Modulation of the conductance-voltage relationship of the BK Ca channel by mutations at the putative flexible interface between two RCK domains

Calcium-dependent gating of the large-conductance Ca²⁺-activated K⁺ (BK_Ca) channel is conferred by the large cytosolic carboxyl terminus containing two domains of the regulator of K⁺ conductance (RCK) and the high-affinity Ca²⁺-binding site (the Ca²⁺-bowl). In our previous study, we located the putative second RCK domain (RCK2) and demonstrated that it interacts directly with RCK1 via a hydrophobic “assembly interface”. In this study, we tested the structural model of the other interface, the “flexible interface”, by strategically positioning charge pairs across the putative interface. Several charge mutations on RCK2 affected the voltage-dependent activation of the channel. In particular, the Gly-to-Asp substitution at position 803 profoundly affected channel activation by stabilizing the open conformation of the channel with minimal effects on its Ca²⁺ affinity and voltage sensitivity. Various mutations at Gly-803 shifted the channel's conductance-voltage curve either left or right over a 145-mV range. Since this residue is predicted to be in the first loop of RCK2 these results strongly suggest that this loop plays a critical role in determining the intrinsic equilibrium constant for channel opening, and they support the hypothesis that this loop is part of an interface that mediates conformational coupling between RCK1 and RCK2.     

Measuring the Influence of the BKCa β1 Subunit on Ca2+ Binding to the BKCa Channel

The large-conductance Ca²⁺-activated potassium (BK_Ca) channel of smooth muscle is unusually sensitive to Ca²⁺ as compared with the BK_Ca channels of brain and skeletal muscle. This is due to the tissue-specific expression of the BK_Ca auxiliary subunit β1, whose presence dramatically increases both the potency and efficacy of Ca²⁺ in promoting channel opening. β1 contains no Ca²⁺ binding sites of its own, and thus the mechanism by which it increases the BK_Ca channel''s Ca²⁺ sensitivity has been of some interest. Previously, we demonstrated that β1 stabilizes voltage sensor activation, such that activation occurs at more negative voltages with β1 present. This decreases the work that Ca²⁺ must do to open the channel and thereby increases the channel''s apparent Ca²⁺ affinity without altering the real affinities of the channel''s Ca²⁺ binding sites. To explain the full effect of β1 on the channel''s Ca²⁺ sensitivity, however, we also proposed that there must be effects of β1 on Ca²⁺ binding. Here, to test this hypothesis, we have used high-resolution Ca²⁺ dose–response curves together with binding site–specific mutations to measure the effects of β1 on Ca²⁺ binding. We find that coexpression of β1 alters Ca²⁺ binding at both of the BK_Ca channel''s two types of high-affinity Ca²⁺ binding sites, primarily increasing the affinity of the RCK1 sites when the channel is open and decreasing the affinity of the Ca²⁺ bowl sites when the channel is closed. Both of these modifications increase the difference in affinity between open and closed, such that Ca²⁺ binding at either site has a larger effect on channel opening when β1 is present.     

BK Ca Channels Activating at Resting Potential without Calcium in LNCaP Prostate Cancer Cells

Large-conductance Ca²⁺-dependent K⁺ (BK_Ca) channels are activated by intracellular Ca²⁺ and membrane depolarization in an allosteric manner. We investigated the pharmacological and biophysical characteristics of a BK_Ca-type K⁺ channel in androgen-dependent LNCaP (lymph node carcinoma of the prostate) cells with novel functional properties, here termed BK_L. K⁺ selectivity, high conductance, activation by Mg²⁺ or NS1619, and inhibition by paxilline and penitrem A largely resembled the properties of recombinant BK_Ca channels. However, unlike conventional BK_Ca channels, BK_L channels activated in the absence of free cytosolic Ca²⁺ at physiological membrane potentials; the half-maximal activation voltage was shifted by about −100 mV compared with BK_Ca channels. Half-maximal Ca²⁺-dependent activation was observed at 0.4 μM for BK_L (at −20 mV) and at 4.1 μM for BK_Ca channels (at +50 mV). Heterologous expression of hSlo1 in LNCaP cells increased the BK_L conductance. Expression of hSlo-β1 in LNCaP cells shifted voltage-dependent activation to values between that of BK_L and BK_Ca channels and reduced the slope of the P_open (open probability)-voltage curve. We propose that LNCaP cells harbor a so far unknown type of BK_Ca subunit, which is responsible for the BK_L phenotype in a dominant manner. BK_L-like channels are also expressed in the human breast cancer cell line T47D. In addition, functional expression of BK_L in LNCaP cells is regulated by serum-derived factors, however not by androgens.     

