Inhibition of Ca2+ release channel (ryanodine receptor) activity by sphingolipid bases: mechanism of action

Sphingosine inhibits the activity of the skeletal muscle Ca2+ release channel (ryanodine receptor) and is a noncompetitive inhibitor of [3H]ryanodine binding (Needleman et al., Am. J. Physiol. 272, C1465-1474, 1997). To determine the contribution of other sphingolipids to the regulation of ryanodine receptor activity, several sphingolipid bases were assessed for their ability to alter [3H]ryanodine binding to sarcoplasmic reticulum (SR) membranes and to modulate the activity of the Ca2+ release channel. Three lipids, N,N-dimethylsphingosine, dihydrosphingosine, and phytosphingosine, inhibited [3H]ryanodine binding to both skeletal and cardiac SR membranes. However, the potency of these three lipids and sphingosine was lower in rabbit cardiac membranes when compared to rabbit skeletal muscle membranes and when compared to sphingosine. Like sphingosine, the lipids inhibited [3H]ryanodine binding by greatly increasing the rate of dissociation of bound [3H]ryanodine from SR membranes, indicating that these three sphingolipid bases were noncompetitive inhibitors of [3H]ryanodine binding. These bases also decreased the activity of the Ca2+ release channel incorporated into planar lipid bilayers by stabilizing a long closed state. Sphingosine-1-PO4 and C6 to C18 ceramides of sphingosine had no significant effect on [3H]ryanodine binding to cardiac or skeletal muscle SR membranes. Saturation of the double bond at positions 4-5 decreased the ability of the sphingolipid bases to inhibit [3H]ryanodine binding 2-3 fold compared to sphingosine. In summary, our data indicate that other endogenous sphingolipid bases are capable of modulating the activity of the Ca2+ release channel and as a class possess a common mechanism of inhibition.     

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

BK channels regulate vascular tone by hyperpolarizing smooth muscle in response to fluctuating calcium concentrations. Oestrogen has been reported to lower blood pressure by increasing BK channel open probability through direct binding to the regulatory β1-subunit(s) associated with the channel. The present investigation demonstrates that 17β-oestradiol activates the BK channel complex by increasing the burst duration of channel openings. A subconductance state was observed in 25% of recordings following the addition of 17β-oestradiol and could reflect uncoupling between the pore forming α1-subunit and the regulatory β1-subunit. We also present evidence that more than one β1-subunit is required to facilitate binding of 17β-oestradiol to the channel complex.     

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

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

Large conductance Ca2+-activated K+ (BK) channel: activation by Ca2+ and voltage

Large conductance Ca2+-activated K+ (BK) channels belong to the S4 superfamily of K+ channels that include voltage-dependent K+ (Kv) channels characterized by having six (S1-S6) transmembrane domains and a positively charged S4 domain. As Kv channels, BK channels contain a S4 domain, but they have an extra (S0) transmembrane domain that leads to an external NH2-terminus. The BK channel is activated by internal Ca2+, and using chimeric channels and mutagenesis, three distinct Ca2+-dependent regulatory mechanisms with different divalent cation selectivity have been identified in its large COOH-terminus. Two of these putative Ca2+-binding domains activate the BK channel when cytoplasmic Ca2+ reaches micromolar concentrations, and a low Ca2+ affinity mechanism may be involved in the physiological regulation by Mg2+. The presence in the BK channel of multiple Ca2+-binding sites explains the huge Ca2+ concentration range (0.1 microM-100 microM) in which the divalent cation influences channel gating. BK channels are also voltage-dependent, and all the experimental evidence points toward the S4 domain as the domain in charge of sensing the voltage. Calcium can open BK channels when all the voltage sensors are in their resting configuration, and voltage is able to activate channels in the complete absence of Ca2+. Therefore, Ca2+ and voltage act independently to enhance channel opening, and this behavior can be explained using a two-tiered allosteric gating mechanism.     

