A function for tyrosine phosphorylation of type 1 inositol 1,4,5-trisphosphate receptor in lymphocyte activation

Sustained elevation of intracellular calcium by Ca²⁺ release–activated Ca²⁺ channels is required for lymphocyte activation. Sustained Ca²⁺ entry requires endoplasmic reticulum (ER) Ca²⁺ depletion and prolonged activation of inositol 1,4,5-trisphosphate receptor (IP₃R)/Ca²⁺ release channels. However, a major isoform in lymphocyte ER, IP3R1, is inhibited by elevated levels of cytosolic Ca²⁺, and the mechanism that enables the prolonged activation of IP₃R1 required for lymphocyte activation is unclear. We show that IP₃R1 binds to the scaffolding protein linker of activated T cells and colocalizes with the T cell receptor during activation, resulting in persistent phosphorylation of IP₃R1 at Tyr353. This phosphorylation increases the sensitivity of the channel to activation by IP₃ and renders the channel less sensitive to Ca²⁺-induced inactivation. Expression of a mutant IP3R1-Y353F channel in lymphocytes causes defective Ca²⁺ signaling and decreased nuclear factor of activated T cells activation. Thus, tyrosine phosphorylation of IP3R1-Y353 may have an important function in maintaining elevated cytosolic Ca²⁺ levels during lymphocyte activation.     

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.     

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.     

Apelin-13 Inhibits Large-Conductance Ca2+-Activated K+ Channels in Cerebral Artery Smooth Muscle Cells via a PI3-Kinase Dependent Mechanism

Apelin-13 causes vasoconstriction by acting directly on APJ receptors in vascular smooth muscle (VSM) cells; however, the ionic mechanisms underlying this action at the cellular level remain unclear. Large-conductance Ca²⁺-activated K⁺ (BK_Ca) channels in VSM cells are critical regulators of membrane potential and vascular tone. In the present study, we examined the effect of apelin-13 on BK_Ca channel activity in VSM cells, freshly isolated from rat middle cerebral arteries. In whole-cell patch clamp mode, apelin-13 (0.001-1 μM) caused concentration-dependent inhibition of BK_Ca in VSM cells. Apelin-13 (0.1 µM) significantly decreased BK_Ca current density from 71.25±8.14 pA/pF to 44.52±7.10 pA/pF (n=14 cells, P<0.05). This inhibitory effect of apelin-13 was confirmed by single channel recording in cell-attached patches, in which extracellular application of apelin-13 (0.1 µM) decreased the open-state probability (NP_o) of BK_Ca channels in freshly isolated VSM cells. However, in inside-out patches, extracellular application of apelin-13 (0.1µM) did not alter the NP_o of BK_Ca channels, suggesting that the inhibitory effect of apelin-13 on BK_Ca is not mediated by a direct action on BK_Ca. In whole cell patches, pretreatment of VSM cells with LY-294002, a PI3-kinase inhibitor, markedly attenuated the apelin-13-induced decrease in BK_Ca current density. In addition, treatment of arteries with apelin-13 (0.1 µM) significantly increased the ratio of phosphorylated-Akt/total Akt, indicating that apelin-13 significantly increases PI3-kinase activity. Taken together, the data suggest that apelin-13 inhibits BK_Ca channel via a PI3-kinase-dependent signaling pathway in cerebral artery VSM cells, which may contribute to its regulatory action in the control of vascular tone.     

