HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) All Versions of this Article: 97/1/62    most recent 00700.2006v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via ISI Web of Science (2) Citing Articles via Google Scholar Google Scholar Articles by Sun, X. Articles by Haddad, G. G. Search for Related Content PubMed PubMed Citation Articles by Sun, X. Articles by Haddad, G. G.

-Subunit–Dependent Modulation of hSlo BK Current by Arachidonic Acid

¹Departments of Pediatrics and ²Neuroscience, University of California San Diego, La Jolla, California; ³Department of Physiology, Yale University, New Haven, Connecticut; and ⁴Department of Biology, Clarkson University, Potsdam, New York

Submitted 6 July 2006; accepted in final form 28 September 2006

	ABSTRACT

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION GRANTS ACKNOWLEDGMENTS REFERENCES

 
In this study, we examined the effect of arachidonic acid (AA) on the BK -subunit with or without -subunits expressed in Xenopus oocytes. In excised patches, AA potentiated the hSlo- current and slowed inactivation only when 2/3 subunit was co-expressed. The 2-subunit–dependent modulation by AA persisted in the presence of either superoxide dismutase or inhibitors of AA metabolism such as nordihydroguaiaretic acid and eicosatetraynoic acid, suggesting that AA acts directly rather than through its metabolites. Other cis unsaturated fatty acids (docosahexaenoic and oleic acid) also enhanced hSlo- + 2 currents and slowed inactivation, whereas saturated fatty acids (palmitic, stearic, and caprylic acid) were without effect. Pretreatment with trypsin to remove the cytosolic inactivation domain largely occluded AA action. Intracellularly applied free synthetic 2-ball peptide induced inactivation of the hSlo- current, and AA failed to enhance this current and slow the inactivation. These results suggest that AA removes inactivation by interacting, possibly through conformational changes, with 2 to prevent the inactivation ball from reaching its receptor. Our data reveal a novel mechanism of -subunit–dependent modulation of BK channels by AA. In freshly dissociated mouse neocortical neurons, AA eliminated a transient component of whole cell K⁺ currents. BK channel inactivation may be a specific mechanism by which AA and other unsaturated fatty acids influence neuronal death/survival in neuropathological conditions.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION GRANTS ACKNOWLEDGMENTS REFERENCES

 
Polyunsaturated fatty acids (PUFAs) such as arachidonic acid (AA) and docosahexaenoic acid (DHA) are highly concentrated in the phospholipids of brain tissues and neuronal synapses. The absolute PUFA levels and their relative concentrations are strictly controlled in mammalian neurons, and significant alterations in brain PUFAs have been found in patients with various neurological disorders and neurodegenerative diseases (Berger et al. 2006; Bois et al. 2005; Markesbery 1997). PUFAs have been implicated as substrates in peroxidative damage because of the abundance of double bonds, which constitute ideal sites for reactive oxygen species attack and free radical generation (Phillis and O'Regan 2004). In ischemia/reperfusion, alterations in lipid metabolism, such as activation of phospholipases and release of fatty acids, are key events in a cascade that leads to neuronal death (Arai et al. 2001; Lipton 1999). PUFA treatments have been shown to exert strong neuroprotective effects because of modulation of ion channels and synaptic glutamatergic transmission in hippocampal neurons in both in vitro and in vivo ischemic models (Lauritzen et al. 2000; Strokin et al. 2006).

AA modulates the activities of various ion channels through direct or indirect pathways (Meves 1994). The direct effects are mediated by the interaction between AA and ion channel proteins or through perturbation of plasma membranes. The indirect actions on ion channels are mediated by metabolites of AA through oxygenases or cellular signal transduction pathways. The effects of AA actions during ischemia/reperfusion could be either beneficial or detrimental to neurons. For example, prostaglandins, which are produced from AA by the action of cyclooxygenase, are considered damaging in brain ischemia (Nogawa et al. 1997). Nonenzymatic oxidative metabolism of AA also generates reactive oxygen radicals that contribute to oxidative injury (Phillis and O'Regan 2004).

Large-conductance, calcium-activated potassium (BK) channels are expressed throughout the vertebrate nervous system. Because BK channel activation depends on depolarization and an increased intracellular calcium concentration, they are implicated for negative feedback regulation of Ca²⁺ influx through voltage-gated Ca²⁺ channels and regulation of cellular excitability and neurotransmitter release (Hu et al. 2001; Raffaelli et al. 2004; Sah 1996). On the one hand, therefore, activation of BK channels could serve to protect neurons and reduce the pathological consequences of ischemia and other conditions characterized by accumulation of intracellular calcium and excessive depolarization (Gribkoff et al. 2001; Runden-Pran et al. 2002). On the other hand, activation of BK channels can usher the initiation of K+ leakage during ischemia, which can trigger cellular dehydration and potentially programmed cell death (Lang et al. 2005). Although AA has been shown to activate BK channels in various types of native cells (Bringmann et al. 1998; Clarke et al. 2002; Denson et al. 2000), the molecular interaction between BK and AA has not yet been clarified. For example, it is not known whether AA modulates directly the pore-forming -subunit or alters modulatory accessory subunits or other associated components.

BK channels are composed of four homologous pore-forming -subunits, along with optional accessory subunits that regulate many important aspects of BK channel function. To date, four mammalian -subunits have been identified in humans (KCNMB1-4, Brenner et al. 2000; Knaus et al. 1994; Meera et al. 2000; Uebele et al. 2000; Wallner et al. 1999; Weiger et al. 2000; Xia et al. 2000), each of which confers unique functional properties on the resulting BK channels. In particular, both 2- and 3-subunits result in kinetically distinct inactivating BK channels (Wallner et al. 1999; Xia et al. 2000). In the brain, there seems to be a widespread expression of Slo -subunit and h4. Expression of 2/3 is less robust and probably more specific to certain cell types, making them an intriguing target with respect to selective neuronal function. Virtually no h1 mRNA can be detected in the CNS.