Activation of BKCa Channels Mediates Hippocampal Neuronal Death After Reoxygenation and Reperfusion

Excessive K⁺ efflux promotes central neuronal apoptosis; however, the type of potassium channel that mediates K⁺ efflux in response to different apoptosis-inducing stimuli is still unknown. It is hypothesized that the activation of large-conductance Ca²⁺-activated K⁺ channels (BK_Ca) mediates hypoxia/reoxygenation (H/R)- and ischemia/reperfusion (I/R)-induced neuronal apoptosis. Rat hippocampal neuronal cultures underwent apoptosis after reoxygenation, as assessed by morphologic observation, terminal deoxynucleotidyl transferase dUTP nick end labeling staining, and caspase-3 activation. Single-channel recordings revealed upregulation of BK_Ca channel activity 6 h after reoxygenation, which might be caused by elevated cytosolic Ca²⁺. The K⁺ ionophore valinomycin and the BK_Ca channel opener NS1619 induced neuronal apoptosis. Transfection of the BK_Ca channel α subunit into Chinese hamster ovary (CHO-K1) cells, which do not express endogenous K⁺ channels, or into neurons will induce cell apoptosis, indicating that the opening of the BK_Ca channel serves as a pivotal event in mediating cell apoptosis. The specific BK_Ca channel blockers charybdotoxin and iberiotoxin and the nonselective K⁺ channel blocker tetraethylammonium at concentrations more specific to the BK_Ca channel were neuroprotective. The A-type potassium channel blocker 4-aminopyridine and apamin, a small-conductance Ca²⁺-activated K⁺ channel blocker, were not protective. This result suggests the involvement of the BK_Ca channel in H/R-induced apoptosis. Similarly, specific BK_Ca channel blockers also showed neuroprotection in neurons subjected to oxygen-glucose deprivation/reoxygenation or animals subjected to forebrain ischemia–reperfusion. These results demonstrate that the over-activity of BK_Ca channels mediates hippocampal neuronal damage induced by H/R in vitro and I/R in vivo.     

Modulation of the Conductance-Voltage Relationship of the BKCa Channel by Shortening the Cytosolic Loop Connecting Two RCK Domains

Calcium-dependent gating of large-conductance calcium-activated potassium (BK_Ca) channels is mediated by the intracellular carboxyl terminus, which contains two domains of regulator of K⁺ conductance (RCK). In mammalian BK_Ca channels, the two RCK domains are separated by a protein segment of 101 residues that is poorly conserved in evolution and predicted to have no regular secondary structures. We investigated the functional importance of this loop using a series of deletion mutations. We found that the length, rather than the specific sequence at the central region of the segment, is critical for the functionality of the channel. As the length of the loop is progressively shorted, the conductance-voltage relationship gradually shifts toward more positive voltages with a minimum length of 70 amino acids, in an apparent response to increased tension within the loop. Thus, the functional activity of the BK_Ca channel can be modulated by altering the tension of this loop region.     