Modulation of the mitochondrial large-conductance calcium-regulated potassium channel by polyunsaturated fatty acids

Polyunsaturated fatty acids (PUFAs) and their metabolites can modulate several biochemical processes in the cell and thus prevent various diseases. PUFAs have a number of cellular targets, including membrane proteins. They can interact with plasma membrane and intracellular potassium channels. The goal of this work was to verify the interaction between PUFAs and the most common and intensively studied mitochondrial large conductance Ca²⁺-regulated potassium channel (mitoBK_Ca). For this purpose human astrocytoma U87 MG cell lines were investigated using a patch-clamp technique. We analyzed the effects of arachidonic acid (AA); eicosatetraynoic acid (ETYA), which is a non-metabolizable analog of AA; docosahexaenoic acid (DHA); and eicosapentaenoic acid (EPA). The open probability (P_o) of the channel did not change significantly after application of 10 μM ETYA. P_o increased, however, after adding 10 μM AA. The application of 30 μM DHA or 10 μM EPA also increased the P_o of the channel. Additionally, the number of open channels in the patch increased in the presence of 30 μM EPA. Collectively, our results indicate that PUFAs regulate the BK_Ca channel from the inner mitochondrial membrane.     

Modulation of voltage-dependent Ca channel current by arachidonic acid and other long-chain fatty acids in rabbit intestinal smooth muscle.

The effects of arachidonic acid (AA) and other long-chain fatty acids on voltage-dependent Ca channel current (ICa) were investigated, with the whole cell patch clamp method, in longitudinal smooth muscle cells of rabbit ileum. 10-30 microM AA caused a gradual depression of ICa. The inhibitory effect of AA was not prevented by indomethacin (10 microM) (an inhibitor of cyclooxygenase) or nordihydroguaiaretic acid (10 microM) (an inhibitor of lipoxygenase). 1-(5-Isoquinolinesulfonyl)-2-methylpiperazine (H7; 25-50 microM) or staurosporine (2 microM) (inhibitors of protein kinase C) did not block the AA-induced inhibition of ICa, and application of phorbol ester (a protein kinase C activator) (phorbol-12,13-dibutyrate, 0.2 microM) did not mimic the AA action. Some other cis-unsaturated fatty acids (palmitoleic, linoleic, and oleic acids) were also found to depress ICa, while a trans-unsaturated fatty acid (linolelaidic acid) and saturated fatty acids (capric, lauric, myristic, and palmitic acids) had no inhibitory effects on ICa. Myristic acid consistently increased the amplitude of ICa at negative membrane potentials. The present results suggest the possible role of AA, and perhaps other fatty acids, in the physiological and/or pathological modulation of ICa in smooth muscle.     

Effects of fatty acids on BK channels in GH(3) cells

Ca²⁺-activated K⁺ (BK) channels inGH₃ cells are activated by arachidonic acid (AA). Becausecytosolic phospholipase A₂ can produce other unsaturatedfree fatty acids (FFA), we examined the effects of FFA on BK channelsin excised patches. Control recordings were made at several holdingpotentials. The desired FFA was added to the bath solution, and thevoltage paradigm was repeated. AA increased the activity of BK channelsby 3.6 ± 1.6-fold. The cis FFA, palmitoleic, oleic,linoleic, linolenic, eicosapentaenoic, and the triple bond analog ofAA, eicosatetraynoic acid, all increased BK channel activity, whereasstearic (saturated) or the trans isomers elaidic,linolelaidic, and linolenelaidic had no effect. The cisunsaturated FFA shifted the open probability vs. voltage relationshipsto the left without a change in slope, suggesting no change in thesensitivity of the voltage sensor. Measurements of membrane fluidityshowed no correlation between the change of membrane fluidity and thechange in BK channel activation. In addition, AA effects on BK channelswere unaffected in the presence of N-acetylcysteine.Arachidonyl-CoA, a membrane impermeable analog of AA, activateschannels when applied to the cytosolic surface of excised patches,suggesting an effect of FFAs from the cytosolic surface of BK channels.Our data imply a direct interaction between cis FFA and theBK channel protein.