In this study, we used the Xenopus oocyte expression system and expressed the hSlo--subunit alone or in conjunction with the 2–4 subunits that have been shown to be present in the brain. We show that AA alters -subunit modulation of BK channel inactivation and potentiates steady-state conductance in the presence of 2/3. AA does not induce a significant response of BK channels in the absence of or in the presence of 4. The -subunit–dependent modulation of BK channels by AA may have important implications on neuronal function under pathophysiological conditions.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION GRANTS ACKNOWLEDGMENTS REFERENCES

 
All animals used in this study were handled in compliance with the United States public service policy on care and use of laboratory animals and National Institutes of Health guide for the care and use of laboratory animals, as well as institutional animal care and use committee guidelines.

Preparation of oocytes

Female Xenopus laevis, the African clawed frog, were purchased from Nasco. Frogs were anesthetized with 0.2% tricaine methanesulphonate (MS 222). Clumps of oocytes were surgically removed and washed in Ca²⁺-free ND96 medium containing (in mM) 82.5 NaCl, 2 KCl, 1.8 MgCl₂, and 5 HEPES, pH 7.4. Single oocytes were dissociated with 1.5 mg/ml type II collagenase (Sigma, St. Louis, MO) in Ca²⁺-free ND96 solution at 18°C for 2 h to remove follicle cells. The oocytes were stored in ND96 incubation medium containing (in mM) 96 NaCl, 2 KCl, 1.8 CaCl₂, 1.8 MgCl₂, and 5 HEPES, pH 7.4, and kept at 18°C for 2 h before RNA injection.

Synthesis of cRNA

pcDNA3-hSlo was used for synthesis of cRNA. pBF-h2 and pBF-h3 were provided by Dr. Christopher Lingle, and pcDNA3-h4 was from Dr. Ligia Toro. The plasmids were first linearized with an appropriate restriction enzyme: XbaI for pcDNA3-hSlo, MluI for pBF-h2 and h3, and NotI for pcDNA3-h4. In vitro transcription of each individual cRNA was performed using mMESSAGE mMACHINE T7 (hSlo and 4 constructs) or SP6 (2 and 3 constructs) Kit (Ambion, Austin, TX) in the presence of the cap analog m7G(5')G according to the manufacturer's instructions. The cRNA products were purified with RNeasy affinity column (Qiagen, Valencia, CA), and aliquots were kept at –80°C.

Microinjection into oocytes

hSlo--subunit was injected at 10–15 ng/oocyte, and was injected at 1:2 ratios by weight to ensure a molar excess of -subunits over subunits. Oocytes were injected with 50 nl cRNA encoding for 1) -subunits alone; 2) + 2–4; and 3) -subunits alone. Control oocytes were injected with the same volume of water. Injected oocytes were incubated at 18°C in ND96 incubation medium and were used for eletrophysiological experiments 3–7 days after injection. During this period, Xenopus oocytes after mRNA injection gave us enough channel expression in healthy oocytes.

Preparation of neocortical neurons

Mice were used between 9 and 15 days of age. The method for cell isolation was similar to that described previously (Sun et al. 2003). In brief, mice were killed by decapitation. The brain was removed rapidly and chilled in 0–4°C oxygenated isolation buffer containing (in mM) 120 NaCl, 5 KCl, 1 CaCl₂, 10 HEPES, and 25 glucose (pH adjusted to 7.0 with NaOH). Neocortical slices were incubated for 20 min with oxygenated isolation buffer containing Protease (type XIV from Streptomyces griseus, Sigma P-5147, 0.5 mg/ml, at 32°C). Slices were washed in oxygenated buffer and maintained for 3 h. Immediately before recording, a cortical slice was dissociated by gentle trituration with fire-polished Pasteur pipettes. The cells were collected and placed into a plastic petri dish and were allowed to settle for several minutes and adhere to the petri dish before superfusion was initiated. Recordings were obtained only from pyramidal-shaped neurons that had a single thick proximal dendrite and did not show any visible evidence of injury.

Electrophysiological recordings

Recording pipettes were made from filament borosilicate capillary glass (1.2 mm OD, 0.69 mm ID; Warner, Hamden, CT), using a Flaming/Brown micropipette puller (model P-87, Sutter Instrument, Novato, CA). The pipettes were fire-polished and had resistances of 5–8 M when filled with solutions listed below. Experiments were conducted at room temperature (22–25°C).

Voltage-clamp experiments were performed in Xenopus oocytes from which the vitelline membrane had been manually removed after shrinkage in a hyperosmotic medium containing (in mM) 200 K-gluconate, 20 KCl, 1.0 MgCl₂, 10 EGTA, and 10 HEPES, pH 7.4. Currents were recorded in either inside-out or outside-out patches. Both the pipette and the bathing solution contained (in mM) 116 K-gluconate, 4 KCl, and 10 HEPES, pH 7.2. Extracellular solutions contained 1 mM MgCl₂. Test solutions bathing the cytoplasmic face of the plasma membrane contained 0 (5 mM EGTA and no added Ca²⁺), 25, or 100 µM CaCl₂.

Whole cell K⁺ currents in mouse neocortical neurons were recorded using the same methods as previous studies (Sun et al. 2003). Briefly, pipette solutions contained (in mM) 110 K-gluconate, 10 KCl, 5 NaCl, 2 MgCl₂, 10 HEPES, 0.5 EGTA, 1 ATP, 0.2 GTP, and 0.1 leupeptin, with pH adjusted to 7.4. To record calcium-activated potassium channel currents, cells were superfused with a HEPES solution containing (in mM) 140 NaCl, 3 KCl, 1 CaCl₂, 1 MgCl₂, 10 HEPES, 0.0005 TTX, and 1 4-AP, 0.005 glibenclamide, and 10 glucose (pH 7.4). TTX, 4-AP, and glibenclamide were routinely included in the K⁺ current recording.