Type 1 IP3 receptors activate BKCa channels via local molecular coupling in arterial smooth muscle cells

Plasma membrane large-conductance Ca²⁺-activated K⁺ (BK_Ca) channels and sarcoplasmic reticulum inositol 1,4,5-trisphosphate (IP₃) receptors (IP₃Rs) are expressed in a wide variety of cell types, including arterial smooth muscle cells. Here, we studied BK_Ca channel regulation by IP₃ and IP₃Rs in rat and mouse cerebral artery smooth muscle cells. IP₃ activated BK_Ca channels both in intact cells and in excised inside-out membrane patches. IP₃ caused concentration-dependent BK_Ca channel activation with an apparent dissociation constant (K_d) of ∼4 µM at physiological voltage (−40 mV) and intracellular Ca²⁺ concentration ([Ca²⁺]_i; 10 µM). IP₃ also caused a leftward-shift in BK_Ca channel apparent Ca²⁺ sensitivity and reduced the K_d for free [Ca²⁺]_i from ∼20 to 12 µM, but did not alter the slope or maximal P_o. BAPTA, a fast Ca²⁺ buffer, or an elevation in extracellular Ca²⁺ concentration did not alter IP₃-induced BK_Ca channel activation. Heparin, an IP₃R inhibitor, and a monoclonal type 1 IP₃R (IP₃R1) antibody blocked IP₃-induced BK_Ca channel activation. Adenophostin A, an IP₃R agonist, also activated BK_Ca channels. IP₃ activated BK_Ca channels in inside-out patches from wild-type (IP₃R1^+/+) mouse arterial smooth muscle cells, but had no effect on BK_Ca channels of IP₃R1-deficient (IP₃R1^−/−) mice. Immunofluorescence resonance energy transfer microscopy indicated that IP₃R1 is located in close spatial proximity to BK_Ca α subunits. The IP₃R1 monoclonal antibody coimmunoprecipitated IP₃R1 and BK_Ca channel α and β1 subunits from cerebral arteries. In summary, data indicate that IP₃R1 activation elevates BK_Ca channel apparent Ca²⁺ sensitivity through local molecular coupling in arterial smooth muscle cells.     

SLO-2 isoforms with unique Ca2+- and voltage-dependence characteristics confer sensitivity to hypoxia in C. elegans

Slo channels are large conductance K⁺ channels that display marked differences in their gating by intracellular ions. Among them, the Slo1 and C. elegans SLO-2 channels are gated by calcium (Ca²⁺), while mammalian Slo2 channels are activated by both sodium (Na⁺) and chloride (Cl⁻). Here, we report that SLO-2 channels, SLO-2a and a novel N-terminal variant isoform, SLO-2b, are activated by Ca²⁺ and voltage, but in contrast to previous reports they do not exhibit Cl⁻ sensitivity. Most importantly, SLO-2 provides a unique case in the Slo family for sensing Ca²⁺ with the high-affinity Ca²⁺ regulatory site in the RCK1 but not the RCK2 domain, formed through interactions with residues E319 and E487 (that correspond to D362 and E535 of Slo1, respectively). The SLO-2 RCK2 domain lacks the Ca²⁺ bowl structure and shows minimal Ca²⁺ dependence. In addition, in contrast to SLO-1, SLO-2 loss-of-function mutants confer resistance to hypoxia in C. elegans. Thus, the C. elegans SLO-2 channels possess unique biophysical and functional properties.     

Caveolin-1 Facilitates the Direct Coupling between Large Conductance Ca2+-activated K+ (BKCa) and Cav1.2 Ca2+ Channels and Their Clustering to Regulate Membrane Excitability in Vascular Myocytes

