) to ion channel activation

Glioma cells prominently express a unique splice variant of a large conductance, calcium-activated potassium channel (BK channel). These channels transduce changes in intracellular calcium to changes of K(+) conductance in the cells and have been implicated in growth control of normal and malignant cells. The Ca(2+) increase that facilitates channel activation is thought to occur via activation of intracellular calcium release pathways or influx of calcium through Ca(2+)-permeable ion channels. We show here that BK channel activation involves the activation of inositol 1,4,5-triphosphate receptors (IP(3)R), which localize near BK channels in specialized membrane domains called lipid rafts. Disruption of lipid rafts with methyl-beta-cyclodextrin disrupts the functional association of BK channel and calcium source resulting in a >50% reduction in K(+) conductance mediated by BK channels. The reduction of BK current by lipid raft disruption was overcome by the global elevation of intracellular calcium through inclusion of 750 nm Ca(2+) in the pipette solution, indicating that neither the calcium sensitivity of the channel nor their overall number was altered. Additionally, pretreatment of glioma cells with 2-aminoethoxydiphenyl borate to inhibit IP(3)Rs negated the effect of methyl-beta-cyclodextrin, providing further support that IP(3)Rs are the calcium source for BK channels. Taken together, these data suggest a privileged association of BK channels in lipid raft domains and provide evidence for a novel coupling of these Ca(2+)-sensitive channels to their second messenger source.     

A molecular switch for specific stimulation of the BKCa channel by cGMP and cAMP kinase

The cGMP and the cAMP pathways control smooth muscle tone by regulation of BK(Ca) (BK) channel activity. BK channels show considerable diversity and plasticity in their regulation by cyclic nucleotide-dependent protein kinases. The underlying molecular mechanisms are unclear but may involve expression of splice variants of the BK channel alpha subunit. Three isoforms, BK(A), BK(B), and BK(C), which were cloned from tracheal smooth muscle, differed only in their C terminus. When expressed in HEK293 cells, cGMP kinase (cGK) but not cAMP kinase (cAK) stimulated the activity of BK(A) and BK(B) by shifting the voltage dependence of the channel to more negative potentials. In contrast, BK(C) was exclusively stimulated by cAK. BK(C) lacks a C-terminal tandem phosphorylation motif for protein kinase C (PKC) with Ser(1151) and Ser(1154). Mutation of this motif in BK(A) switched channel regulation from cGK to cAK. Furthermore, inhibition of PKC in excised patches from cells expressing BK(A) abolished the stimulatory effect of cGK but allowed channel stimulation by cAK. cAK and cGK phosphorylated the channel at different sites. Thus, phosphorylation/dephosphorylation by PKC determines whether the BK channel is stimulated by cGK or cAK. The molecular mechanisms may be relevant for smooth muscle relaxation by cAMP and cGMP.     

Palmitoylation and Membrane Association of the Stress Axis Regulated Insert (STREX) Controls BK Channel Regulation by Protein Kinase C

Large-conductance, calcium- and voltage-gated potassium (BK) channels play an important role in cellular excitability by controlling membrane potential and calcium influx. The stress axis regulated exon (STREX) at splice site 2 inverts BK channel regulation by protein kinase A (PKA) from stimulatory to inhibitory. Here we show that palmitoylation of STREX controls BK channel regulation also by protein kinase C (PKC). In contrast to the 50% decrease of maximal channel activity by PKC in the insertless (ZERO) splice variant, STREX channels were completely resistant to PKC. STREX channel mutants in which Ser(700), located between the two regulatory domains of K(+) conductance (RCK) immediately downstream of the STREX insert, was replaced by the phosphomimetic amino acid glutamate (S700E) showed a ～50% decrease in maximal channel activity, whereas the S700A mutant retained its normal activity. BK channel inhibition by PKC, however, was effectively established when the palmitoylation-mediated membrane-anchor of the STREX insert was removed by either pharmacological inhibition of palmitoyl transferases or site-directed mutagenesis. These findings suggest that STREX confers a conformation on BK channels where PKC fails to phosphorylate and to inhibit channel activity. Importantly, PKA which inhibits channel activity by disassembling the STREX insert from the plasma membrane, allows PKC to further suppress the channel gating independent from voltage and calcium. Our results present an important example for the cross-talk between ion channel palmitoylation and phosphorylation in regulation of cellular excitability.     