     

Modulation of adenylate cyclase activity of fibroblasts by free fatty acids and phospholipids

The effect of certain lipids on adenylate cyclase activity [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] from fibroblasts in culture has been investigated. The unsaturated fatty acids, as well as lysolecithin, were found to act as potent inhibitors of fibroblast adenylate cyclase activity. Increasing the degree of unsaturation increases the extent of inhibition noted at a given fatty acid concentration. The inhibitory effect of the unsaturated fatty acids or lysolecithin is not selective for a specific function of the adenylate cyclase system since basal, and hormone- or fluoride-stimulated cyclase activities are inhibited to the same extent. The fatty acid-inactivated state of fibroblast adenylate cyclase is not readily reversed for enzyme activity is not restored when arachidonate-treated membranes are washed with Tris buffer containing 10 mm EDTA, 0.15 mm albumin, or 0.15 m KCl. Previous studies have shown that the adenylate cyclase system from Moloney sarcoma virus-transformed NRK (MNRK) cells is not stimulated by the addition of GTP or hormones. Of interest is the present finding that the addition of unsaturated fatty acids, or lysolecithin, over a narrow concentration range (0.1 – 0.2 mm) leads to partial restoration of GTP activation of MNRK cyclase activity. Hormonal responsiveness to l-epinephrine or prostaglandin E₁ is not restored to the MNRK enzyme with fatty acid or lysolecithin treatment.     

Membrane stretch and cytoplasmic Ca2+ independently modulate stretch-activated BK channel activity

Large conductance Ca²⁺-activated K⁺ (BK) channels are responsible for changes in chemical and physical signals such as Ca²⁺, Mg²⁺ and membrane potentials. Previously, we reported that a BK channel cloned from chick heart (SAKCaC) is activated by membrane stretch. Molecular cloning and subsequent functional characterization of SAKCaC have shown that both the membrane stretch and intracellular Ca²⁺ signal allosterically regulate the channel activity via the linker of the gating ring complex. Here we investigate how these two gating principles interact with each other. We found that stretch force activated SAKCaC in the absence of cytoplasmic Ca²⁺. Lack of Ca²⁺ bowl (a calcium binding motif) in SAKCaC diminished the Ca²⁺-dependent activation, but the mechanosensitivity of channel was intact. We also found that the abrogation of STREX (a proposed mechanosensing apparatus) in SAKCaC abolished the mechanosensitivity without altering the Ca²⁺ sensitivity of channels. These observations indicate that membrane stretch and intracellular Ca²⁺ could independently modulate SAKCaC activity.     

Modulation of Ca2+ release channel activity from sarcoplasmic reticulum by annexin VI (67-kDa calcimedin)

The effect of annexin VI (67-kDa calcimedin) on the activity of the Ca2+ release channel was studied using heavy sarcoplasmic reticulum membranes reconstituted into planar bilayers. Annexin VI, in a range of 5-40 nM, modified the gating behavior of the Ca2+ release channel by increasing the probability of opening by 2.7-fold and the mean open time by 82-fold relative to controls. Annexin VI caused no change in the slope conductance of the channel. The modulatory effect of annexin VI on the activity of Ca2+ release channels was Ca2+ dependent, and the annexin VI-modified channel was sensitive to both ruthenium red and ryanodine. The effect of annexin VI was observed when this protein was added specifically to the trans chamber, which corresponds to the luminal side of sarcoplasmic reticulum as determined by the ATP activation of the channel. In addition, differential extraction studies demonstrated that some annexin VI is localized within the lumen of the isolated heavy sarcoplasmic reticulum vesicles prepared by several different procedures. Annexin VI did not modify, from either the cis or trans chambers, the activity of K+ or Cl- channels from sarcoplasmic reticulum or the dihydropyridine sensitive Ca2+ channel from transverse tubules. In addition, the 38-kDa core proteolytic fragments of annexin VI had no effect on the Ca2+ release channel activity. Annexin VI is therefore a candidate for a physiological modulator of the Ca2+ release channel and as such, may play an important role in the excitation-contraction coupling.     

Modulation of endothelium function by fatty acids

Molecular and Cellular Biochemistry - The endothelium acts as the barrier that prevents circulating lipids such as lipoproteins and fatty acids into the arterial wall; it also regulates normal...     