L-type voltage-dependent Ca²⁺ channels (LVDCC) and large conductance Ca²⁺-activated K⁺ channels (BK_Ca) are the major factors defining membrane excitability in vascular smooth muscle cells (VSMCs). The Ca²⁺ release from sarcoplasmic reticulum through ryanodine receptor significantly contributes to BK_Ca activation in VSMCs. In this study direct coupling between LVDCC (Cav1.2) and BK_Ca and the role of caveoline-1 on their interaction in mouse mesenteric artery SMCs were examined. The direct activation of BK_Ca by Ca²⁺ influx through coupling LVDCC was demonstrated by patch clamp recordings in freshly isolated VSMCs. Using total internal reflection fluorescence microscopy, it was found that a large part of yellow fluorescent protein-tagged BK_Ca co-localized with the cyan fluorescent protein-tagged Cav1.2 expressed in the plasma membrane of primary cultured mouse VSMCs and that the two molecules often exhibited FRET. It is notable that each BKα subunit of a tetramer in BK_Ca can directly interact with Cav1.2 and promotes Cav1.2 cluster in the molecular complex. Furthermore, caveolin-1 deficiency in knock-out (KO) mice significantly reduced not only the direct coupling between BK_Ca and Cav1.2 but also the functional coupling between BK_Ca and ryanodine receptor in VSMCs. The measurement of single cell shortening by 40 mm K⁺ revealed enhanced contractility in VSMCs from KO mice than wild type. Taken together, caveolin-1 facilitates the accumulation/clustering of BK_Ca-LVDCC complex in caveolae, which effectively regulates spatiotemporal Ca²⁺ dynamics including the negative feedback, to control the arterial excitability and contractility.     

Structural Determinants of Phosphatidylinositol 4,5-Bisphosphate (PIP2) Regulation of BK Channel Activity through the RCK1 Ca2+ Coordination Site

Big or high conductance potassium (BK) channels are activated by voltage and intracellular calcium (Ca²⁺). Phosphatidylinositol 4,5-bisphosphate (PIP₂), a ubiquitous modulator of ion channel activity, has been reported to enhance Ca²⁺-driven gating of BK channels, but a molecular understanding of this interplay or even of the PIP₂ regulation of this channel''s activity remains elusive. Here, we identify structural determinants in the KDRDD loop (which follows the αA helix in the RCK1 domain) to be responsible for the coupling between Ca²⁺ and PIP₂ in regulating BK channel activity. In the absence of Ca²⁺, RCK1 structural elements limit channel activation through a decrease in the channel''s PIP₂ apparent affinity. This inhibitory influence of BK channel activation can be relieved by mutation of residues that (a) connect either the RCK1 Ca²⁺ coordination site (Asp³⁶⁷ or its flanking basic residues in the KDRDD loop) to the PIP₂-interacting residues (Lys³⁹² and Arg³⁹³) found in the αB helix or (b) are involved in hydrophobic interactions between the αA and αB helix of the RCK1 domain. In the presence of Ca²⁺, the RCK1-inhibitory influence of channel-PIP₂ interactions and channel activity is relieved by Ca²⁺ engaging Asp³⁶⁷. Our results demonstrate that, along with Ca²⁺ and voltage, PIP₂ is a third factor critical to the integral control of BK channel activity.     

Functional validation of Ca2 +-binding residues from the crystal structure of the BK ion channel

BK channels are dually regulated by voltage and Ca^2 +, providing a cellular mechanism to couple electrical and chemical signalling. Intracellular Ca^2 + concentration is sensed by a large cytoplasmic region in the channel known as “gating ring”, which is formed by four tandems of regulator of conductance for K⁺ (RCK1 and RCK2) domains. The recent crystal structure of the full-length BK channel from Aplysia californica has provided new information about the residues involved in Ca^2 + coordination at the high-affinity binding sites located in the RCK1 and RCK2 domains, as well as their cooperativity. Some of these residues have not been previously studied in the human BK channel. In this work we have investigated, through site directed mutagenesis and electrophysiology, the effects of these residues on channel activation by voltage and Ca^2 +. Our results demonstrate that the side chains of two non-conserved residues proposed to coordinate Ca^2 + in the A. californica structure (G523 and E591) have no apparent functional role in the human BK Ca^2 + sensing mechanism. Consistent with the crystal structure, our data indicate that in the human channel the conserved residue R514 participates in Ca^2 + coordination in the RCK1 binding site. Additionally, this study provides functional evidence indicating that R514 also interacts with residues E902 and Y904 connected to the Ca^2 + binding site in RCK2. Interestingly, it has been proposed that this interaction may constitute a structural correlate underlying the cooperative interactions between the two high-affinity Ca^2 + binding sites regulating the Ca^2 + dependent gating of the BK channel. This article is part of a Special Issue entitled: Beyond the Structure-Function Horizon of Membrane Proteins edited by Ute Hellmich, Rupak Doshi and Benjamin McIlwain.     