Potassium (BK(Ca)) currents are reduced in microvascular smooth muscle cells from insulin-resistant rats

Insulin resistance (IR) syndrome is associated with impaired vascular relaxation; however, the underlying pathophysiology is unknown. Potassium channel activation causes vascular smooth muscle hyperpolarization and relaxation. The present study determined whether a reduction in large conductance calcium- and voltage-activated potassium (BK(Ca)) channel activity contributes to impaired vascular relaxation in IR rats. BK(Ca) channels were characterized in mesenteric microvessels from IR and control rats. Macroscopic current density was reduced in myocytes from IR animals compared with controls. In addition, inhibition of BK(Ca) channels with tetraethylammonium (1 mM) or iberiotoxin (100 nM) was greater in myocytes from control (70%) compared with IR animals (approximately 20%). Furthermore, activation of BK(Ca) channels with NS-1619 was three times more effective at increasing outward current in cells from control versus IR animals. Single channel and Western blot analysis of BK(Ca) channels revealed similar conductance, amplitude, voltage sensitivity, Ca2+ sensitivity, and expression density between the two groups. These data provide the first direct evidence that microvascular potassium currents are reduced in IR and suggest a molecular mechanism that could account for impaired vascular relaxation in IR.     

Cysteine String Protein Limits Expression of the Large Conductance,Calcium-Activated K+ (BK) Channel

Large-conductance, calcium-activated K⁺ (BK) channels are widely distributed throughout the nervous system and play an essential role in regulation of action potential duration and firing frequency, along with neurotransmitter release at the presynaptic terminal. We have previously demonstrated that select mutations in cysteine string protein (CSPα), a presynaptic J-protein and co-chaperone, increase BK channel expression. This observation raised the possibility that wild-type CSPα normally functions to limit neuronal BK channel expression. Here we show by Western blot analysis of transfected neuroblastoma cells that when BK channels are present at elevated levels, CSPα acts to reduce expression. Moreover, we demonstrate that the accessory subunits, BKβ4 and BKβ1 do not alter CSPα-mediated reduction of expressed BKα subunits. Structure-function analysis reveals that the N-terminal J-domain of CSPα is critical for the observed regulation of BK channels levels. Finally, we demonstrate that CSPα limits BK current amplitude, while the loss-of-function homologue CSPα_HPD-AAA increases BK current. Our observations indicate that CSPα has a role in regulating synaptic excitability and neurotransmission by limiting expression of BK channels.     

Selectivity filter gating in large-conductance Ca(2+)-activated K+ channels

Membrane voltage controls the passage of ions through voltage-gated K (K(v)) channels, and many studies have demonstrated that this is accomplished by a physical gate located at the cytoplasmic end of the pore. Critical to this determination were the findings that quaternary ammonium ions and certain peptides have access to their internal pore-blocking sites only when the channel gates are open, and that large blocking ions interfere with channel closing. Although an intracellular location for the physical gate of K(v) channels is well established, it is not clear if such a cytoplasmic gate exists in all K(+) channels. Some studies on large-conductance, voltage- and Ca(2+)-activated K(+) (BK) channels suggest a cytoplasmic location for the gate, but other findings question this conclusion and, instead, support the concept that BK channels are gated by the pore selectivity filter. If the BK channel is gated by the selectivity filter, the interactions between the blocking ions and channel gating should be influenced by the permeant ion. Thus, we tested tetrabutyl ammonium (TBA) and the Shaker "ball" peptide (BP) on BK channels with either K(+) or Rb(+) as the permeant ion. When tested in K(+) solutions, both TBA and the BP acted as open-channel blockers of BK channels, and the BP interfered with channel closing. In contrast, when Rb(+) replaced K(+) as the permeant ion, TBA and the BP blocked both closed and open BK channels, and the BP no longer interfered with channel closing. We also tested the cytoplasmically gated Shaker K channels and found the opposite behavior: the interactions of TBA and the BP with these K(v) channels were independent of the permeant ion. Our results add significantly to the evidence against a cytoplasmic gate in BK channels and represent a positive test for selectivity filter gating.     