Analyzing and Quantifying the Gain-of-Function Enhancement of IP3 Receptor Gating by Familial Alzheimer’s Disease-Causing Mutants in Presenilins

Familial Alzheimer’s disease (FAD)-causing mutant presenilins (PS) interact with inositol 1,4,5-trisphosphate (IP₃) receptor (IP₃R) Ca²⁺ release channels resulting in enhanced IP₃R channel gating in an amyloid beta (Aβ) production-independent manner. This gain-of-function enhancement of IP₃R activity is considered to be the main reason behind the upregulation of intracellular Ca²⁺ signaling in the presence of optimal and suboptimal stimuli and spontaneous Ca²⁺ signals observed in cells expressing mutant PS. In this paper, we employed computational modeling of single IP₃R channel activity records obtained under optimal Ca²⁺ and multiple IP₃ concentrations to gain deeper insights into the enhancement of IP₃R function. We found that in addition to the high occupancy of the high-activity (H) mode and the low occupancy of the low-activity (L) mode, IP₃R in FAD-causing mutant PS-expressing cells exhibits significantly longer mean life-time for the H mode and shorter life-time for the L mode, leading to shorter mean close-time and hence high open probability of the channel in comparison to IP₃R in cells expressing wild-type PS. The model is then used to extrapolate the behavior of the channel to a wide range of IP₃ and Ca²⁺ concentrations and quantify the sensitivity of IP₃R to its two ligands. We show that the gain-of-function enhancement is sensitive to both IP₃ and Ca²⁺ and that very small amount of IP₃ is required to stimulate IP₃R channels in the presence of FAD-causing mutant PS to the same level of activity as channels in control cells stimulated by significantly higher IP₃ concentrations. We further demonstrate with simulations that the relatively longer time spent by IP₃R in the H mode leads to the observed higher frequency of local Ca²⁺ signals, which can account for the more frequent global Ca²⁺ signals observed, while the enhanced activity of the channel at extremely low ligand concentrations will lead to spontaneous Ca²⁺ signals in cells expressing FAD-causing mutant PS.     

cGMP/Protein Kinase G Signaling Suppresses Inositol 1,4,5-Trisphosphate Receptor Phosphorylation and Promotes Endoplasmic Reticulum Stress in Photoreceptors of Cyclic Nucleotide-gated Channel-deficient Mice

Photoreceptor cyclic nucleotide-gated (CNG) channels play a pivotal role in phototransduction. Mutations in the cone CNG channel subunits CNGA3 and CNGB3 are associated with achromatopsia and cone dystrophies. We have shown endoplasmic reticulum (ER) stress-associated apoptotic cone death and increased phosphorylation of the ER Ca²⁺ channel inositol 1,4,5-trisphosphate receptor 1 (IP₃R1) in CNG channel-deficient mice. We also presented a remarkable elevation of cGMP and an increased activity of the cGMP-dependent protein kinase (protein kinase G, PKG) in CNG channel deficiency. This work investigated whether cGMP/PKG signaling regulates ER stress and IP₃R1 phosphorylation in CNG channel-deficient cones. Treatment with PKG inhibitor and deletion of guanylate cyclase-1 (GC1), the enzyme producing cGMP in cones, were used to suppress cGMP/PKG signaling in cone-dominant Cnga3^−/−/Nrl^−/− mice. We found that treatment with PKG inhibitor or deletion of GC1 effectively reduced apoptotic cone death, increased expression levels of cone proteins, and decreased activation of Müller glial cells. Furthermore, we observed significantly increased phosphorylation of IP₃R1 and reduced ER stress. Our findings demonstrate a role of cGMP/PKG signaling in ER stress and ER Ca²⁺ channel regulation and provide insights into the mechanism of cone degeneration in CNG channel deficiency.     

Regulation of transient receptor potential melastatin 4 channel by sarcoplasmic reticulum inositol trisphosphate receptors: Role in human detrusor smooth muscle function