Membrane potential and membrane currents were recorded with an Axopatch 200A amplifier (Axon Instruments, Foster City, CA). Current signals were filtered at 2–5 kHz and digitized at 20–50 kHz. Stimulus generation and data acquisition were controlled with the Clampex program of the pClamp8 software package (Axon Instruments).

Drugs and solutions

All salts and chemicals were obtained from Sigma (St. Louis, MO). Stock solutions of AA were 100 mM either in dimethyl sulfoxide (DMSO), or water (sodium salt). Stock solutions of DHA, oleic acid, palmitic acid, elaidic acid, stearic acid, caprylic acid, nordihydroguaiaretic acid (NDGA), and eicosatetraynoic acid (ETYA) were all 100 mM in DMSO. Lysophosphatidylinositol (sodium salt) stock solution was 20 mM in water, lysophosphatidylcholine was 20 mM in methanol, and sphingosine was 50 mM in DMSO. Superoxide dismutase (SOD) stock solution was dissolved in medium at 25,000 U/ml; aliquots of stock solutions were kept at –80°C and were later diluted with bath solution before use. Final concentration of DMSO was <1:2,000, a concentration that was found to have no effect in control studies without drugs.

2 "ball" peptides consisting of 26 N-terminal amino acids (MFIWTSGRTSSSYRHDEKRNIYQKIR) were synthesized (Invitrogen). The peptides were dissolved to a concentration of 10 mM in 50 mM Tris · Cl (pH 7; Wallner et al. 1999). Aliquots of stock solution were kept at –20°C and were further diluted in the bath solution before experiments.

Data analysis

Whole cell K⁺ currents were recorded using the same protocol as we have done previously (Sun et al. 2003). Currents were evoked by depolarizing steps (–60 to +50 mV) from a holding potential of –70 mV for 200 ms in 10-mV steps, with 5-s intervals between sweeps.

Oocyte inside-out and outside-out patches were held at a potential of –70 mV before hyperpolarizing to –140 mV for 150 ms to remove inactivation. Membrane patches were depolarized stepwise from –100 to +120 mV in 20-mV steps for the duration of 200 ms with 5-s intervals between sweeps (unless otherwise indicated). In some cases, the hyperpolarizing prepulses were omitted for recording noninactivating currents ( alone or + 4), and the data were grouped because no difference was found in the data obtained. In a few cases, short hyperpolarizing postpulses after the depolarizing steps were applied before stepping back to –70 mV. Conductance-voltage (G-V) relations were generated by measuring values at the end of the voltage steps for the sustained current and at the peak values during voltage steps for the peak current at a given activation potential. G-V curves for activation were fit with a Boltzmann equation using Origin program. Steady-state inactivation curves were generated by stepping to potentials between –140 and +40 mV (in 20-mV increments) for 2 s before a 200-ms test step to +100 mV. Peak current activated at +100 mV is plotted as a function of conditioning potential. Data are presented as means ± SE. Student's t-test is used for statistical significance.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION GRANTS ACKNOWLEDGMENTS REFERENCES

 
Effects of AA on hSlo- channels with or without subunits expressed in Xenopus oocytes

Inside-out or outside-out patches taken from Xenopus oocytes expressing the hSlo--subunit alone, or the -subunit plus one of the 2-4-subunits, were examined to determine the steady-state and kinetic properties of BK channels. Patches were exposed to 25 µM intracellular Ca²⁺ (unless otherwise indicated), a value that was sufficient for adequate activation of the BK channels. Patches pulled from oocytes that were not injected or injected with either H₂O or -subunits alone showed no evidence of channel activity under our recording conditions and were used as negative controls (n = 33 patches). Currents obtained from channels composed of only the -subunit displayed no inactivation over the course of the pulses (n = 44 patches; Fig. 1A). However, channels composed of either + 2 (n = 120 patches; Fig. 1B) or + 3 (n = 30 patches; Fig. 1C) inactivated to varying degrees throughout the depolarization protocols. Currents obtained from channels composed of + 4 did not inactivate (n = 22 patches; Fig. 1D). Application of AA (10–30 µM) did not significantly alter hSlo- channel activity in the absence of -subunits (n = 7 patches; Fig. 1A), but markedly increased both the peak and steady-state currents in the presence of 2 (n = 14 patches; Fig. 2B) or 3 subunits (n = 9 patches; Fig. 1C). AA did not significantly alter maximal conductance of the steady-state current produced by co-expression of hSlo- channels with 4 subunits (n = 10 patches; Fig. 1D).

View larger version (32K): [in this window] [in a new window]   FIG. 1. Effects of arachidonic acid (AA) on hSlo- channels with or without -subunit. Current recordings were obtained in symmetrical K+-gluconate containing 25 µM Ca2+ (intracellular). Patches were held at –70 mV before undergoing a hyperpolarizing step to –140 mV to remove inactivation, immediately followed by 200-ms depolarizing steps from a range of potentials (–100 to +120 mV) in 20-mV steps. A: representative noninactivating hSlo- current traces were taken before and during AA (30 µM). G-V curves of the steady-state hSlo- currents were measured at the end of command steps (200 ms) before and during AA (10–30 µM, n = 7 patches). B: representative current traces from co-expression of hSlo- and -2-subunits were recorded before and during AA (30 µM). G-V curves were measured at the peak of the current before and during application of AA (10–30 µM, n = 14 patches). C: representative current traces of hSlo- + 3 currents were recorded before and during application of 10 µM AA. G-V curves were measured at the end of command steps (n = 9 patches). D: representative current traces show currents resulting from co-expression of and 4, taken before and during application of 10 µM AA. G-V curves were measured at the end of command steps (n = 10 patches). Values are expressed as means ± SE.

