FK506 binding protein associated with the calcium release channel (ryanodine receptor).

The calcium release channel (CRC)/ryanodine receptor (RyRec) has been identified as the foot structure of the sarcoplasmic reticulum (SR) and provides the pathway for calcium efflux required for excitation-contraction coupling in skeletal muscle. The CRC has previously been reported to consist of four identical 565-kDa protomers. We now report the identification of a 12-kDa protein which is tightly associated with highly purified RyRec from rabbit skeletal muscle SR. N-terminal amino acid sequencing and cDNA cloning demonstrates that the 12-kDa protein from fast twitch skeletal muscle is the binding protein for the immunosuppressant drug FK506. In humans, FK506 binds to the 12-kDa FK506-binding protein (FKBP12) and blocks calcium-dependent T cell activation. We find that FKBP12 and the RyRec are tightly associated in skeletal muscle SR on the basis of: 1) co-purification through sequential heparin-agarose, hydroxylapatite, and size exclusion chromatography columns; 2) coimmunoprecipitation of the RyRec and FKBP12 with anti-FKBP12 antibodies; and 3) subcellular localization of both proteins to the terminal cisternae of the SR, and not in the longitudinal tubules of SR, in fast twitch skeletal muscle. The molar ratio of FKBP12 to RyRec in highly purified RyRec preparations is approximately 1:4, indicating that one FKBP12 molecule is associated with each calcium release channel/foot structure.     

Identification of the receptor for omega-conotoxin in brain. Probable components of the calcium channel

Recently omega-conotoxin GVIA was shown to specifically block neuronal and other calcium channels. In this work, an azidonitrobenzoyl derivative of mono-[125I]iodo-omega-conotoxin GVIA was used to identify the components of its receptor site in synaptic plasma membrane by photoaffinity labeling. Components of Mr approximately equal to 310,000, approximately equal to 230,000, and 34,000 were specifically photolabeled. The characteristics of photolabeling of these three components were consistent with those of the specific binding of omega-conotoxin GVIA to synaptic plasma membrane with respect to the effects of metal ions, conventional calcium antagonists, and an agonist (1,4-dihydropyridines, verapamil, and diltiazem, etc.), omega-conotoxins GVIIA and GVIIB. Furthermore, the distribution of these three components in subcellular fractions from rat brain as estimated by photolabeling was in good agreement with that of the specific binding of omega-conotoxin GVIA to its receptor. These findings indicate that the components of Mr approximately equal to 310,000, approximately equal to 240,000, and 34,000 are the receptor for omega-conotoxin GVIA and suggest that these components are constituents of the voltage-sensitive calcium channel in brain. No specific photolabeling was observed in the plasma membrane of human erythrocytes, probably indicating the absence of the receptor for omega-conotoxin GVIA in the membrane.     

Characterization of a stretch‐activated potassium channel in chondrocytes

Chondrocytes possess the capacity to transduce load‐induced mechanical stimuli into electrochemical signals. The aim of this study was to functionally characterize an ion channel activated in response to membrane stretch in isolated primary equine chondrocytes. We used patch‐clamp electrophysiology to functionally characterize this channel and immunohistochemistry to examine its distribution in articular cartilage. In cell‐attached patch experiments, the application of negative pressures to the patch pipette (in the range of 20–200 mmHg) activated ion channel currents in six of seven patches. The mean activated current was 45.9 ± 1.1 pA (n = 4) at a membrane potential of 33 mV (cell surface area approximately 240 µm²). The mean slope conductance of the principal single channels resolved within the total stretch‐activated current was 118 ± 19 pS (n = 6), and reversed near the theoretical potassium equilibrium potential, E_K+, suggesting it was a high‐conductance potassium channel. Activation of these high‐conductance potassium channels was inhibited by extracellular TEA (K_d approx. 900 µM) and iberiotoxin (K_d approx. 40 nM). This suggests that the current was largely carried by BK‐like potassium (MaxiK) channels. To further characterize these BK‐like channels, we used inside‐out patches of chondrocyte membrane: we found these channels to be activated by elevation in bath calcium concentration. Immunohistochemical staining of equine cartilage samples with polyclonal antibodies to the α1‐ and β1‐subunits of the BK channel revealed positive immunoreactivity for both subunits in superficial zone chondrocytes. These experiments support the hypothesis that functional BK channels are present in chondrocytes and may be involved in mechanotransduction and chemotransduction. J. Cell. Physiol. 223: 511–518, 2010. © 2010 Wiley‐Liss, Inc.     