Quantification and distribution of big conductance Ca2+-activated K+ channels in kidney epithelia

Big conductance Ca2+ activated K+ channels (BK channels) is an abundant channel present in almost all kind of tissue. The accurate quantity and especially the precise distribution of this channel in kidney epithelia are, however, still debated. The aim of the present study has therefore been to examine the presence of BK channels in kidney epithelia and determine the actual number and distribution of these channels. For this purpose, a selective peptidyl ligand for BK channels called iberiotoxin or the radiolabeled double mutant analog 125I-IbTX-D19Y/Y36F has been employed. The presence of BK channels were determined by a isotope flux assay where up to 44% of the total K+ channel activity could be inhibited by iberiotoxin indicating that BK channels are widely present in kidney epithelia. Consistent with these functional studies, 125I-IbTX-D19Y/Y36F binds to membrane vesicles from outer cortex, outer medulla and inner medulla with Bmax values (in fmol/mg protein) of 6.8, 2.6 and 21.4, respectively. These studies were performed applying rabbit kidney epithelia tissue. The distinct distribution of BK channels in both rabbit and rat kidney epithelia was confirmed by autoradiography and immunohistochemical studies. In cortical collecting ducts, BK channels were exclusively located in principal cells while no channels could be found in intercalated cells. The abundant and distinct distribution in kidney epithelia talks in favor for BK channels being important contributors in maintaining salt and water homeostasis.     

Membrane trafficking of large conductance Ca2+- and voltage-activated K+ (BK) channels is regulated by Rab4 GTPase

The large conductance voltage- and Ca²⁺-activated K⁺ (BK) channel is a major ionic determinant of vascular tone, vasodilation, and blood pressure. The activity of BK channels is regulated in part by membrane presentation. Rab GTPase (Rab) regulates important cellular processes, including ion channel membrane trafficking. We hypothesize that Rab4a participates in the regulation of BK channel α-subunit (BK-α) membrane trafficking. We found that vascular BK-α interacts physically with Rab4a. Co-expression of dominant-negative Rab4a reduced BK-α surface expression, whereas that of constitutively-active Rab4a augmented BK-α surface presentation. These novel findings suggest that vascular BK channel membrane expression is regulated by Rab4a through channel membrane trafficking.     

Quantification and distribution of big conductance Ca-activated K channels in kidney epithelia

Big conductance Ca²⁺ activated K⁺ channels (BK channels) is an abundant channel present in almost all kind of tissue. The accurate quantity and especially the precise distribution of this channel in kidney epithelia are, however, still debated. The aim of the present study has therefore been to examine the presence of BK channels in kidney epithelia and determine the actual number and distribution of these channels. For this purpose, a selective peptidyl ligand for BK channels called iberiotoxin or the radiolabeled double mutant analog ¹²⁵I-IbTX-D19Y/Y36F has been employed. The presence of BK channels were determined by a isotope flux assay where up to 44% of the total K⁺ channel activity could be inhibited by iberiotoxin indicating that BK channels are widely present in kidney epithelia. Consistent with these functional studies, ¹²⁵I-IbTX-D19Y/Y36F binds to membrane vesicles from outer cortex, outer medulla and inner medulla with B_max values (in fmol/mg protein) of 6.8, 2.6 and 21.4, respectively. These studies were performed applying rabbit kidney epithelia tissue. The distinct distribution of BK channels in both rabbit and rat kidney epithelia was confirmed by autoradiography and immunohistochemical studies. In cortical collecting ducts, BK channels were exclusively located in principal cells while no channels could be found in intercalated cells. The abundant and distinct distribution in kidney epithelia talks in favor for BK channels being important contributors in maintaining salt and water homeostasis.     