Identification and characterization of Ca2+-activated K+ channels in granulosa cells of the human ovary

Background  

Granulosa cells (GCs) represent a major endocrine compartment of the ovary producing sex steroid hormones. Recently, we identified in human GCs a Ca²⁺-activated K⁺ channel (K_Ca) of big conductance (BK_Ca), which is involved in steroidogenesis. This channel is activated by intraovarian signalling molecules (e.g. acetylcholine) via raised intracellular Ca²⁺ levels. In this study, we aimed at characterizing 1. expression and functions of K_Ca channels (including BK_Ca beta-subunits), and 2. biophysical properties of BK_Ca channels.     

Comparative effects of H+ and Ca2+ on large-conductance Ca2+- and voltage-gated Slo1 K+ channels

Large-conductance Ca²⁺- and voltage-gated Slo1 BK channels are allosterically activated by depolarization and intracellular ligands such as Ca²⁺. Of the two high-affinity Ca²⁺ sensors present in the channel, the RCK1 sensor also mediates H⁺-dependent activation of the channel. In this study, we examined the comparative mechanisms of the channel activation by Ca²⁺ and H⁺. Steady-state macroscopic conductance-voltage measurements as well as single-channel openings at negative voltages where voltage-sensor activation is negligible showed that at respective saturating concentrations Ca²⁺ is more effective in relative stabilization of the open conformation than H⁺. Calculations using the Debye-Hückel formulation suggest that small structural changes in the RCK1 sensor, on the order of few angstroms, may accompany the H⁺-mediated opening of the channel. While the efficacy of H+ in activation of the channel is less than that of Ca²⁺, H⁺ more effectively accelerates the activation kinetics when examined at the concentrations equipotent on macroscopic voltage-dependent activation. The RCK1 sensor therefore is capable of transducing the nature of the ligand bound and transmits qualitatively different information to the channel's permeation gate.     

Role of the beta1 subunit in large-conductance Ca(2+)-activated K(+) channel gating energetics. Mechanisms of enhanced Ca(2+) sensitivity

Over the past few years, it has become clear that an important mechanism by which large-conductance Ca²⁺-activated K⁺ channel (BK_Ca) activity is regulated is the tissue-specific expression of auxiliary β subunits. The first of these to be identified, β1, is expressed predominately in smooth muscle and causes dramatic effects, increasing the apparent affinity of the channel for Ca²⁺ 10-fold at 0 mV, and shifting the range of voltages over which the channel activates −80 mV at 9.1 μM Ca²⁺. With this study, we address the question: which aspects of BK_Ca gating are altered by β1 to bring about these effects: Ca²⁺ binding, voltage sensing, or the intrinsic energetics of channel opening? The approach we have taken is to express the β1 subunit together with the BK_Ca α subunit in Xenopus oocytes, and then to compare β1''s steady state effects over a wide range of Ca²⁺ concentrations and membrane voltages to those predicted by allosteric models whose parameters have been altered to mimic changes in the aspects of gating listed above. The results of our analysis suggest that much of β1''s steady state effects can be accounted for by a reduction in the intrinsic energy the channel must overcome to open and a decrease in its voltage sensitivity, with little change in the affinity of the channel for Ca²⁺ when it is either open or closed. Interestingly, however, the small changes in Ca²⁺ binding affinity suggested by our analysis (K_c 7.4 μM → 9.6 μM; K_o = 0.80 μM → 0.65 μM) do appear to be functionally important. We also show that β1 affects the mSlo conductance–voltage relation in the essential absence of Ca²⁺, shifting it +20 mV and reducing its apparent gating charge 38%, and we develop methods for distinguishing between alterations in Ca²⁺ binding and other aspects of BK_Ca channel gating that may be of general use.     