线粒体ATP敏感钾通道与钙激活钾通道激活对正常与缺血脑线粒体通透性转变的影响

目的：明确线粒体ATP敏感钾通道与钙激活钾通道对正常和缺血脑线粒体渗透性转变的作用。方法：实验采用分光光度法,在分离的线粒体上分别观察两种线粒体钾通道激动剂对正常与缺血脑线粒体肿胀的影响。结果：在正常脑线粒体,diazoxide与NSl619能有效抑制由钙诱导的线粒体氏20下降,但其效应可被atractyloside所阻断。与正常相比,缺血损伤后的脑线粒体在钙离子诱导下线粒体A520下降较快,diazoxide与NS1619仍可抑制由钙诱导的线粒体A520下降,其作用同样为atractykxside所阻断。结论：线粒体ATP敏感钾通道与钙激活钾通道激活在离体条件均具有保护脑线粒体的作用,其作用可能是通过影响线粒体通透性转变而实现。     

The discovery of a novel calcium channel blocker related to the structure of potassium channel opener cromakalim

During our studies aimed at the identification of novel analogs of the potassium channel opener cromakalim (3), we serendipitously observed pyranoquinoline 4 to possess pure calcium channel blocking activity. The results of the studies conducted to confirm the calcium channel blocking mechanism of 4 are reported in this paper.     

The sigma receptor as a ligand-regulated auxiliary potassium channel subunit

The sigma receptor is a novel protein that mediates the modulation of ion channels by psychotropic drugs through a unique transduction mechanism depending neither on G proteins nor protein phosphorylation. The present study investigated sigma receptor signal transduction by reconstituting responses in Xenopus oocytes. Sigma receptors modulated voltage-gated K+ channels (Kv1.4 or Kv1.5) in different ways in the presence and absence of ligands. Association between Kv1.4 channels and sigma receptors was demonstrated by coimmunoprecipitation. These results indicate a novel mechanism of signal transduction dependent on protein-protein interactions. Domain accessibility experiments suggested a structure for the sigma receptor with two cytoplasmic termini and two membrane-spanning segments. The ligand-independent effects on channels suggest that sigma receptors serve as auxiliary subunits to voltage-gated K+ channels with distinct functional interactions, depending on the presence or absence of ligand.     

Effects of calcium channel blockers on potassium homeostasis.

The known effects of calcium channel blockers on various aspects of potassium homeostasis are reviewed. Regulation of potassium homeostasis requires both renal and external handling mechanisms. Signaling by calcium appears to mediate both of these. Calcium channels have been identified in adrenal glomerulosa cells, and cellular calcium entry has been demonstrated in vitro to be necessary for the synthesis and secretion of aldosterone. Calcium channel antagonists such as verapamil and nifedipine, at pharmacologic doses, can block aldosterone production. In vivo, however, only chronic administration of verapamil appears to attenuate aldosterone responsiveness to angiotensin II. Chronic administration of nifedipine does not have a dramatic effect on aldosterone production following potassium loading. Other studies have demonstrated improved extrarenal potassium disposal following treatment with calcium channel blocking agents. Clinically, there are no reports of either hyperkalemia or hypokalemia with the routine therapeutic use of these agents given alone. This review was prompted by the development of hyperkalemia in a patient with chronic renal failure following the initiation of therapy with the calcium channel blocker diltiazem: however, numerous other etiologies may also have contributed to the development of hyperkalemia in this case. Review of the data indicates that current evidence implicating this class of drugs in the pathogenesis of disordered potassium regulation remains equivocal.     