View larger version (13K): [in this window] [in a new window]   FIG. 2. AA enhances hSlo- + 2 currents and slows inactivation. Macroscopic hSlo- + 2 currents were recorded from either inside-out or outside-out patches. Currents were evoked by 200-ms voltage steps to +80 mV. A: superimposed traces were taken before (1), during (2), and after bath application of 30 µM AA (3) at the time-points indicated in plots of time-course of peak current (Ipeak) and inactivation time constant (). Inset: channel activities were recorded before (top) and during (bottom) AA (10 µM) application. B: percent change in Ipeak and ratios of inactivation time constants (AA/control) were plotted as a function of AA concentrations (1–50 µM, n = 3–16 patches). Values are expressed as means ± SE.

A prepulse potential of –140 mV was used in our study to relieve inactivation of hSlo- + 2 currents and give maximal currents. The full steady-state inactivation curves were shown in Fig. 3. Patches were held at 0 mV before stepping to different conditional potentials (–140 to +40 mV) for 2 s. Peak current elicited at +100 mV from each conditioning potential was normalized to the peak current elicited from –140 mV. AA (20 µM) increased the maximal available current (Fig. 3A) and shifted the fractional availability of hSlo- + 2 currents to more positive potentials (V_0.5 from –93.5 ± 2.0 to –56.7 ± 7.6 mV, n = 8 patches; Fig. 3B).

View larger version (13K): [in this window] [in a new window]   FIG. 3. Effects of AA on steady-state inactivation properties of hSlo- + 2 currents. Macroscopic hSlo- + 2 currents were recorded from inside-out patches. From a holding potential of 0 mV, patches were stepped to potentials between –140 and +40 mV for 2 s before a 200-ms test step to +100 mV. A: representative traces were taken before and during bath application of AA (20 µM). B: normalized fractional availability is plotted as a function of conditioning potential. AA, 20 µM, n = 8 patches. Values are means ± SE.

View larger version (23K): [in this window] [in a new window]   FIG. 4. Effects of AA on hSlo- + 2 currents in the absence of intracellular Ca2+. Macroscopic hSlo- + 2 currents were recorded from inside-out patches at Ca2+ free intracellular solutions (5 mM EGTA and no added CaCl2). Currents were evoked by 200-ms voltage steps ranging from –20 to +140 mV. A: representative current traces of hSlo- currents were taken before and during bath application of AA (30 µM). G-V curves of the steady-state hSlo- currents were measured at the end of command steps (n = 3 patches). B: representative current traces of hSlo- + 2 currents were taken before and during AA (30 µM). G-V curves were measured at the peak of current (n = 4 patches). Values are means ± SE.

AA ACTS DIRECTLY AND NOT THROUGH ITS METABOLITES. Some ion channels are activated by the oxidation products of fatty acids rather than by the parent fatty acid itself. This can happen nonenzymatically or through the action of oxygenases. Because we used excised patches in this study, soluble cytoplasmic signaling components are unlikely to be involved in AA action. To determine whether superoxide anions generated from AA metabolism play a role in the activation of BK channels by AA, we examined the effect of AA in the presence of SOD, a known scavenger of superoxide free radicals. SOD alone did not significantly alter hSlo- +2 currents, and bath application of AA, in the presence of SOD, induced a potentiation of peak current and slowing of inactivation (n = 6 patches; Fig. 5). These data suggest that superoxide radicals are not critically involved in the AA action.

View larger version (13K): [in this window] [in a new window]   FIG. 5. AA action on hSlo- + 2 currents is not mediated by its metabolites. Currents were activated by voltage steps to +80 mV. A: representative current traces recorded from an outside-out patch before and during bath application of superoxide dismutase (SOD; 100 U/ml), a superoxide free radical scavenger, and followed by AA (30 µM). B: superimposed current traces taken from an inside-out patch before and after eicosatetraynoic acid (ETYA; a cyclooxygenase and lipoxygenase inhibitor, 20 µM) and followed by AA (20 µM) in the presence of SOD (50 U/ml). C and D: summary of effects of AA on hSlo- + 2 peak current amplitude (Ipeak, C), expressed as percent change in Ipeak, and inactivation time constant (, D), expressed as ratios of the inactivation time constants (AA/control), in the presence or absence of various inhibitors of AA metabolism. Values are means ± SE. AA (30 µM): n = 8 patches; SOD (100 U/ml) + AA (30 µM): n = 6 patches; SOD (50–100 U/ml) + ETYA (20–30 µM): n = 5 patches; SOD (50–100 U/ml) + ETYA (20–30 µM) + AA (20–30 µM): n = 4 patches; NDGA (10 µM) + AA (30 µM): NDGA, nordihydroguaiaretic acid, a nonspecific lipoxygenase inhibitor, n = 5 patches.