We recently reported key physiologic roles for Ca²⁺-activated transient receptor potential melastatin 4 (TRPM4) channels in detrusor smooth muscle (DSM). However, the Ca²⁺-signaling mechanisms governing TRPM4 channel activity in human DSM cells are unexplored. As the TRPM4 channels are activated by Ca²⁺, inositol 1,4,5-trisphosphate receptor (IP₃R)-mediated Ca²⁺ release from the sarcoplasmic reticulum represents a potential Ca²⁺ source for TRPM4 channel activation. We used clinically-characterized human DSM tissues to investigate the molecular and functional interactions of the IP₃Rs and TRPM4 channels. With in situ proximity ligation assay (PLA) and perforated patch-clamp electrophysiology, we tested the hypothesis that TRPM4 channels are tightly associated with the IP₃Rs and are activated by IP₃R-mediated Ca²⁺ release in human DSM. With in situ PLA, we demonstrated co-localization of the TRPM4 channels and IP₃Rs in human DSM cells. As the TRPM4 channels and IP₃Rs must be located within close apposition to functionally interact, these findings support the concept of a potential Ca²⁺-mediated TRPM4-IP₃R regulatory mechanism. To investigate IP₃R regulation of TRPM4 channel activity, we sought to determine the consequences of IP₃R pharmacological inhibition on TRPM4 channel-mediated transient inward cation currents (TICCs). In freshly-isolated human DSM cells, blocking the IP₃Rs with the selective IP₃R inhibitor xestospongin-C significantly decreased TICCs. The data suggest that IP₃Rs have a key role in mediating the Ca²⁺-dependent activation of TRPM4 channels in human DSM. The study provides novel insight into the molecular and cellular mechanisms regulating TRPM4 channels by revealing that TRPM4 channels and IP₃Rs are spatially and functionally coupled in human DSM.     

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.     

N-terminal Isoforms of the Large-conductance Ca2+-activated K+ Channel Are Differentially Modulated by the Auxiliary β1-Subunit

The large-conductance Ca²⁺-activated K⁺ (BK_Ca) channel is essential for maintaining the membrane in a hyperpolarized state, thereby regulating neuronal excitability, smooth muscle contraction, and secretion. The BK_Ca α-subunit has three predicted initiation codons that generate proteins with N-terminal ends starting with the amino acid sequences MANG, MSSN, or MDAL. Because the N-terminal region and first transmembrane domain of the α-subunit are required for modulation by auxiliary β1-subunits, we examined whether β1 differentially modulates the N-terminal BK_Ca α-subunit isoforms. In the absence of β1, all isoforms had similar single-channel conductances and voltage-dependent activation. However, whereas β1 did not modulate the voltage-activation curve of MSSN, β1 induced a significant leftward shift of the voltage activation curves of both the MDAL and MANG isoforms. These shifts, of which the MDAL was larger, occurred at both 10 μm and 100 μm Ca²⁺. The β1-subunit increased the open dwell times of all three isoforms and decreased the closed dwell times of MANG and MDAL but increased the closed dwell times of MSSN. The distinct modulation of voltage activation by the β1-subunit may be due to the differential effect of β1 on burst duration and interburst intervals observed among these isoforms. Additionally, we observed that the related β2-subunit induced comparable leftward shifts in the voltage-activation curves of all three isoforms, indicating that the differential modulation of these isoforms was specific to β1. These findings suggest that the relative expression of the N-terminal isoforms can fine-tune BK_Ca channel activity in cells, highlighting a novel mechanism of BK_Ca channel regulation.     

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.     

Gene Knock-Outs of Inositol 1,4,5-Trisphosphate Receptors Types 1 and 2 Result in Perturbation of Cardiogenesis

Background

Inositol 1,4,5-trisphosphate receptors (IP₃R1, 2, and 3) are intracellular Ca²⁺ release channels that regulate various vital processes. Although the ryanodine receptor type 2, another type of intracellular Ca²⁺ release channel, has been shown to play a role in embryonic cardiomyocytes, the functions of the IP₃Rs in cardiogenesis remain unclear.