BK(Ca) channel activation by membrane-associated cGMP kinase may contribute to uterine quiescence in pregnancy

Weinvestigated the influence of pregnancy on large-conductancecalcium-activated potassium channel (BK_Ca) activity(NP_o) and on channel expression in membranes ofisolated human myometrial smooth muscle cells.NP_o in inside-out patches was higher in pregnant myometria (PM) compared with nonpregnant myometria (NPM), and thehalf-maximal activation potential was shifted by 39 mV to more negativepotentials. This effect was not due to an enhanced BK_Cachannel expression. In the presence of cAMP kinase (PKA) or cGMP kinase(PKG), NP_o increased in patches from PMbut decreased in those from NPM. Western blot analysis and use of aspecific PKG inhibitor (1 µM KT-5823) verified the existence of apartially active membrane-associated PKG. Inhibition of PKA by100 nM PKI, the inhibitory peptide of PKA, had no effect onNP_o. 8-p-Chlorophenylthio-cGMP (8-pCPT-cGMP) hyperpolarized cells from PM. This effect wasabolished by iberiotoxin, a specific blocker of BK_Cachannels. It is concluded that an endogenous, membrane-bound PKG inmyometrial cells specifically enhances BK_Ca channelactivity during pregnancy and thus may contribute to uterine quiescenceduring pregnancy.

     

Menthol increases human glioblastoma intracellular Ca2+, BK channel activity and cell migration

This study examined the effect of menthol, an agonist for transient receptor potential melastatin 8 (TRPM8) ion channels, to increase intracellular Ca²⁺ concentration, [Ca²⁺]_i, in human glioblastoma cells (DBTRG cells), which resulted in activation of the large-conductance Ca²⁺-activated K⁺ membrane ion channels (BK channels). Voltage ramps applied over 300 ms from -100 to 100 mV resulted in membrane currents with marked inwardly- and outwardly-rectifying components. Paxilline (2 μM) abolished the outwardly-rectifying current. Outwardly-rectifying on-cell patch currents were increased markedly by menthol (100 μM) added to the bath. The estimated on-cell conductance of these channels was 253 pS. Kinetic analysis showed that added menthol increased channel open probability and mean open frequency after 5 min. In a similar time course menthol increased [Ca²⁺]_i, and this increase was abolished either by added paxilline, tetraethylammonium ion or by Ca²⁺-free external solution. Finally, menthol stimulated the rate of DBTRG cell migration into scratch wounds made in confluent cells, and this also was inhibited by paxilline or by tetraethylammonium ion. We conclude that menthol, a TRPM8 agonist, increases DBTRG cell [Ca²⁺]_i that in turn activates membrane BK ion channels. Inhibition of BK channels by paxilline reverses menthol-stimulated increase of [Ca²⁺]_i and of cell migration. Thus, BK channels function to maintain elevations in [Ca²⁺]_i needed to sustain increases in DBTRG cell migration.     

Modulation of hSlo BK current inactivation by fatty acid esters of CoA

Lipid metabolism influences membrane proteins, including ion channels, in health and disease. Fatty acid esters of CoA are important intermediates in fatty acid metabolism and lipid biosynthesis. In the present study, we examined the effect of acyl-CoAs on hSlo BK currents. Arachidonoyl-CoA (C₂₀-CoA) induced β2-dependent inhibition of hSlo -α current when applied intracellularly but not extracellularly. This action was also mimicked by other long-chain acyl-CoAs such as oleoyl-CoA (C₁₈-CoA) and palmitoyl-CoA (C₁₆-CoA), but not acetyl-CoA (C₂-CoA, shorter chain), suggesting that the length of acyl chains, rather than CoA headgroups, is critical. When hSlo -α inactivation was induced by a free synthetic cationic β2 NH2-terminus inactivation ball peptide, long-chain acyl-CoAs inhibited hSlo -α current and facilitated inactivation. The precursor fatty acids also facilitated the ball peptide-induced inactivation in a chain length-dependent manner, whereas sphingosine (positively charged) slowed this inactivation. When the β2-induced inactivation was compared with that of the ball peptide, there was a negative shift in the steady state inactivation, slower recovery, and a reduced voltage-dependence of inactivation onset. These data suggest that electrostatic interactions with the cytosolic inactivation domain of β2 mediate acyl-CoA modulation of BK currents. BK channel inactivation may be a specific target for lipid modulation in physiological and pathophysiological conditions.     

