©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Concomitant Acceleration of the Activation and Inactivation Kinetics of the Human Delayed Rectifier K Channel (Kv1.1) by Ca-independent Phospholipase A(*)

(Received for publication, November 4, 1994; and in revised form, December 13, 1994)

Rose A. Gubitosi-Klug (1) Shan Ping Yu (4) Dennis W. Choi (4) Richard W. Gross (1) (2) (3)(§)

From the  (1)Division of Bioorganic Chemistry and Molecular Pharmacology, Departments of Medicine, (2)Chemistry, (3)Molecular Biology & Pharmacology, and (4)Neurology, Washington University School of Medicine, St. Louis, Missouri 63110

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

The electrophysiologic sequelae of arachidonic acid release mediated by the major phospholipase A(2) (PLA(2)) in electrically active tissues (i.e. the 40-kDa Ca-independent PLA(2)) were assessed in Sf9 cells expressing the human recombinant delayed rectifier K channel Kv1.1. Intracellular administration of Ca-independent PLA(2) increased the rate of activation of the macroscopic current (from = 6.25 ± 0.76 ms to


INTRODUCTION

Ca-independent PLA(2)(^1)is the predominant phospholipase activity in two prominent electrically active organs, heart and brain(1, 2, 3) . The specialized membrane compartments mediating electrical activity in these tissues are highly enriched in phospholipids containing arachidonic acid esterified at the sn-2 position(4, 5, 6) . Since a variety of eicosanoid metabolites have been implicated in ion channel regulation(7, 8, 9, 10) , Ca-independent PLA(2) has assumed an as yet unproven role as an enzymic modulator of ion channel function. More recently, arachidonic acid per se has been implicated in the regulation of ion channel function(11, 12, 13, 14, 15, 16, 17) , and the effects of unoxidized arachidonic acid in these systems are believed to be the direct result of the interaction of arachidonic acid with target proteins. However, these phenomena have been explored in cells that extensively oxidize arachidonic acid, and the importance of arachidonic acid itself as an effector has only been implied indirectly through pharmacologic inhibition of arachidonic acid oxidation. Furthermore, the interaction of exogenously administered arachidonic acid (existing as polydisperse micelles in aqueous solution) with membranes is complex, and in no case has the in situ generation of arachidonic acid by an intracellular phospholipase been shown to modulate ion channel function.

To clarify the role of arachidonic acid per se on ion channel function, we compared effects of either exogenously administered arachidonic acid or endogenously generated arachidonic acid (released by the major intracellular PLA(2) in brain and heart (i.e. Ca-independent PLA(2)(2, 3) ) on human recombinant neuronal delayed rectifier K channel, Kv1.1 (HuKI) (18) expressed in Sf9 cells, which do not produce oxidized eicosanoid metabolites. We now report that either in situ generation of arachidonic acid by intracellular administration of Ca-independent PLA(2) or exogenous application of arachidonic acid (but not other fatty acids) results in profound increases in the kinetics of Kv1.1 macroscopic current activation and inactivation.


MATERIALS AND METHODS

Expression of Kv1.1

cDNA encoding the Kv1.1 was subcloned into the baculovirus transfer plasmid, pVL1393, which was inserted through homologous recombination into wild-type baculovirus genome to produce the desired recombinant K channel virus. An aliquot of the purified recombinant virus was kindly provided by Dr. Alexander Kamb(18, 19) . Cells were infected for 40 h at a multiplicity of infection = 6 for recordings.

Electrophysiologic Measurements in Sf9 Cells

Whole-cell voltage clamp recordings of Sf9 cells were performed utilizing similar conditions and techniques to those previously described(19, 20) . Data were acquired, stored, and analyzed utilizing the Macintosh-based software Pulse/Pulsefit. Activation and inactivation time constants were determined through best fit analysis of monoexponential equations. The indicated fatty acids were dissolved in Me(2)SO and diluted into extracellular solution to a final concentration, unless otherwise stated, of 20 µM, with a final Me(2)SO concentration of less than 0.1%.

Metabolism of Arachidonic Acid in Sf9 Cells

The metabolism of arachidonic acid in Sf9 cells was examined by incubating Sf9 cells with 1 µCi of [5,6,8,9,11,12,14,15-^3H]arachidonic acid (DuPont NEN) in Me(2)SO for 1, 5, 10, or 60 min. Incubations were terminated by the addition of acidified methanol, and metabolites were extracted utilizing a modified Bligh and Dyer extraction (acidified to pH 4.5) (21) . After aliquots of the CHCl(3) extracts were applied to Whatman Silica Gel 60 TLC plates (Whatman), metabolites were resolved utilizing a mobile phase comprised of chloroform/methanol/acetic acid/water (90/8/1/0.8, v/v) and were subsequently visualized by fluorography(22) .