DOUBLE BONDS IN FREE FATTY ACID STRUCTURE PLAY A ROLE. AA is a cis-polyunsaturated fatty acid (20:4, cis-5,8,11,14) with negative charge. To examine the fatty acid specificity of the activation of hSlo- + 2 currents, we tested a range of fatty acids. Besides AA, other cis-unsaturated fatty acids tested included docosahexaenoic acid, which is a long-chain polyunsaturated fatty acid that is also highly enriched in the brain (DHA, 22:6, cis-4,7,10,13,16,19), and oleic acid, which is a cis-monounsaturated fatty acid (OA, 18:1, cis-9). Both DHA (20 µM, n = 6 patches) and OA (5–30 µM, n = 8 patches) mimicked AA action to reversibly enhance hSlo- + 2 currents and slow inactivation (Fig. 6). Elaidic acid (EA, 18:1, trans-9, 30 µM), which is a trans-configurational isomer of 9-octadecenoic acid, similarly altered the current, although to a lesser extent than OA, which is a cis configuration of 9-octadecenoic acid (n = 5 patches, P < 0.05; Fig. 6, C and D). None of the saturated fatty acids with various acyl chain lengths [stearic acid (SA), 18:0, 30 µM; palmitic acid (PA), 16:0, 30 µM; caprylic acid (CA), 8:0, 30 µM] induced significant changes in the hSlo- + 2 currents (n = 3–4 patches; Fig. 6, C and D). Among all the fatty acids tested, OA is the most potent to enhance hSlo- + 2 currents (Figs. 2 and 6, A and B). We also tested other lipid compounds with different charges, including lysophosphatidylinositol (LPI, negative), lysophosphatidycholine (LPC, neutral), and sphingosine (Sph, positive). None of these molecules mimicked AA action to enhance BK currents (n = 3–10 patches; Fig. 6, C and D). For example, sphingosine markedly inhibited BK currents both in the presence and absence of 2 subunits (77 ± 1.4 and 60 ± 4.0% reduction of peak hSlo-+ 2 and hSlo- alone currents, respectively, n = 4 patches). Our data suggest that double bonds seem to be necessary for the action of fatty acids to activate BK channels in the presence of 2.

View larger version (23K): [in this window] [in a new window]   FIG. 6. Effects of fatty acids and lipids on hSlo- + 2 currents. Macroscopic hSlo- + 2 currents were recorded from inside-out patches. Currents were activated by voltage steps to +80 mV. A and B: representative current traces and time-course plots of peak current (Ipeak) and inactivation time constant () recorded before and during bath application of docosahexaenoic acid (DHA; 20 µM; A) and oleic acid (OA; 5 µM; B). C and D: summary of effects of various fatty acids and lipids on hSlo- + 2 current peak amplitude (percent change in Ipeak; C) and inactivation kinetics (ratios of inactivation time constants /control; D). Values are means ± SE. AA (30 µM): n = 8 patches; DHA (20 µM): n = 6 patches; OA (10 µM): n = 6 patches; elaidic acid (EA, 30 µM): n = 5 patches; stearic acid (SA, 30 µM): n = 4 patches; palmitic acid (PA, 30 µM): n = 3 patches; caprylic acid (CA, 30 µM): n = 3 patches; lysophosphatidylinositol (LPI, 10 µM): n = 3 patches; lysophosphatidycholine (LPC, 3–10 µM): n = 10 patches; sphingosine (Sph, 10 µM): n = 4 patches.

View larger version (11K): [in this window] [in a new window]   FIG. 7. AA alters trypsin-sensitive inactivation of hSlo- + 2 currents. Macroscopic hSlo- + 2 currents were recorded from inside-out patches. A: representative current traces taken before, after trypsin exposure (0.1 mg/ml), and followed by AA (30 µM). Currents were evoked by a series of depolarizing steps (–80 to +80 mV, in 20-mV steps) from a holding potential of –70 mV followed by a hyperpolarizing –140 mV. B: I-V curves were measured at the peak of current shown in A. Inset: plot of time-course of Ipeak (pA) from the same recording as in A.

AA ACTS ON THE -SUBUNIT TO PREVENT BALL-RECEPTOR INTERACTION.

View larger version (14K): [in this window] [in a new window]   FIG. 8. AA fails to slow inactivation of hSlo- current induced by a free 2 ball peptide. Macroscopic currents were evoked by 200-ms voltage steps to +80 mV from inside-out patches. Representative current traces of hSlo- (A) and plots of time-course of peak current (Ipeak) and inactivation time constant (; B) were recorded before and during additive application of 1 µM 2 ball peptide and 30 µM AA.

Under certain pathophysiological conditions such as ischemia/reperfusion, brain levels of many fatty acids, including unsaturated fatty acid such as AA, DHA, and OA, increase rapidly (Lipton 1999). Small changes caused by any individual fatty acids may induce major changes in BK channel kinetics. For example, lower concentrations of AA (5 µM, 15 ± 4% change in I_peak, n = 7 patches) and OA (3 µM, 27 ± 2% change in I_peak, n = 5 patches) individually had only mild effects on hSlo- + 2 currents (Fig. 9A). However, when coapplied, the effects they induced at these concentrations were dramatic (89 ± 26% change in I_peak, n = 3 patches; Fig. 9A).

View larger version (13K): [in this window] [in a new window]   FIG. 9. Physiological implications of AA action on BK channels. A: macroscopic hSlo- + 2 currents were recorded from inside-out patches. Currents were evoked by 200-ms voltage steps to +80 mV. Superimposed traces were taken before and during additive application of 5 µM AA and 3 µM OA (left) or 3 µM OA and 5 µM AA (right). B: whole cell outward currents in mouse neocortical neurons were recorded in the presence of 4-AP (1 mM), TTX (0.5 µM), and glybenclamide (5 µM). Currents were activated by depolarizing voltage steps to 0 mV from a holding potential of –70 mV. Superimposed traces were taken before and during bath application of AA (10 µM).

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION GRANTS ACKNOWLEDGMENTS REFERENCES

 
We report here on a novel mechanism of -subunit–dependent modulation of hSlo- channels by AA. AA enhanced hSlo- current and slowed inactivation only when 2/3 subunits were co-expressed. The AA action on hSlo- + 2 currents was mediated directly by AA rather than by its metabolites and was not mediated by a direct interaction of AA with the inactivation ball. The AA action is also not mediated by interacting with the ball receptor site on the -subunit. We suggest that AA may remove inactivation by interacting with the 2 to prevent the inactivation ball from reaching its receptor.

