Increased Expression of the Large Conductance, Calcium-Activated K+ (BK) Channel in Adult-Onset Neuronal Ceroid Lipofuscinosis.

Cysteine string protein (CSPα) is a presynaptic J protein co-chaperone that opposes neurodegeneration. Mutations in CSPα (i.e., Leu115 to Arg substitution or deletion (Δ) of Leu116) cause adult neuronal ceroid lipofuscinosis (ANCL), a dominantly inherited neurodegenerative disease. We have previously demonstrated that CSPα limits the expression of large conductance, calcium-activated K+ (BK) channels in neurons, which may impact synaptic excitability and neurotransmission. Here we show by western blot analysis that expression of the pore-forming BKα subunit is elevated ~2.5 fold in the post-mortem cortex of a 36-year-old patient with the Leu116∆ CSPα mutation. Moreover, we find that the increase in BKα subunit level is selective for ANCL and not a general feature of neurodegenerative conditions. While reduced levels of CSPα are found in some postmortem cortex specimens from Alzheimer's disease patients, we find no concomitant increase in BKα subunit expression in Alzheimer's specimens. Both CSPα monomer and oligomer expression are reduced in synaptosomes prepared from ANCL cortex compared with control. In a cultured neuronal cell model, CSPα oligomers are short lived. The results of this study indicate that the Leu116∆ mutation leads to elevated BKα subunit levels in human cortex and extend our initial work in rodent models demonstrating the modulation of BKα subunit levels by the same CSPα mutation. While the precise sequence of pathogenic events still remains to be elucidated, our findings suggest that dysregulation of BK channels may contribute to neurodegeneration in ANCL.

Some assembly required: SOCE and Orai1 channels couple to NFAT transcriptional activity via calmodulin and calcineurin.

Advances in our ability to monitor the temporal and spatial dynamics of intracellular second messengers such as Ca(2+) and cyclic nucleotides at millisecond and sub-micron levels of resolution have greatly increased our understanding of cellular signal transduction mechanisms. Thus, it is now well appreciated that second messengers can rise and fall within discrete regions of the intracellular compartment, as opposed to global changes, and on a time scale determined by the local collection of signaling molecules responsible for the synthesis and degradation/re-uptake of the second messenger. Efforts to identify the components of such macromolecular signaling domains have revealed the presence of hormone receptors, modifying enzymes and scaffolding proteins that tend to assemble and organize these complexes. Emerging evidence now suggests that these signal transduction entities need not be pre-existing, static complexes within the cell, but in fact, may dynamically assemble in response to a specific stimulus. Such an arrangement would thus allow key signaling molecules to be trafficked where they are needed, thereby allowing a cell to utilize these resources more effectively. On the flip side, having such molecules constantly remain within a single cellular domain would facilitate rapid signaling responses and help maintain fidelity of the pathway.

A pharmacologic activator of endothelial KCa channels enhances coronary flow in the hearts of type 2 diabetic rats.

Endothelial dysfunction is a common early pathogenic event in patients with type 2 diabetes (T2D) who exhibit cardiovascular disease. In the present study, we have examined the effect of SKA-31, a positive modulator of endothelial Ca(2+)-activated K(+) (KCa) channels, on total coronary flow in isolated hearts from Goto-Kakizaki rats, a non-obese model of T2D exhibiting metabolic syndrome. Total coronary flow and left ventricular developed pressure were monitored simultaneously in isolated, spontaneously beating Langendorff-perfused hearts. Acute administrations of bradykinin (BK) or adenosine (ADO) increased coronary flow, but responses were significantly blunted in diabetic hearts at 10-12 and 18-20weeks of age compared with age-matched Wistar controls, consistent with the presence of endothelial dysfunction. In contrast, SKA-31 dose-dependently (0.01-5µg) increased total coronary flow to comparable levels in both control and diabetic rat hearts at both ages. Flow responses to sodium nitroprusside were not different between control and diabetic hearts, suggesting normal arterial smooth muscle function. Importantly, exposure to a sub-threshold concentration of SKA-31 (i.e. 0.3µM) rescued the impaired BK and ADO-evoked vasodilatory responses in diabetic hearts. Endothelial KCa channel activators may thus help to preserve coronary flow in diabetic myocardium.

Cigarette smoke and calcium conspire to impair CFTR function in airway epithelia.