Methodology/Principal Findings

We found that IP₃R1^−/−-IP₃R2^−/− double-mutant mice died in utero with developmental defects of the ventricular myocardium and atrioventricular (AV) canal of the heart by embryonic day (E) 11.5, even though no cardiac defect was detectable in IP₃R1^−/− or IP₃R2^−/− single-mutant mice at this developmental stage. The double-mutant phenotype resembled that of mice deficient for calcineurin/NFATc signaling, and NFATc was inactive in embryonic hearts from the double knockout-mutant mice. The double mutation of IP₃R1/R2 and pharmacologic inhibition of IP₃Rs mimicked the phenotype of the AV valve defect that result from the inhibition of calcineurin, and it could be rescued by constitutively active calcineurin.

Conclusions/Significance

Our results suggest an essential role for IP₃Rs in cardiogenesis in part through the regulation of calcineurin-NFAT signaling.     

Functional Inositol 1,4,5-Trisphosphate Receptors Assembled from Concatenated Homo- and Heteromeric Subunits

Vertebrate genomes code for three subtypes of inositol 1,4,5-trisphosphate (IP₃) receptors (IP₃R1, -2, and -3). Individual IP₃R monomers are assembled to form homo- and heterotetrameric channels that mediate Ca²⁺ release from intracellular stores. IP₃R subtypes are regulated differentially by IP₃, Ca²⁺, ATP, and various other cellular factors and events. IP₃R subtypes are seldom expressed in isolation in individual cell types, and cells often express different complements of IP₃R subtypes. When multiple subtypes of IP₃R are co-expressed, the subunit composition of channels cannot be specifically defined. Thus, how the subunit composition of heterotetrameric IP₃R channels contributes to shaping the spatio-temporal properties of IP₃-mediated Ca²⁺ signals has been difficult to evaluate. To address this question, we created concatenated IP₃R linked by short flexible linkers. Dimeric constructs were expressed in DT40–3KO cells, an IP₃R null cell line. The dimeric proteins were localized to membranes, ran as intact dimeric proteins on SDS-PAGE, and migrated as an ∼1100-kDa band on blue native gels exactly as wild type IP₃R. Importantly, IP₃R channels formed from concatenated dimers were fully functional as indicated by agonist-induced Ca²⁺ release. Using single channel “on-nucleus” patch clamp, the channels assembled from homodimers were essentially indistinguishable from those formed by the wild type receptor. However, the activity of channels formed from concatenated IP₃R1 and IP₃R2 heterodimers was dominated by IP₃R2 in terms of the characteristics of regulation by ATP. These studies provide the first insight into the regulation of heterotetrameric IP₃R of defined composition. Importantly, the results indicate that the properties of these channels are not simply a blend of those of the constituent IP₃R monomers.     

Selective down-regulation of IP3receptor subtypes by caspases and calpain during TNF α -induced apoptosis of human T-lymphoma cells

There are at least three types of inositol 1,4,5-trisphosphate receptor (IP₃R) [IP₃-gated Ca²⁺channels], which are expressed in different cell types and mammalian tissues. In this study, we have identified three IP₃R subtypes in human Jurkat T-lymphoma cells. All three subtypes have a molecular mass of about 260 kDa, and display Ca²⁺channel properties in an IP₃-dependent manner. We have also demonstrated that TNFα promotes the activity of different proteases (e.g. caspase-8, caspase-3 and calpain), alters the TCR-mediated Ca²⁺response and subsequently induces apoptosis in Jurkat cells. During the first 6 h of incubation with TNFα, several IP₃R subtype-related changes occur (e.g. proteolysis of IP₃R subtypes, inhibition of IP₃binding and impairment of IP₃-mediated Ca²⁺flux) concomitantly with an elevation of protease (caspase-8, caspase-3 and calpain) activity. Furthermore, the caspase inhibitor, Z-VAD-fmk, significantly reduces TNFα-mediated perturbation of IP₃R1 and IP₃R2 (but not IP₃R3) function; whereas the calpain inhibitor I, ALLN, is capable of blocking the inhibitory effect of TNFα on IP₃R3 function. These findings suggest that IP₃R1 and IP₃R2 serve as cellular substrates for caspases, and IP₃R3 is a substrate for calpain. We propose that the selective down-regulation of IP₃R subtype-mediated Ca²⁺function by caspase-dependent and calpain-sensitive mechanisms may be responsible for the early onset of the apoptotic signal by TNFα in human T-cells.     