IP3 decreases coronary artery tone via activating the BKCa channel of coronary artery smooth muscle cells in pigs

Large conductance Ca²⁺-activated K⁺ channel (BK_Ca) is a potential target for coronary artery-relaxing medication, but its functional regulation is largely unknown. Here, we report that inositol trisphosphate (IP3) activated BK_Ca channels in isolated porcine coronary artery smooth muscle cells and by which decreased the coronary artery tone. Both endogenous and exogenous IP3 increased the spontaneous transient outward K⁺ currents (STOC, a component pattern of BK_Ca currents) in perforated and regular whole-cell recordings, which was dependent on the activity of IP3 receptors. IP3 also increased the macroscopic currents (MC, another component pattern of BK_Ca currents) via an IP3 receptor- and sarcoplasmic Ca²⁺ mobilization-independent pathway. In inside-out patch recordings, direct application of IP3 to the cytosolic side increased the open probability of single BK_Ca channel in an IP3 receptor-independent manner. We conclude that IP3 is an activator of BK_Ca channels in porcine coronary smooth muscle cells and exerts a coronary artery-relaxing effect. The activation of BK_Ca channels by IP3 involves the enhancement of STOCs via IP3 receptors and stimulation of MC by increasing the Ca²⁺ sensitivity of the channels.     

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.     

Functional and molecular consequences of ionizing irradiation on large conductance Ca2+-activated K+ channels in rat aortic smooth muscle cells

AimsThe goal of this study was to evaluate the influence of γ-irradiation on Ca²⁺-activated K⁺ channel (BK_Ca) function and expression in rat thoracic aorta.Main methodsAortic cells or tissues were studied by the measurement of force versus [Ca²⁺]_i, patch-clamp technique, and RT-PCR.Key findingsStimulation of smooth muscle cells with depolarizing voltage steps showed expression of outward K⁺ currents. Paxilline, an inhibitor of BK_Ca channels, decreased outward K⁺ current density. Outward currents in smooth muscle cells obtained from irradiated animals 9 and 30 days following radiation exposure demonstrated a significant decrease in K⁺ current density. Paxilline decreased K⁺ current in cells obtained 9 days, but was without effect 30 days after irradiation suggesting the absence of BK_Ca channels. Aortic tissue from irradiated animals showed progressively enhanced contractile responses to phenylephrine in the post-irradiation period of 9 and 30 days. The concomitant Ca²⁺ transients were significantly smaller, as compared to tissues from control animals, 9 days following irradiation but were increased above control levels 30 days following irradiation. Irradiation produced a decrease in BK_Ca α- and β₁-subunit mRNA levels in aortic smooth muscle cells suggesting that the vasorelaxant effect of these channels may be diminished.SignificanceThese results suggest that the enhanced contractility of vascular tissue from animals exposed to radiation may result from an increase in myofilament Ca²⁺ sensitivity in the early post-irradiation period and a decrease in BK_Ca channel expression in the late post-irradiation period.     

Exercise training changes the gating properties of large-conductance Ca2+-activated K+ channels in rat thoracic aorta smooth muscle cells

Large-conductance Ca²⁺-activated K⁺ (BK_Ca) channels play a critical role in regulating the cellular excitability in response to change in blood flow. It has been demonstrated that vascular BK_Ca channel currents in both humans and rats are increased after exercise training. This up-regulation of the BK_Ca channel activity in arterial myocytes may represent a cellular compensatory mechanism of limiting vascular reactivity to exercise training. However, the underlying mechanisms are not fully understood. In the present study, we examined the single channel activities and kinetics of the BK_Ca channels in rat thoracic aorta smooth muscle cells. We showed that exercise training significantly increased the open probability (Po), decreased the mean closed time and increased the mean open time, and the sensitivity to Ca²⁺ and voltage without altering the unitary conductance and the K⁺ selectivity. Our results suggest a novel mechanism by which exercise training increases the K⁺ currents by changing the BK_Ca channel activities and kinetics.     