To maintain health and function in response to inhaled environmental irritants and toxins, the lungs and airways depend upon an innate defense system that involves the secretion of mucus (i.e., mucin, salts, and water) by airway epithelium onto the apical surface to trap foreign particles. Airway mucus is then transported in an oral direction via ciliary beating and coughing, which helps to keep the airways clear. CFTR (cystic fibrosis transmembrane conductance regulator) is a cAMP-regulated Cl(-) channel in the apical membrane of epithelium that contributes to salt and water secretion onto the luminal surface of airways, thereby ensuring that secreted mucus is sufficiently hydrated for movement along the epithelial surface. Dehydration of airway mucus, as occurs in cystic fibrosis, results in a more viscous, less mobile secretion that compromises the lung's innate defense system by facilitating a build-up of foreign particles and bacterial growth. Related to this situation is chronic obstructive pulmonary disease (COPD), which is a leading cause of death globally. A major cause of COPD is cigarette smoking, which has been reported to decrease the cellular levels of CFTR in airway epithelia. In their recent article, Rasmussen and coworkers now report that exposure to cigarette smoke elevates cytosolic free Ca(2+) in airway epithelium, leading to decreased surface localization and cellular expression of CFTR and reduced levels of secreted airway surface liquid. Blocking this increase in cytosolic Ca(2+) largely prevented CFTR loss in airway epithelium and surprisingly, cellular lysosomes appear to be a major source for smoke-induced Ca(2+) elevation.

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, BKß4 and BKß1 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 CSPαHPD-AAA increases BK current. Our observations indicate that CSPα has a role in regulating synaptic excitability and neurotransmission by limiting expression of BK channels.

Built for speed: molecular properties of the voltage sensor domain underlie the rapid activation of voltage-gated Na(+) channels compared with Shaker-type K(+) channels.

Many of us were taught in high school biology that the action potential waveform in nerves and other excitable tissues was generated by an initial rapid influx of external Na(+) ions across the plasma membrane, followed by an outward movement of intracellular K(+) ions. The former event, mediated by voltage-gated Na(+) channels, is responsible for the fast depolarizing upstroke of the action potential, while voltage-gated K+ channels are responsible for the subsequent repolarizing phase, which largely controls action potential duration. Although Hodgkin and Huxley described the fundamental importance of this sequential activation process more than 60 y ago, the molecular and structural details underlying the faster activation of voltage-gated Na(+) (Nav) vs. K(+) (Kv) channels have yet to be fully resolved.

The large conductance, calcium-activated K+ (BK) channel is regulated by cysteine string protein.

Large-conductance, calcium-activated-K(+) (BK) channels are widely distributed throughout the nervous system, where they regulate action potential duration and firing frequency, along with presynaptic neurotransmitter release. Our recent efforts to identify chaperones that target neuronal ion channels have revealed cysteine string protein (CSPα) as a key regulator of BK channel expression and current density. CSPα is a vesicle-associated protein and mutations in CSPα cause the hereditary neurodegenerative disorder, adult-onset autosomal dominant neuronal ceroid lipofuscinosis (ANCL). CSPα null mice show 2.5 fold higher BK channel expression compared to wild type mice, which is not seen with other neuronal channels (i.e. Cav2.2, Kv1.1 and Kv1.2). Furthermore, mutations in either CSPα's J domain or cysteine string region markedly increase BK expression and current amplitude. We conclude that CSPα acts to regulate BK channel expression, and consequently CSPα-associated changes in BK activity may contribute to the pathogenesis of neurodegenerative disorders, such as ANCL.

SKA-31, a novel activator of SK(Ca) and IK(Ca) channels, increases coronary flow in male and female rat hearts.

AIMS: Endothelial SK(Ca) and IK(Ca) channels play an important role in the regulation of vascular function and systemic blood pressure. Based on our previous findings that small molecule activators of SK(Ca) and IK(Ca) channels (i.e. NS309 and SKA-31) can inhibit myogenic tone in isolated resistance arteries, we hypothesized that this class of compounds may induce effective vasodilation in an intact vascular bed, such as the coronary circulation. METHODS AND RESULTS: In a Langendorff-perfused, beating rat heart preparation, acute bolus administrations of SKA-31 (0.01-5 µg) dose-dependently increased total coronary flow (25-30%) in both male and female hearts; these responses were associated with modest, secondary increases in left ventricular (LV) systolic pressure and heart rate. SKA-31 evoked responses in coronary flow, LV pressure, and heart rate were qualitatively comparable to acute responses evoked by bradykinin (1 µg) and adenosine (10 µg). In the presence of apamin and TRAM-34, selective blockers of SK(Ca) and IK(Ca) channels, respectively, SKA-31 and bradykinin-induced responses were largely inhibited, whereas the adenosine-induced changes were blocked by ∼40%; TRAM-34 alone produced less inhibition. Sodium nitroprusside (SNP, 0.2 µg bolus dose) evoked changes in coronary flow, LV pressure, and heart rate were similar to those induced by SKA-31, but were unaffected by apamin + TRAM-34. The NOS inhibitor L-NNA reduced bradykinin- and adenosine-evoked changes, but did not affect responses to either SKA-31 or SNP. CONCLUSION: Our study demonstrates that SKA-31 can rapidly and reversibly induce dilation of the coronary circulation in intact functioning hearts under basal flow and contractility conditions.