Stimulation of Inositol 1,4,5-Trisphosphate (IP3) Receptor Subtypes by Adenophostin A and Its Analogues

Inositol 1,4,5-trisphosphate receptors (IP₃R) are intracellular Ca²⁺ channels. Most animal cells express mixtures of the three IP₃R subtypes encoded by vertebrate genomes. Adenophostin A (AdA) is the most potent naturally occurring agonist of IP₃R and it shares with IP₃ the essential features of all IP₃R agonists, namely structures equivalent to the 4,5-bisphosphate and 6-hydroxyl of IP₃. The two essential phosphate groups contribute to closure of the clam-like IP₃-binding core (IBC), and thereby IP₃R activation, by binding to each of its sides (the α- and β-domains). Regulation of the three subtypes of IP₃R by AdA and its analogues has not been examined in cells expressing defined homogenous populations of IP₃R. We measured Ca²⁺ release evoked by synthetic adenophostin A (AdA) and its analogues in permeabilized DT40 cells devoid of native IP₃R and stably expressing single subtypes of mammalian IP₃R. The determinants of high-affinity binding of AdA and its analogues were indistinguishable for each IP₃R subtype. The results are consistent with a cation-π interaction between the adenine of AdA and a conserved arginine within the IBC α-domain contributing to closure of the IBC. The two complementary contacts between AdA and the α-domain (cation-π interaction and 3″-phosphate) allow activation of IP₃R by an analogue of AdA (3″-dephospho-AdA) that lacks a phosphate group equivalent to the essential 5-phosphate of IP₃. These data provide the first structure-activity analyses of key AdA analogues using homogenous populations of all mammalian IP₃R subtypes. They demonstrate that differences in the Ca²⁺ signals evoked by AdA analogues are unlikely to be due to selective regulation of IP₃R subtypes.     

Pacemaking through Ca2+ stores interacting as coupled oscillators via membrane depolarization

This study presents an investigation of pacemaker mechanisms underlying lymphatic vasomotion. We tested the hypothesis that active inositol 1,4,5-trisphosphate receptor (IP₃R)-operated Ca²⁺ stores interact as coupled oscillators to produce near-synchronous Ca²⁺ release events and associated pacemaker potentials, this driving action potentials and constrictions of lymphatic smooth muscle. Application of endothelin 1 (ET-1), an agonist known to enhance synthesis of IP₃, to quiescent lymphatic smooth muscle syncytia first enhanced spontaneous Ca²⁺ transients and/or intracellular Ca²⁺ waves. Larger near-synchronous Ca²⁺ transients then occurred leading to global synchronous Ca²⁺ transients associated with action potentials and resultant vasomotion. In contrast, blockade of L-type Ca²⁺ channels with nifedipine prevented ET-1 from inducing near-synchronous Ca²⁺ transients and resultant action potentials, leaving only asynchronous Ca²⁺ transients and local Ca²⁺ waves. These data were well simulated by a model of lymphatic smooth muscle with: 1), oscillatory Ca²⁺ release from IP₃R-operated Ca²⁺ stores, which causes depolarization; 2), L-type Ca²⁺ channels; and 3), gap junctions between cells. Stimulation of the stores caused global pacemaker activity through coupled oscillator-based entrainment of the stores. Membrane potential changes and positive feedback by L-type Ca²⁺ channels to produce more store activity were fundamental to this process providing long-range electrochemical coupling between the Ca²⁺ store oscillators. We conclude that lymphatic pacemaking is mediated by coupled oscillator-based interactions between active Ca²⁺ stores. These are weakly coupled by inter- and intracellular diffusion of store activators and strongly coupled by membrane potential. Ca²⁺ store-based pacemaking is predicted for cellular systems where: 1), oscillatory Ca²⁺ release induces depolarization; 2), membrane depolarization provides positive feedback to induce further store Ca²⁺ release; and 3), cells are interconnected. These conditions are met in a surprisingly large number of cellular systems including gastrointestinal, lymphatic, urethral, and vascular tissues, and in heart pacemaker cells.     