Modulation of a variety of voltage-dependent potassium channels by AA is well documented (Meves 1994). AA has been shown to activate BK channels in various native cell types (Bringmann et al. 1998; Clarke et al. 2002; Denson et al. 2000). However, it was not clear whether AA modulates the pore-forming -subunits, some associated proteins, or the -subunits. AA activation of BK channel activities could be a result of AA producing a shift in the voltage/or Ca²⁺ dependence, making it easier for channels to open, or a result of AA removing an inactivation mechanism. In this study, we showed that, in the presence of hSlo 2- or 3-subunits, AA potentiates a steady-state BK current by modulating the -subunit–dependent inactivation. AA failed to significantly alter BK current in the absence of the -subunit. Our data provide an explanation for the observation that BK channel is activated by AA in native cells where 2/3 is present.

In this study, the action of AA on hSlo- + 2 currents is membrane-delimited because our data were obtained from excised patches. Although lipoxygenases could potentially be present in the excised membrane patch (Hagmann et al. 1993; Rouzer and Kargman 1988), inhibitors of AA metabolism did not have much effect on AA action. Furthermore, oleic acid, which is not a substrate for the AA metabolic pathways that yield bioactive compounds, induced a similar response to that of AA. These observations provide evidence that metabolites are not necessary for AA action. Additional data that indicate that the effect of AA is a direct one include our observation that nonenzymatic metabolites of AA do not mediate its action. For example, the AA-induced response was not abolished by adding the superoxide free radical scavenger SOD, indicating that the action is not through the generation of reactive oxygen radicals. Thus our data indicate that AA interacts directly either with the channel protein or the lipid membrane to alter hSlo- + 2 currents.

Our results from the trypsin pretreatment experiments have indicated that the action of AA is likely to be based on a trypsin-sensitive "ball-and-chain" mechanism, which involves the cytosolic N terminus of the -subunits (Solaro and Lingle 1992; Wallner et al. 1999). The action of AA was largely occluded by prior exposure to trypsin, suggesting that AA acts to relieve the cytosolic inactivation. AA failed to slow inactivation induced by the cytosolic free ball peptide, suggesting that AA does not directly interact with the ball domain itself to relieve inactivation. Our data with various fatty acids and lipids show that there is a modest chemical specificity in the interaction between fatty acids and BK channel protein. Double bonds play a critical role in the action of fatty acids. All the unsaturated fatty acids tested in our study with different chain conformations, including the cis polyunsaturated fatty acid (AA, 20:4n-6 and DHA, 22:6n-3) and the cis mono-unsaturated fatty acid (OA, 18:1n-9), induced a similar response, whereas saturated fatty acids with different acyl chain lengths (SA, 18:0; PA, 16:0; CA, 8:0) and other lipids with different charges were not able to mimick AA action. Furthermore, OA was more potent than AA and DHA to enhance hSlo- + 2 currents, which is consistent with their potency to promote hexagonal phase formation of fatty acid-phosphatidylethanolamine mixtures (Epand et al. 1991). Lysophosphatides, which have a different shape (smaller head with larger tail) from unsaturated fatty acids, were not able to mimick AA action. Thus our results can be rationalized by considering the effective shape of unsaturated fatty acids in which double bonds make the cross-section of the hydrophobic region larger than that of the polar carboxyl head. When incorporated in bilayers, these molecules promote a hexagonal negative curvature (Cullis and Kruijff 1979; Epand et al. 1991; Perozo et al. 2002). We hypothesize that a conformational change of the -subunit, as a result of membrane deformation induced by AA and other unsaturated fatty acids, may prevent the cytosolic inactivation ball from reaching its target.

Our findings may have implications in certain pathophysiological conditions. In the brain, the level of free AA and other fatty acids dramatically increases as a result of increased phospholipase A2 activity in ischemia/reperfusion (Lipton 1999; Phillis and O'Regan 2004). First, it should be noted that the concentrations of AA used are not outside the range of AA concentrations that can exist in the brain during such pathophysiologic conditions as stroke and ischemia (Lipton 1999). In addition, the action of such free fatty acids may not be based on a singular interaction with one fatty acid in such conditions. Indeed, we show in this work that small concentrations of more than one fatty acid may lead to major changes that amplify the effect of each alone. Because 1) a high BK channel density is detected in the cortex and at the axonal terminals (Knaus et al. 1996; Wanner et al. 1999; Weiger et al. 2000) and 2) BK channels in cortical neurons exhibit inactivation (Smith and Ashford 2000; Sun et al. 2003), an increased AA and other unsaturated fatty acids during ischemia/reperfusion may alter K⁺ efflux and release of neurotransmitters such as glutamate by activating BK channels in neocortical neurons. However, free reactive oxygen radicals from oxidative metabolism of AA can also exert opposite effects on BK channels (Tang et al. 2004), especially during reoxygenation. Therefore it is possible that AA may have opposing effects, and additional experiments are needed in this area to decipher the temporal results induced by AA and its metabolism in pathophysiological conditions.

In summary, we discovered that -dependent BK channel inactivation is a specific molecular target for AA and other unsaturated fatty acids. The pronounced slowing of inactivation kinetics may be a specific mechanism by which AA and unsaturated fatty acids modulate K⁺ efflux, neuronal excitability, and neurotransmitter release, and thus influence neuronal death/survival in various neuropathological conditions.

	GRANTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION GRANTS ACKNOWLEDGMENTS REFERENCES

 
This work was supported by National Institute of Neurological Diseases and Strok Grant 1P01 NS-42202.

	ACKNOWLEDGMENTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION GRANTS ACKNOWLEDGMENTS REFERENCES

 
We appreciate the technical assistance of O. Gavrialov and Drs. X. Gu and V. Verselis. The 4-subunit was provided by Dr. L. Toro and 2 and 3 were obtained from Dr. C. Lingle.