多不饱和脂肪酸对钾通道的调控作用及机理

多不饱和脂肪酸具有包括离子通道在内的众多作用靶点,通过作用于这些靶点,可以有效保护免疫系统、神经系统和心血管系统的功能,在一定程度上保护人体健康。电压门控钾离子通道家族K_V7通道和大电导钙离子激活的钾离子通道(BK_Ca)广泛表达于机体的各类组织中,具有重要的生理或病理功能。本综述围绕K_V7和BK_Ca通道,根据对已有报道的汇总,多不饱和脂肪酸可以增大K_V7和BK_Ca通道的电流幅值,其中对K_V7通道电流的影响主要是改变其电压依赖特性和最大电导值,而对BK_Ca通道电流的影响主要是改变其孔道区域关闭态的构象。此外,多不饱和脂肪酸对K_V7和BK_Ca通道功能的调节也会受到共表达的辅助亚基影响,但相关机制有待进一步阐明。深入理解多不饱和脂肪酸对K_V7和BK_Ca通道调节作用效果和分子机制,有助于全面理解K_V7和BK     

Electrophysiology of human cardiac atrial and ventricular telocytes

Telocytes (TCs) with exceptionally long cellular processes of telopodes have been described in human epicardium to act as structural supporting cells in the heart. We examined myocardial chamber‐specific TCs identified in atrial and ventricular fibroblast culture using immunocytochemistry and studied their electrophysiological property by whole‐cell patch clamp. Atrial and ventricular TCs with extended telopodes and alternating podoms and podomers that expressed CD34, c‐Kit and PDGFR‐β were identified. These cells expressed large conductance Ca²⁺‐activated K⁺ current (BK_Ca) and inwardly rectifying K⁺ current (IK_ir), but not transient outward K⁺ current (I_to) and ATP‐sensitive potassium current (K_ATP). The active channels were functionally competent with demonstrated modulatory response to H₂S and transforming growth factor (TGF)‐β1 whereby H₂S significantly inhibited the stimulatory effect of TGF‐β1 on current density of both BK_Ca and IK_ir. Furthermore, H₂S attenuated TGF‐β1‐stimulated KCa1.1/Kv1.1 (encode BK_Ca) and Kir2.1 (encode IK_ir) expression in TCs. Our results show that functionally competent K⁺ channels are present in human atrial and ventricular TCs and their modulation may have significant implications in myocardial physiopathology.     

Mechanisms of the Inhibitory Action of Neurotransmitters on Smooth Muscles

Nonadrenergic inhibitory and excitatory junction potentials (IJP and EJP) in the intestinal smooth muscle cells are of a complex transmitter and ion nature. The IJP consist of two components; the initial, fast, component is of a purinergic nature. Low-conductance Ca²⁺-dependent potassium channels (SK_(Ca)) are involved in generation of the initial component of IJP because this component can be specifically and reversibly blocked by apamin. Probably, local Ca²⁺ release from the InsP₃-sensitive store can be a link between the P2Y receptors and activation of the SK_(Ca) channels because inhibition of the activity of phospholipase C (PLC) decreases IJP. The second, slow, component of IJP is nitric oxide-dependent. Such a component of IJP develops due to activation of high-conductance Ca²⁺-dependent potassium channels (BK_(Ca)) because this component can be blocked by TEA and charybdotoxin. The release of Ca²⁺ from the ryanodine-sensitive store is responsible for activation of the BK_(Ca) channels and generation of the second component of IJP. Thus, it appears that Ca²⁺ released from one of the intracellular stores can activate only a certain type of the Ca²⁺-dependent K⁺ channels involved in the generation of IJP.