Administration of Ca-independent Phospholipase A(2)

First, the endogenous arachidonic acid content of Sf9 cells was enhanced by supplementing medium with 5 µM arachidonic acid for 24 h prior to baculovirus infection as well as during the 40-h infection interval. Confirmation of an increase in arachidonic acid-containing phospholipids was obtained by electrospray mass spectroscopy of a Bligh and Dyer phospholipid extract of aliquots of the Sf9 cells just prior to recording(23) . Unincorporated fatty acid was removed by exhaustive washing with Grace's insect media containing 1 mg/ml bovine serum albumin (Cohn Fraction V, Sigma). Ca-independent PLA(2) (purified from canine myocardium through the affinity chromatography step as described previously(2) ) was diluted 1:40 into intracellular or extracellular recording solution just prior to the experiments. The bromoenol lactone suicide substrate (E)-6-(bromomethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran-2-one (BEL) was prepared and purified as described previously(24) , reconstituted in ethanol, and added to a vial containing purified Ca-independent PLA(2) at a final concentration of 10 µM. The BEL and Ca-independent PLA(2) mixture was incubated for 5 min on ice and then diluted into intracellular recording solution.


RESULTS

Electrophysiologic Characteristics of Kv1.1 Expressed in Sf9 Cells

Expression of the human brain cDNA sequence encoding the delayed rectifier K channel Kv1.1 in Sf9 cells resulted in the appearance of a tetraethylammonium-sensitive, slowly inactivating, K-selective, voltage-dependent current, which was not present in uninfected cells (compare A to B in Fig. 1). Routinely, maximum currents of 1.91 ± 0.54 nA were induced by a 120-mV depolarizing step (from -80 mV to +40 mV). The observed currents were stable for >1 h.


Figure 1: Bidirectional modulation of Kv1.1 macroscopic current by arachidonic acid (AA). Whole-cell, voltage clamp recordings with depolarizing voltage steps from -40 to +40 mV (20-mV increments) from a holding potential of -80 mV were performed on uninfected (A) and infected (B-G) Sf9 cells expressing Kv1.1. A, uninfected Sf9 cell recording; B, Kv1.1-infected Sf9 cell recording; C, macroscopic current 2 min postapplication of arachidonic acid (20 µM); D, macroscopic current 5 min postapplication of arachidonic acid (20 µM). Noticeably, arachidonic acid application results in inactivation ( = 76.6 ± 1.4 ms at +40 mV, n = 8). E, macroscopic current 5 min after perfusion with albumin-containing extracellular solution; F, comparison of activation phase and activation rate constants of Kv1.1 macroscopic current at +40 mV before ( = 5.73 ± 0.88 ms, n = 8) and after ( = 1.91 ± 0.39 ms, n = 8) arachidonic acid application. The overall decrease in the time constant of activation was statistically significant (p < 0.005). G, Kv1.1 current-voltage relationship prior to arachidonic acid treatment (circle), peak current-voltage relationship 5 min after application of arachidonic acid (bullet), and steady-state current-voltage relationship 5 min after application of arachidonic acid (times). Leak subtraction of current traces was performed prior to analysis. Errorbars are within symbol boundaries if not seen. Data are representative of eight independent experiments.



Arachidonic Acid-induced Bidirectional Alterations in Kv1.1 Macroscopic Current

Superfusion of Sf9 cells with buffer containing arachidonic acid (5-20 µM) resulted in several prominent electrophysiologic effects including: 1) an increase in rate of activation of the macroscopic voltage-dependent current (Fig. 1, B-D, and superposition of pre- and postarachidonic acid application in Fig. 1F); 2) an increase in rate of inactivation of the macroscopic current (Fig. 1, B-D); and 3) a decrease in peak and steady-state K current (compare B to D in Fig. 1; also Fig. 1G). Iterative application of arachidonic acid (every 10 min) and subsequent washout resulted in sequential alterations in electrophysiologic properties and their subsequent return to near control values (Fig. 1E). Application of arachidonic acid did not shift the range of voltage sensitivity of Kv1.1 (Fig. 1G).

Structure-Activity Relationships of Fatty Acid-mediated Alterations in Kv1.1 Macroscopic Currents

Perfusion of cells with radiolabeled arachidonic acid resulted in the incorporation of arachidonic acid into cellular phospholipids without evidence of oxidized arachidonic acid metabolites (Fig. 2A). This result is in agreement with previous results demonstrating that arachidonic acid was not oxidized into eicosanoid metabolites in Sf9 cells(25) . Thus, the observed effects were mediated by arachidonic acid and not its oxidized metabolites.