Two-pore domain potassium channels: variation on a structural theme.

The ability of cells to reliably fire action potentials is critically dependent upon the maintenance of a hyperpolarized resting potential, which allows voltage-gated Na(+) and Ca(2+) channels to recover from inactivation and open in response to a subsequent stimulus. Hodgkin and Huxley first recognized the functional importance a small, steady outward leak of K(+) ions to the resting potential, action potential generation and cellular excitability, and we now appreciate the contribution of inward rectifier-type K(+) channels (Kir or KCNJ channels) to this process. More recently, however, it has become evident that two-pore domain K(+) (K2P) channels also contribute to the steady outward leak of K(+) ions, and thus, maintenance of the resting potential. Molecular cloning efforts have demonstrated that K2P channel exist in yeast to humans, and represent a major branch in the K(+) channel superfamily. Humans express 15 types of K2P channels, which are grouped into six subfamilies, based on similarities in amino acid sequence and functional properties. Although K2P channels are not voltage-gated, due to the absence of a canonical voltage sensor domain, their activity can be regulated by a variety of stimuli, including mechanical force, polyunsaturated fatty acids (PUFAs) (e.g., arachidonic acid), volatile anesthetics, acidity/pH, pharmacologic agents, heat and signaling events, such as phosphorylation and protein-protein interactions. K2P channels thus represent important regulators of cellular excitability by virtue of their impact on the resting potential, and as such, have garnered considerable attention in recent years.

Purinergic P2Y2 receptors mediate rapid Ca(2+) mobilization, membrane hyperpolarization and nitric oxide production in human vascular endothelial cells.

In blood vessels, stimulation of the vascular endothelium by the Ca(2+)-mobilizing agonist ATP initiates a number of cellular events that cause relaxation of the adjacent smooth muscle layer. Although vascular endothelial cells are reported to express several subtypes of purinergic P2Y and P2X receptors, the major isoform(s) responsible for the ATP-induced generation of vasorelaxant signals in human endothelium has not been well characterized. To address this issue, ATP-evoked changes in cytosolic Ca(2+), membrane potential and acute nitric oxide production were measured in isolated human umbilical vein endothelial cells (HUVECs) and profiled using established P2X and P2Y receptor probes. Whereas selective P2X agonist (i.e. α,ß-methyl ATP) and antagonists (i.e. TNP-ATP and PPADS) could neither mimic nor block the observed ATP-evoked cellular responses, the specific P2Y receptor agonist UTP functionally reproduced all the ATP-stimulated effects. Furthermore, both ATP and UTP induced intracellular Ca(2+) mobilization with comparable EC(50) values (i.e. 1-3µM). Collectively, these functional and pharmacological profiles strongly suggest that ATP acts primarily via a P2Y2 receptor sub-type in human endothelial cells. In support, P2Y2 receptor mRNA and protein were readily detected in isolated HUVECs, and siRNA-mediated knockdown of endogenous P2Y2 receptor protein significantly blunted the cytosolic Ca(2+) elevations in response to ATP and UTP, but did not affect the histamine-evoked response. In summary, these results identify the P2Y2 isoform as the major purinergic receptor in human vascular endothelial cells that mediates the cellular actions of ATP linked to vasorelaxation.

Alpha5beta1 integrin engagement increases large conductance, Ca2+-activated K+ channel current and Ca2+ sensitivity through c-src-mediated channel phosphorylation.

Large conductance, calcium-activated K(+) (BK) channels are important regulators of cell excitability and recognized targets of intracellular kinases. BK channel modulation by tyrosine kinases, including focal adhesion kinase and c-src, suggests their potential involvement in integrin signaling. Recently, we found that fibronectin, an endogenous alpha5beta1 integrin ligand, enhances BK channel current through both Ca(2+)- and phosphorylation-dependent mechanisms in vascular smooth muscle. Here, we show that macroscopic currents from HEK 293 cells expressing murine BK channel alpha-subunits (mSlo) are acutely potentiated following alpha5beta1 integrin activation. The effect occurs in a Ca(2+)-dependent manner, 1-3 min after integrin engagement. After integrin activation, normalized conductance-voltage relations for mSlo are left-shifted at free Ca(2+) concentrations >or=1 microm. Overexpression of human c-src with mSlo, in the absence of integrin activation, leads to similar shifts in mSlo Ca(2+) sensitivity, whereas overexpression of catalytically inactive c-src blocks integrin-induced potentiation. However, neither integrin activation nor c-src overexpression potentiates current in BK channels containing a point mutation at Tyr-766. Biochemical tests confirmed the critical importance of residue Tyr-766 in integrin-induced channel phosphorylation. Thus, BK channel activity is enhanced by alpha5beta1 integrin activation, likely through an intracellular signaling pathway involving c-src phosphorylation of the channel alpha-subunit at Tyr-766. The net result is increased current amplitude, enhanced Ca(2+) sensitivity, and rate of activation of the BK channel, which would collectively promote smooth muscle hyperpolarization in response to integrin-extracellular matrix interactions.