Estradiol rapidly induces the translocation and activation of the intermediate conductance calcium activated potassium channel in human eccrine sweat gland cells

Background and aims

Steroid hormones target K⁺ channels as a means of regulating electrolyte and fluid transport. In this study, ion transporter targets of Estradiol (E2) were investigated in the human eccrine sweat gland cell line NCL-SG3.

Results

Whole cell patch-clamp studies revealed E2 (10 nM) rapidly activates a whole cell K⁺ conductance, which is abolished by clotrimazole (30 μM), an inhibitor of the intermediate conductance calcium activated K⁺ channel (IK_Ca). The estrogen receptor (ER) antagonist ICI 182, 780 had no effect on this E2 activated K⁺ conductance, suggesting an estrogen receptor independent mechanism of activation. Confocal microscopy studies revealed under basal conditions that the IK_Ca channel is located within the cell cytoplasm and in the presence of E2, rapidly translocates to both the apical and basolateral membrane. In the presence of E2, tyrosine phosphorylation of calmodulin, which is known to regulate trafficking of the IK_Ca channel, is increased, and treatment of cells with the calmodulin inhibitor trifluoperazine (TFP) prevents the E2-induced translocation.

Conclusions

Estradiol rapidly regulates a K⁺ conductance through the IK_Ca channel in an estrogen receptor independent manner. E2 stimulates the translocation of IK_Ca to the cell membrane in a calmodulin dependent manner, representing a novel paradigm of estrogen action in sweat gland epithelial cells.     

Molecular rearrangements of the Kv2.1 potassium channel termini associated with voltage gating

The voltage-gated Kv2.1 channel is composed of four identical subunits folded around the central pore and does not inactivate appreciably during short depolarizing pulses. To study voltage-induced relative molecular rearrangements of the channel, Kv2.1 subunits were genetically fused with enhanced cyan fluorescent protein and/or enhanced yellow fluorescent protein, expressed in COS1 cells, and investigated using fluorescence resonance energy transfer (FRET) microscopy combined with patch clamp. Fusion of fluorophores to either or both termini of the Kv2.1 monomer did not significantly affect the gating properties of the channel. FRET between the N- and C-terminal tags fused to the same or different Kv2.1 monomers decreased upon activation of the channel by depolarization from -80 to +60 mV, suggesting voltage-gated relative rearrangement between the termini. Because FRET between the Kv2.1 N- or C-terminal tags and the membrane-trapped EYFP(N)-PH pleckstrin homology domains did not change on depolarization, voltage-gated relative movements between the Kv2.1 termini occurred in a plane parallel to the plasma membrane, within a distance of 1-10 nm. FRET between the N-terminal tags did not change upon depolarization, indicating that the N termini do not rearrange relative to each other, but they could either move cooperatively with the Kv2.1 tetramer or not move at all. No FRET was detected between the C-terminal tags. Assuming their randomized orientation in the symmetrically arranged Kv2.1 subunits, C termini may move outwards in order to produce relative rearrangements between N and C termini upon depolarization.     

Phosphoproteins associated with the regulation of a specific potassium channel in the identified Aplysia neuron R15