	FOOTNOTES

 
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Address for reprint requests and other correspondence: G. G. Haddad, Dept. of Pediatrics, Univ. of California San Diego, 9500 Gilman Dr., La Jolla, CA 92037-0735 (E-mail: ghaddad{at}ucsd.edu)

	REFERENCES

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION GRANTS ACKNOWLEDGMENTS REFERENCES

 
Arai K, Ikegaya Y, Nakatani Y, Kudo I, Nishiyama N, Matsuki N. Phospholipase A2 mediates ischemic injury in the hippocampus: a regional difference of neuronal vulnerability. Eur J Neurosci 13: 2319–2323, 2001.[CrossRef][ISI][Medline]

Berger GE, Smesny S, Ammigner GP. Bioactive lipids in schizophrenia. Int Rev Psychiatry 18: 85–98, 2006.[CrossRef][ISI][Medline]

Bois TM, Deng C, Huan X-F. Membrane phospholipid composition, alterations in neurotransmitter systems and schizophrenia. Prog Neuropsychopharmacol Biol Psychiatry 29: 878–888, 2005.[CrossRef][Medline]

Brenner R, Jegla TJ, Wickenden A, Liu Y, Aldrich RW. Cloning and functional characterization of novel large conductance calcium-activated potassium channel beta subunits, hKCNMB3 and hKCNMB4. J Biol Chem 275: 6453–6461, 2000.[Abstract/Free Full Text]

Bringmann A, Skatchkov SN, Biedermann B, Faude F, Reichenbach A. Alterations of potassium channel activity in retinal Muller glial cells induced by arachidonic acid. Neuroscience 86: 1291–1306, 1998.[CrossRef][ISI][Medline]

Clarke AL, Petrou S, Walsh JV Jr, Singer JJ. Modulation of BK(Ca) channel activity by fatty acids: structural requirements and mechanism of action. Am J Physiol Cell Physiol 283: C1441–C1453, 2002.[Abstract/Free Full Text]

Cullis PR, De Kruijff B. Lipid polymorphism and the functional roles of lipids in biological membranes. Biochim Biophys Acta 559: 399–420, 1979.[Medline]

Denson DD, Wang X, Worrell RT, Eaton DC. Effects of fatty acids on BK channels in GH3 cells. Am J Physiol Cell Physiol 279: C1211–C1219, 2000.[Abstract/Free Full Text]

Epand RM, Epand RF, Ahmed N, Chen R. Promotion of hexagonal phase formation and lipid mixing by fatty acids with varying degrees of unsaturation. Chem Phys Lipids 57: 75–80, 1991.[CrossRef][ISI][Medline]

Gribkoff VK, Starrett JE Jr, Dworetzky SI, Hewawasam P, Boissard CG, Cook DA, Frantz SW, Heman K, Hibbard JR, Huston K, Johnson G, Krishnan BS, Kinney GG, Lombardo LA, Meanwell NA, Molinoff PB, Myers RA, Moon SL, Ortiz A, Pajor L, Pieschl RL, Post-Munson DJ, Signor LJ, Srinivas N, Taber MT, Thalody G, Trojnacki JT, Wiener H, Yeleswaram K, Yeola SW. Targeting acute ischemic stroke with a calcium-sensitive opener of maxi-K potassium channels. Nat Med 7: 471–477, 2001.[CrossRef][ISI][Medline]

Hagmann W, Kagawa D, Renaud C, Honn KV. Activity and protein distribution of 12-lipoxygenase in HEL cells: induction of membrane-association by phorbol ester TPA, modulation of activity by glutathione and 13-HPODE, and Ca²⁺-dependent translocation to membranes. Prostaglandins 46: 471–477, 1993.[CrossRef][ISI][Medline]

Hu H, Shao LR, Chavoshy S, Gu N, Trieb M, Behrens R, Laake P, Pongs O, Knaus HG, Ottersen OP, Storm JF. Presynaptic Ca²⁺-activated K⁺ channels in glutamatergic hippocampal terminals and their role in spike repolarization and regulation of transmitter release. J Neurosci 21: 9585–9597, 2001.[Abstract/Free Full Text]

Knaus HG, Folander K, Garcia-Calvo M, Garcia ML, Kaczorowski GJ, Smith M, Swanson R. Primary sequence and immunological characterization of beta-subunit of high conductance Ca²⁺-activated K⁺ channel from smooth muscle. J Biol Chem 269: 17274–17278, 1994.[Abstract/Free Full Text]

Knaus HG, Schwarzer C, Koch RO, Eberhart A, Kaczorowski GJ, Glossmann H, Wunder F, Pongs O, Garcia ML, Sperk G. Distribution of high-conductance Ca²⁺-activated K⁺ channels in rat brain: targeting to axons and nerve terminals. J Neurosci 16: 955–963, 1996.[Abstract/Free Full Text]

Lang F, Foller M, Lang KS, Lang PA, Ritter M, Gulbins E, Vereninov A, Huber SM. Ion channels in cell proliferation and apoptotic cell death. J Membr Biol 205: 147–157, 2005.[CrossRef][ISI][Medline]

Lauritzen I, Blondeau N, Haurteaux C, Widmann C, Romey G, Lazdunski M. Polyunsaturated fatty acids are potent neuroprotectors. EMBO J 19: 1784–1793, 2000.[CrossRef][ISI][Medline]

Lipton P. Ischemic cell death in brain neurons. Physiol Rev 79: 1431–1568, 1999.[Abstract/Free Full Text]

Markesbery WR. Oxidative stress hypothesis in Alzheimer's disease. Free Radic Biol Med 23: 134–147, 1997.[CrossRef][ISI][Medline]