Hsp40 couples with the CSPalpha chaperone complex upon induction of the heat shock response.

In response to a conditioning stress, the expression of a set of molecular chaperones called heat shock proteins is increased. In neurons, stress-induced and constitutively expressed molecular chaperones protect against damage induced by ischemia and neurodegenerative diseases, however the molecular basis of this protection is not known. Here we have investigated the crosstalk between stress-induced chaperones and cysteine string protein (CSPalpha). CSPalpha is a constitutively expressed synaptic vesicle protein bearing a J domain and a cysteine rich "string" region that has been implicated in the long term functional integrity of synaptic transmission and the defense against neurodegeneration. We have shown previously that the CSPalpha chaperone complex increases isoproterenol-mediated signaling by stimulating GDP/GTP exchange of Galpha(s). In this report we demonstrate that in response to heat shock or treatment with the Hsp90 inhibitor geldanamycin, the J protein Hsp40 becomes a major component of the CSPalpha complex. Association of Hsp40 with CSPalpha decreases CSPalpha-CSPalpha dimerization and enhances the CSPalpha-induced increase in steady state GTP hydrolysis of Galpha(s). This newly identified CSPalpha-Hsp40 association reveals a previously undescribed coupling of J proteins. In view of the crucial importance of stress-induced chaperones in the protection against cell death, our data attribute a role for Hsp40 crosstalk with CSPalpha in neuroprotection.

RDJ2 (DNAJA2) chaperones neural G protein signaling pathways.

A number of structurally divergent proteins with J domains, called J proteins, interact with and activate the ATPase of Hsp70s, thereby harnessing the ATPase activity for conformational work on target proteins. The precise role of most mammalian J proteins remains undefined. In this paper, we demonstrate that transient expression of the J protein, Rdj2, in HEK 293 cells increased cellular cyclic adenosine monophosphate (cAMP) levels in the presence of the beta-adrenergic agonist isoproterenol. In CNS-derived catecholaminergic neuronal cell line (CAD) neuroblastoma cells, expression of Rdj2 increased isoproterenol-stimulated phosphorylation of cAMP response element binding protein (CREB). Moreover, we have characterized the binding properties of Rdj2 and observed a direct interaction between Rdj2 and receptor-coupled trimeric GTP-binding proteins (G proteins). We further show that the composition of the Rdj2-chaperone complex and the cysteine string protein (CSPalpha)-chaperone complex, another J protein, is distinct. Our data demonstrate that Rdj2 modulates G protein signaling and further suggest that chaperoning G proteins is an emerging theme of the J protein network.

Openers of SKCa and IKCa channels enhance agonist-evoked endothelial nitric oxide synthesis and arteriolar vasodilation.

Recent data have led us to hypothesize that selective activation of endothelial small- and/or intermediate-conductance, calcium-activated potassium channels (SK(Ca) and IK(Ca) channels, respectively) by the opener compounds NS309 and DCEBIO would augment stimulated nitric oxide (NO) synthesis and vasodilation in resistance arteries. Experimentally, ATP-evoked changes in membrane potential, cytosolic Ca(2+), and NO synthesis were recorded by patch clamp and microfluorimetry in single human endothelial cells. Agonist-evoked inhibition of myogenic tone in isolated, pressurized arterioles from rat cremaster skeletal muscle was analyzed by video microscopy. NS309 and DCEBIO enhanced ATP-evoked membrane hyperpolarization and cytosolic Ca(2+) transients, along with acute NO synthesis in isolated endothelial cells. The acetylcholine-mediated inhibition of myogenic tone (IC(50)=237 nM) was left-shifted in the presence of NS309 and DCEBIO (10, 100, and 1000 nM) to IC(50) values of 101, 78, and 43 nM; endothelial denudation inhibited this drug effect. L-NAME attenuated the acetylcholine-induced inhibition of myogenic tone but did not interfere with NS309 and DCEBIO-evoked vasodilation. Collectively, our data demonstrate that drug-induced enhancement of endothelial SK(Ca) and IK(Ca) channel activities represents a novel cellular mechanism to increase vasodilation of small-resistance arterioles, thereby highlighting these channels as potential therapeutic targets in cardiovascular disease states associated with compromised NO signaling.