EETs relax airway smooth muscle via an EpDHF effect: BK(Ca) channel activation and hyperpolarization

Epoxyeicosatrienoic acids (EETs) are produced from arachidonic acid via the cytochrome P-450 epoxygenase pathway. EETs are able to modulate smooth muscle tone by increasing K(+) conductance, hence generating hyperpolarization of the tissues. However, the molecular mechanisms by which EETs induce smooth muscle relaxation are not fully understood. In the present study, the effects of EETs on airway smooth muscle (ASM) were investigated using three electrophysiological techniques. 8,9-EET and 14,15-EET induced concentration-dependent relaxations of the ASM precontracted with a muscarinc agonist (carbamylcholine chloride), and these relaxations were partly inhibited by 10 nM iberiotoxin (IbTX), a specific large-conductance Ca(2+)-activated K(+) (BK(Ca)) channel blocker. Moreover, 3 microM 8,9- or 14,15-EET induced hyperpolarizations of -12 +/- 3.5 and -16 +/- 3 mV, with EC(50) values of 0.13 and 0.14 microM, respectively, which were either reversed or blocked on addition of 10 nM IbTX. These results indicate that BK(Ca) channels are involved in hyperpolarization and participate in the relaxation of ASM. In addition, complementary experiments demonstrated that 8,9- and 14,15-EET activate reconstituted BK(Ca) channels at low free Ca(2+) concentrations without affecting their unitary conductance. These increases in channel activity were IbTX sensitive and correlated well with the IbTX-sensitive hyperpolarization and relaxation of ASM. Together these results support the view that, in ASM, the EETs act through an epithelium-derived hyperpolarizing factorlike effect.     

Modulation of T-cell signalling by non-esterified fatty acids

Polyunsaturated fatty acids (PUFAs) have been shown to be immunosuppressive. In particular, they can decrease important T-cell functions that may have a profound impact on the acquired immune response. Several mechanisms may explain the immunosuppressive properties of PUFAs. Here we review the mechanisms by which they interfere with T-cell activation. PUFAs affect lipid rafts composition and function that play an essential role in T-cell signalling. The possible physiological and pathological significances of this immunomodulation by PUFAs are discussed. Further mechanistic studies and randomized controlled clinical trials are needed to assess more accurately their effects in healthy and pathological states.     

The NH2 terminus of RCK1 domain regulates Ca2+-dependent BK(Ca) channel gating

Large conductance, voltage- and Ca2+-activated K+ (BK(Ca)) channels regulate blood vessel tone, synaptic transmission, and hearing owing to dual activation by membrane depolarization and intracellular Ca2+. Similar to an archeon Ca2+-activated K+ channel, MthK, each of four alpha subunits of BK(Ca) may contain two cytosolic RCK domains and eight of which may form a gating ring. The structure of the MthK channel suggests that the RCK domains reorient with one another upon Ca2+ binding to change the gating ring conformation and open the activation gate. Here we report that the conformational changes of the NH2 terminus of RCK1 (AC region) modulate BK(Ca) gating. Such modulation depends on Ca2+ occupancy and activation states, but is not directly related to the Ca2+ binding sites. These results demonstrate that AC region is important in the allosteric coupling between Ca2+ binding and channel opening. Thus, the conformational changes of the AC region within each RCK domain is likely to be an important step in addition to the reorientation of RCK domains leading to the opening of the BK(Ca) activation gate. Our observations are consistent with a mechanism for Ca2+-dependent activation of BK(Ca) channels such that the AC region inhibits channel activation when the channel is at the closed state in the absence of Ca2+; Ca2+ binding and depolarization relieve this inhibition.