The neurotransmitter serotonin (5HT) activates a specific K+ conductance in the identified Aplysia neuron R15. This response to 5HT has been shown previously to be mediated by cAMP and cAMP-dependent protein phosphorylation. We have measured protein phosphorylation within neuron R15 in vivo, following the intracellular injection of [gamma-32P]ATP, and have demonstrated that 5HT modulates the phosphorylation of a number of proteins in R15. The present study was undertaken to determine which of these phosphoproteins are closely associated with, and may be responsible for, the K+ conductance increase. Treatment of neuron R15 with a cAMP analog produces some but not all of the 5HT-induced phosphoprotein changes, indicating that some are not cAMP-dependent and thus can be dissociated from the cAMP-dependent K+ conductance increase. Similar results are obtained by intracellular injection of the adenylate cyclase inhibitor guanosine 5'-O-(2-thiodiphosphate), which completely blocks the 5HT-evoked K+ conductance increase but fails to block some of the 5HT-induced phosphorylation changes. Examination of the phosphoprotein pattern at short times after 5HT application has demonstrated that some of the phosphoprotein changes, but not others, are closely associated in time with the appearance of the physiological response. These and other pharmacological and kinetic experiments have allowed the identification of two phosphoproteins, of Mr = 29,000 and 70,000, which cannot be dissociated from the 5HT-induced K+ conductance increase whatever the experimental manipulation. Thus, one or both of these phosphoproteins may be involved in the regulation of the 5HT-sensitive K+ channel in neuron R15.     

Comparison of effects of a potassium channel opener BRL34915, a specific potassium ionophore valinomycin and calcium channel blockers on endothelin-induced vascular contraction

The effects of a potassium (K+) channel opener BRL34915 and a specific K+ ionophore valinomycin on vasoconstriction induced by endothelin (ET) were compared with those of calcium (Ca2+) channel blockers, nicardipine and verapamil, using helical strips from rat thoracic aorta. ET induced potent and persistent contraction in control solution and similar but smaller contraction in Ca2+-free solution. BRL34915 and valinomycin inhibited the ET-induced contraction dose-dependently in control solution, but not in Ca2+-free solution. The ET-induced contraction was also inhibited by nicardipine and verapamil, though less strongly. On the other hand, high K+ (35 mM)-induced vasoconstriction was strongly inhibited by nicardipine and verapamil, but not by BRL34915 or valinomycin. These results support the idea that the extracellular Ca2+-dependent component of the ET-induced contraction may be mediated by Ca2+ influx by a route other than voltage-dependent Ca2+-channels.     

Auxiliary subunits: essential components of the voltage-gated calcium channel complex

Voltage-gated calcium channels are important mediators of several physiological processes, including neuronal excitability and muscle contraction. At the molecular level, the channels are composed of four subunits--the pore forming alpha(1) subunit and the auxiliary alpha(2)delta, beta and gamma subunits. The auxiliary subunits modulate the trafficking and the biophysical properties of the alpha(1) subunit. In the past several years there has been an acceleration of our understanding of the auxiliary subunits, primarily because of their molecular characterization and the availability of spontaneous and targeted mouse mutants. These studies have revealed the crucial role of the subunits in the functional effects that are mediated by voltage-gated calcium channels.     

Mapping the receptor site for charybdotoxin, a pore-blocking potassium channel inhibitor.

The Shaker K+ channel belongs to a family of structurally related voltage-activated cation channels that play a central role in cellular electrical signaling. By studying multiple site-directed mutants of the Shaker K+ channel, a region that forms the binding site for a pore-blocking scorpion toxin has been identified. The region contains a sequence that is highly conserved among cloned K+ channels and may contribute to the formation of the ion conduction pore.     

Electrophysiological characterization of a schistosome transient receptor potential channel activated by praziquantel