Meera P, Wallner M, Toro L. A neuronal beta subunit (KCNMB4) makes the large conductance, voltage- and Ca²⁺-activated K⁺ channel resistant to charybdotoxin and iberiotoxin. Proc Natl Acad Sci USA 97: 5562–5567, 2000.[Abstract/Free Full Text]

Meves H. Modulation of ion channels by arachidonic acid. Prog Neurobiol 43: 175–186, 1994.[CrossRef][ISI][Medline]

Nogawa S, Zhang F, Ross ME, Iadecola C. Cyclo-oxygenase-2 gene expression in neurons contributes to ischemic brain damage. J Neurosci 17: 2746–2755, 1997.[Abstract/Free Full Text]

Perozo E, Kloda A, Cortes DM, Martinac B. Physical principles underlying the transduction of bilayer deformation forces during mechanosensitive channel gating. Nat Struct Biol 9: 696–703, 2002.[CrossRef][ISI][Medline]

Phillis JW, O'Regan MH. A potentially critical role of phospholipases in central nervous system ischemic, traumatic, and neurodegenerative disorders. Brain Res Rev 44: 13–47, 2004.[CrossRef][Medline]

Raffaelli G, Saviane C, Mohajerani MH, Pedarzani P, Cherubini E. BK potassium channels control transmitter release at CA3-CA3 synapses in the rat hippocampus. J Physiol 557: 147–157, 2004.[Abstract/Free Full Text]

Rouzer CA, Kargman S. Translocation of 5-lipoxygenase to the membrane in human leukocytes challenged with ionophore A23187. J Biol Chem 263: 10980–10988, 1988.[Abstract/Free Full Text]

Runden-Pran E, Haug FM, Storm JF, Ottersen OP. BK channel activity determines the extent of cell degeneration after oxygen and glucose deprivation: a study in organotypical hippocampal slice cultures. Neuroscience 112: 277–288, 2002.[CrossRef][ISI][Medline]

Sah P. Ca²⁺-activated K⁺ currents in neurons: types, physiological roles and modulation. Trends Neurosci 19: 150–154, 1996.[CrossRef][ISI][Medline]

Smith MA, Ashford MLJ. Inactivation of large-conductance, calcium-activated potassium channels in rat cortical neurons. Neuroscience 95: 33–50, 2000.[CrossRef][ISI][Medline]

Solaro CR, Lingle CJ. Trypsin-sensitive, rapid inactivation of a calcium-activated potassium channel. Science 25: 1694–1698, 1992.

Strokin M, Chechneva O, Reymann KG, Reiser G. Neuroprotection of rat hippocampal slices exposed to oxygen-glucose deprivation by enrichment with docosahexaenoic acid and by inhibition of hydrolysis of docosahexaenoic acid-containing phospholipids by calcium independent phospholipase A2. Neuroscience 140: 547–553, 2006.[CrossRef][ISI][Medline]

Sun X, Gu XQ, Haddad GG. Calcium influx via L- and N-type calcium channels activates a transient large-conductance Ca²⁺-activated K⁺ current in mouse neocortical pyramidal neurons. J Neurosci 23: 3639–3648, 2003.[Abstract/Free Full Text]

Tang XD, Garcia ML, Heinemann SH, Hoshi T. Reactive oxygen species impair Slo1 BK channel function by altering cysteine-mediated calcium sensing. Nat Struct Mol Biol 11: 171–178, 2004.[CrossRef][ISI][Medline]

Uebele VN, Lagrutta A, Wade T, Figueroa DJ, Liu Y, McKenna E, Austin CP, Bennett PB, Swanson R. Cloning and functional expression of two families of beta-subunits of the large conductance calcium-activated K⁺ channel. J Biol Chem 275: 23211–23218, 2000.[Abstract/Free Full Text]

Wallner M, Meera P, Toro L. Molecular basis of fast inactivation in voltage and Ca²⁺-activated K⁺ channels: a transmembrane beta-subunit homolog. Proc Natl Acad Sci USA 96: 4137–4142, 1999.[Abstract/Free Full Text]

Wanner SG, Koch RO, Koschak A, Trieb M, Garcia ML, Kaczorowski GJ, Knaus HG. High-conductance calcium-activated potassium channels in rat brain: pharmacology, distribution, and subunit composition. Biochemistry 38: 5392–5400, 1999.[CrossRef][Medline]

Weiger TM, Holmqvist MH, Levitan IB, Clark FT, Sprague S, Huang WJ, Ge P, Wang C, Lawson D, Jurman ME, Glucksmann MA, Silos-Santiago I, DiStefano PS, Curtis R. A novel nervous system beta subunit that downregulates human large conductance calcium-dependent potassium channels. J Neurosci 20: 3563–3570, 2000.[Abstract/Free Full Text]

Xia XM, Ding JP, Zeng XH, Duan KL, Lingle CJ. Rectification and rapid activation at low Ca²⁺ of Ca²⁺-activated, voltage-dependent BK currents: consequences of rapid inactivation by a novel beta subunit. J Neurosci 20: 4890–4903, 2000.[Abstract/Free Full Text]

This article has been cited by other articles:

				P. Zhang, C. Yang, and R. J. Delay Urine Stimulation Activates BK Channels in Mouse Vomeronasal Neurons J Neurophysiol, October 1, 2008; 100(4): 1824 - 1834. [Abstract] [Full Text] [PDF]

				T. Vaithianathan, A. Bukiya, J. Liu, P. Liu, M. Asuncion-Chin, Z. Fan, and A. Dopico Direct Regulation of BK Channels by Phosphatidylinositol 4,5-Bisphosphate as a Novel Signaling Pathway J. Gen. Physiol., July 1, 2008; 132(1): 13 - 28. [Abstract] [Full Text] [PDF]