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.     

Activation of cloned BK<Subscript>Ca</Subscript> channels in nitric oxide-induced apoptosis of HEK293 cells

The large conductance Ca²⁺-activated K⁺ (BK_Ca) channels are highly expressed in vascular smooth muscle cells (VSMCs) and play an essential role in the regulation of various physiological functions. Besides its electrophysiological function in vascular relaxation, BK_Ca has also been reported to be implicated in nitric oxide (NO)-induced apoptosis of VSMCs. However, the molecular mechanism is not clear and has not been determined on cloned channels. The present study was designed to clarify whether activation of cloned BK_Ca channel was involved in NO-induced apoptosis in human embryonic kidney 293 (HEK293) cell. The cDNA encoding the α-subunit of BK_Ca channel, hSloα, was transiently transfected into HEK293 cells. The apoptotic death in HEK-hSloα cells was detected using immunocytochemistry, analysis of fragmented DNA by agarose gel electrophoresis, MTT test, and flow cytometry assays. Whole-cell and single-channel characteristics of HEK-hSloα cells exhibited functional features similar to native BK_Ca channel in VSMCs. Exposuring of HEK- hSloα cells to S-nitroso-N-acetyl-penicillamine increased the hSloα channel activities of whole-cell and single-channel, and then increased percentage of cells undergoing apoptosis. However, blocking hSloα channels with 1 mM tetraethylammonia or 100 nM iberiotoxin significantly decreased the NO-induced apoptosis, whereas 30 μM NS1619, the specific agonist of BK_Ca, independently increased hSloα currents and induced apoptosis. These results indicated that activation of cloned BK_Ca channel was involved in NO-induced apoptosis of HEK293 cells.     

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.     

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.     

Hydrogen peroxide down-regulates inositol 1,4,5-trisphosphate receptor content through proteasome activation

Hydrogen peroxide (H₂O₂) is implicated in the regulation of signaling pathways leading to changes in vascular smooth muscle function. Contractile effects produced by H₂O₂ are due to the phosphorylation of myosin light chain kinase triggered by increases in intracellular calcium (Ca²⁺) from intracellular stores or influx of extracellular Ca²⁺. One mechanism for mobilizing such stores involves the phosphoinositide pathway. Inositol 1,4,5-trisphosphate (IP₃) mobilizes intracellular Ca²⁺ by binding to a family of receptors (IP₃Rs) on the endoplasmic–sarcoplasmic reticulum that act as ligand-gated Ca²⁺ channels. IP₃Rs can be rapidly ubiquitinated and degraded by the proteasome, causing a decrease in cellular IP₃R content. In this study we show that IP₃R₁ and IP₃R₃ are down-regulated when vascular smooth muscle cells (VSMC) are stimulated by H₂O₂, through an increase in proteasome activity. Moreover, we demonstrate that the decrease in IP₃R by H₂O₂ is accompanied by a reduction in calcium efflux induced by IP₃ in VSMC. Also, we observed that angiotensin II (ANGII) induces a decrease in IP₃R by activation of NADPH oxidase and that preincubation with H₂O₂ decreases ANGII-mediated calcium efflux and planar cell surface area in VSMC. The decreased IP₃ receptor content observed in cells was also found in aortic rings, which exhibited a decreased ANGII-dependent contraction after treatment with H₂O₂. Altogether, these results suggest that H₂O₂ mediates IP₃R down-regulation via proteasome activity.