Ion channels have proved to be productive targets for anthelmintic chemotherapy. One example is the recent discovery of a parasitic flatworm ion channel targeted by praziquantel (PZQ), the main clinical therapy used for treatment of schistosomiasis. The ion channel activated by PZQ – a transient receptor potential ion channel of the melastatin subfamily, named TRPM_PZQ – is a Ca²⁺-permeable ion channel expressed in all parasitic flatworms that are PZQ-sensitive. However, little is currently known about the electrophysiological properties of this target that mediates the deleterious action of PZQ on many trematodes and cestodes. Here, we provide a detailed biophysical characterization of the properties of Schistosoma mansoni TRPM_PZQ channel (Sm.TRPM_PZQ) in response to PZQ. Single channel electrophysiological analysis demonstrated that Sm.TRPM_PZQ when activated by PZQ is a non-selective, large conductance, voltage-insensitive cation channel that displays distinct properties from human TRPM paralogs. Sm.TRPM_PZQ is Ca²⁺-permeable but does not require Ca²⁺ for channel gating in response to PZQ. TRPM_PZQ from Schistosoma japonicum (Sj.TRPM_PZQ) and Schistosoma haematobium (Sh.TRPM_PZQ) displayed similar characteristics. Profiling Sm.TRPM_PZQ responsiveness to PZQ has established a biophysical signature for this channel that will aid future investigation of endogenous TRPM_PZQ activity, as well as analyses of endogenous and exogenous regulators of this novel, druggable antiparasitic target.     

Regulation of the ryanodine receptor calcium release channel: a molecular complex system

The thermal behaviour and structural changes associated with the phase transformation of 1,2-dipalmitoyl-sn-glycerol (DPG) were studied by means of simultaneous X-ray diffraction and differential scanning calorimetry. Metastable DPG solid phases are crystallized from the melted sample by thermal quenching. The metastable phase (alpha-phase) formed initially is converted into a stable phase (beta' phase) at approximately 50 degrees C on heating. It was found that the behaviour of the alpha- to beta'-phase transformation depends on the thermal history. DPG solid samples incubated at approximately 3 degrees C for more than 10 h after cooling transformed directly into the beta'-phase with heat release. On the other hand, in the solid samples without incubation, the alpha-phase once melted and then the crystallization of the beta'-phase occurred successively from the melted state.     

Human macrophages contain a stretch-sensitive potassium channel that is activated by adherence and cytokines

A variety of stimuli, including cytokines and adhesion to surfaces and matrix proteins, can regulate macrophage function, in part through changes in Ca²⁺-dependent second messengers. While fluctuation in in-tracellular Ca²⁺ is an important modulator of cellular activation, little attention has been paid to the roles of other ions whose cytoplasmic concentrations can be rapidly regulated by ion channels. To examine the role of ion channels in macrophage function, we undertook patch clamp studies of human culture-derived macrophages grown under serum-free conditions. The major ionic current in these cells was carried by an outwardly rectifying K⁺ channel, which had a single-channel conductance of 229 pS in symmetrical K⁺-rich solution and macroscopic whole-cell conductance of 9.8 nS. These channels opened infrequently in resting cells but were activated immediately by (i) adhesion of mobile cells onto a substrate, (ii) stretch applied to isolated membrane patches in Ca²⁺-free buffers, (iii) intracellular Ca²⁺ (EC₅₀ of 0.4 m), and (iv) the cytokine IL-2. Furthermore, barium and 4-aminopyridine, blockers of this channel, altered the organization and structure of the cytoskeletal proteins actin, tubulin and vimentin. These cytoskeletal changes were associated with reversible alteration to the morphology of the cells. Thus, we have identified an outwardly rectifying K⁺ channel that appeared to be involved in cytokine and adherence-mediated macrophage activation, and in the maintenance of cytoskeletal integrity and cell shape.We thank Ken Wyse and Sue Bennett for excellent technical assistance. This work was supported by the National Health & Medical Research Council of Australia, the National Heart Foundation of Australia, the Clive & Vera Ramaciotti Foundation of Australia, the St Vincent's Hospital Clinic Foundation and a St Vincent's Hospital Research Grant.     