Reversible tyrosine protein phosphorylation regulates large conductance voltage- and calcium-activated potassium channels via cortactin

Large conductance calcium- and voltage-activated potassium (BK) channels assemble as macromolecular signaling complexes and are potently regulated by reversible protein phosphorylation. However, although numerous studies have revealed regulation of BK channels through changes in direct phosphorylation of the pore-forming alpha-subunits the functional role of changes in phosphorylation of defined adapter/signaling proteins within the complex on channel function are essentially not known. Here, we demonstrate that mammalian BK channels are potently regulated by endogenous protein-tyrosine kinase and protein-tyrosine phosphatase activity closely associated with the channel. BK channel regulation was not dependent upon direct phosphorylation of the BK alpha-subunit, rather channel function was controlled by the tyrosine phosphorylation status of the adapter protein cortactin that assembles directly with the BK channel. Our data thus reveal a novel mode for BK channel regulation by reversible tyrosine phosphorylation and strongly support the hypothesis that phosphorylation-dependent regulation of accessory proteins within the BK channel signaling complex represents an important target for control of BK channel function.     

The effect of single cerebroside compounds on activation of BKCa channels

We have previously shown that a mixture of cerebrosides obtained from dried tubers of herb Typhonium giganteum Engl. plays a neuroprotective role in the ischemic brain through its effect on activation of BK(Ca) channels. It is very curious to know whether a single pure cerebroside compound could activate the BK(Ca) channel as well. This study explored the possible effects of pure cerebroside compounds, termitomycesphins A and B, on the BK(Ca) channel activation. Both termitomycesphins A and B activated the BK(Ca) channels at micromole concentration without significant difference. Termitomycesphin A increased the single channel open probability of the BK(Ca) channels in a dose-dependent manner without modifying the single channel conductance. Termitomycesphin A activated BK(Ca) channel more efficiently when it was applied to the cytoplasmic face of the membrane, suggesting that binding site for termitomycesphin A is located at the cytoplasmic side. Termitomycesphin A shifted the voltage-dependent activation curve to less positive membrane potentials and the Ca(2+)-dependent activation curve of the channel upwards, suggesting that termitomycesphin A could activate the channels even without intracellular free Ca(2+). Furthermore, STREX-deleted BK(Ca) channels were completely insensitive to termitomycesphin A, indicating that STREX domain is required for the activation of the BK(Ca) channel. These data provide evidence that termitomycesphins are potent in stimulating the activity of the BK(Ca) channels. As BK(Ca) channels are associated with pathology of many diseases, termitomycesphins might be used as therapeutic agents for treating these diseases through its regulatory effect on the BK(Ca) channels.     

Modulation of the BK channel by estrogens: examination at single channel level

BK channels regulate vascular tone by hyperpolarizing smooth muscle in response to fluctuating calcium concentrations. Oestrogen has been reported to lower blood pressure by increasing BK channel open probability through direct binding to the regulatory beta1-subunit(s) associated with the channel. The present investigation demonstrates that 17beta-oestradiol activates the BK channel complex by increasing the burst duration of channel openings. A subconductance state was observed in 25% of recordings following the addition of 17beta-oestradiol and could reflect uncoupling between the pore forming alpha1-subunit and the regulatory beta1-subunit. We also present evidence that more than one beta1-subunit is required to facilitate binding of 17beta-oestradiol to the channel complex.