Alpha 1E subunit of the R-type calcium channel is associated with myelinogenesis

During myelinogenesis, we found an exceedingly strong, transient expression of the α_1E gene for the R-type voltage-gated calcium channel in CNS white matter. This immunoreactivity appeared in glial cells along specific pathways of the brainstem, cerebellum, and telencephalon. The reactivity followed a wave that progressed from the brainstem at P5, to the cerebellar peduncles by P8, the arbor vitae by P14, and the granular layer by P17. The reactivity-peaked about 3–4 days later and decreased gradually to become negligible in all areas before adulthood. Ultrastructural analysis confirmed that α_1E immunoreactivity was located in oligodendroglial somata, their projections, paranodal wraps and loose myelin sheaths. There was a distinct association of the channel protein reactivity on oligodendroglial membranes in contact with the axon. We propose that glial projections, contacting axons, sense axonal firing through small K⁺ currents and open the high voltage R-type calcium channels to signal myelination.     

alpha-Bungarotoxin-sensitive hippocampal nicotinic receptor channel has a high calcium permeability.

The hippocampal nicotinic acetylcholine receptor (nAChR) is a newly identified ligand-gated ion channel that is blocked by the snake toxin alpha-bungarotoxin (alpha-BGT) and that probably contains the alpha 7 nAChR subunit in its structure. Here its ion selectivity was characterized and compared with that of the N-methyl-D-aspartate (NMDA) receptor channel. The reversal potentials (VR) of acetylcholine- and NMDA-activated whole-cell currents were determined under various ionic conditions. Using ion activities and a Goldman-Hodgkin-Katz equation for VR shifts in the presence of Ca2+, permeability ratios were calculated. For the alpha-BGT-sensitive nAChR, PNa/PCs was close to 1 and Cl- did not contribute to the currents. Changing the [Ca2+]0 from 1 to 10 mM, the VRs of the nAChR and NMDA currents were shifted by +5.6 +/- 0.4 and +8.3 +/- 0.4 mV, respectively, and the nAChR current decay was accelerated. These shifts yielded PCa/PCss of 6.1 +/- 0.5 for the nAChR channel and 10.3 +/- 0.7 for the NMDA channel. Thus, the neuronal alpha-BGT-sensitive nAChR is a cation channel considerably selective to Ca2+ and may mediate a fast rise in intracellular Ca2+ that would increase in magnitude with membrane hyperpolarization.     

The brain ryanodine receptor: a caffeine-sensitive calcium release channel.

The release of stored Ca2+ from intracellular pools triggers a variety of important neuronal processes. Physiological and pharmacological evidence has indicated the presence of caffeine-sensitive intracellular pools that are distinct from the well-characterized inositol 1,4,5,-trisphosphate (IP3)-gated pools. Here we report that the brain ryanodine receptor functions as a caffeine- and ryanodine-sensitive Ca2+ release channel that is distinct from the brain IP3 receptor. The brain ryanodine receptor has been purified 6700-fold with no change in [3H]ryanodine binding affinity and shown to be a homotetramer composed of an approximately 500 kd protein subunit, which is identified by anti-peptide antibodies against the skeletal and cardiac muscle ryanodine receptors. Our results demonstrate that the brain ryanodine receptor functions as a caffeine-sensitive Ca2+ release channel and thus is the likely gating mechanism for intracellular caffeine-sensitive Ca2+ pools in neurons.     

Structure of the skeletal muscle calcium release channel activated with Ca2+ and AMP-PCP.

The functional state of the skeletal muscle Ca2+ release channel is modulated by a number of endogenous molecules during excitation-contraction. Using electron cryomicroscopy and angular reconstitution techniques, we determined the three-dimensional (3D) structure of the skeletal muscle Ca2+ release channel activated by a nonhydrolyzable analog of ATP in the presence of Ca2+. These ligands together produce almost maximum activation of the channel and drive the channel population toward a predominately open state. The resulting 30-A 3D reconstruction reveals long-range conformational changes in the cytoplasmic region that might affect the interaction of the Ca2+ release channel with the t-tubule voltage sensor. In addition, a central opening and mass movements, detected in the transmembrane domain of both the Ca(2+)- and the Ca2+/nucleotide-activated channels, suggest a mechanism for channel opening similar to opening-closing of the iris in a camera diaphragm.