Figure 2: Fatty acid selectivity of the bidirectional modulation of Kv1.1 macroscopic current. A, chloroform extracts of Sf9 cells incubated with [^3H]arachidonic acid for 1 min (lane1), 5 min (lane2), 10 min (lane3), and 60 min (lane4) were applied to Whatman Silica Gel 60 plates and developed in chloroform/methanol/acetic acid/water (90:8:1: 0.8), and radiolabeled metabolites were visualized by fluorography. AA, 12-HETE, 5-HETE, TxB(2), and PL represent the migration positions of arachidonic acid, 12-hydroxyeicosa-5,8,10,14-tetraenoic acid, 5-hydroxyeicosa-6,8,11,14-tetraenoic acid, thromboxane B(2), and phospholipids, respectively. SF and O denote the solvent front and origin, respectively. B, macroscopic Kv1.1 current recordings performed before (-) and 5 min after (+) application of 20 µM docosahexaenoic acid, 15 µM 5-HETE, 20 µM eicosa-5,8,11-trienoic acid, 20 µM eicosa-8,11,14-trienoic acid, and 20 µM methyl arachidonate (using the voltage step paradigm described in Fig. 1). These data are representative of four independent experiments for each fatty acid or fatty acid metabolite.



The bidirectional effects of arachidonic acid on K channel function were reproducibly observed (>15 preparations) but were not seen after perfusion with 20-50 µM palmitic acid, oleic acid (data not shown), and docosahexaenoic acid (Fig. 2B). Furthermore, 5-hydroxyeicosa-6,8,11,14-tetraenoic acid (5-HETE) did not alter the rate of activation or inactivation of Kv1.1 macroscopic current (Fig. 2B). Exposure of cells to eicosa-5,8,11-trienoic acid resulted in a modest increase of the rate of activation and the peak macroscopic current but had no effects on inactivation even at concentrations up to 50 µM. In contrast, eicosa-8,11,14-trienoic acid neither activates nor inactivates Kv1.1 macroscopic currents (Fig. 2B). Finally, application of the methyl ester of arachidonic acid altered neither the activation nor the inactivation of the Kv1.1 current (Fig. 2B).

Ca-independent PLA(2)-mediated Bidirectional Alterations in Kv1.1 Macroscopic Current

To compare the electrophysiologic effects of exogenously applied arachidonic acid (presented to cells as polydisperse micelles) to those of arachidonic acid generated in situ, we studied the effects of Ca-independent PLA(2), the major intracellular phospholipase activity present in two prominent electrically active tissues (heart and brain) on Kv1.1 channel function. Initial experiments demonstrated that intracellular application of PLA(2) did not result in electrophysiologic alterations in Kv1.1 macroscopic current kinetics in cells grown in routine culture medium (see nonsupplemented column, Table 1). Since prior experiments demonstrated the obligatory requirement of arachidonic acid for Kv1.1 modulation (see above), we examined the endogenous fatty acid content of Sf9 cells and demonstrated that Sf9 cells cultured under routine conditions do not contain substantial amounts of arachidonic acid in their choline and ethanolamine glycerophospholipid pools (Fig. 3). Accordingly, we augmented the endogenous arachidonic acid content of Sf9 cells by supplementing the growth medium with 5 µM arachidonic acid. The addition of this small amount of exogenous arachidonic acid resulted in a substantial increase in the endogenous content of arachidonic acid in the major phospholipid classes as determined by electrospray ionization mass spectrometry (e.g. phosphatidylethanolamine (m/z 767), phosphatidylinositol (m/z 886), and phosphatidylcholine (m/z 831 and 833), which each contain arachidonic acid at their sn-2 position) (Fig. 3). Modulation of the individual molecular species present in Sf9 cells did not alter the basal electrophysiologic function of Kv1.1 (Table 1). Intracellular administration of Ca-independent PLA(2) to Sf9 cells previously cultured in media supplemented with arachidonic acid (and exhaustively washed with albumin solution prior to these experiments) resulted in a substantial increase in the rates of activation and inactivation of Kv1.1 (Table 1). Importantly, the increase in the rate of activation and inactivation mediated by highly purified myocardial Ca-independent PLA(2) was blocked by pretreatment of enzyme with the mechanism-based inhibitor, BEL (Table 1). No alteration in the macroscopic current was elicited by BEL alone or was observed in control experiments utilizing buffer alone (data not shown). Extracellular application of Ca-independent PLA(2) to Sf9 cells neither resulted in changes in macroscopic current kinetics nor the measurable release of [^3H]arachidonic acid from Sf9 cells prelabeled with [^3H]arachidonic acid for 48 h (data not shown). Collectively, these results demonstrate that alterations in Kv1.1 electrophysiologic function are induced by the intracellular administration (but not the extracellular application) of cytosolic Ca-independent PLA(2), which catalyzes the hydrolysis of endogenous membrane phospholipids leading to the in situ generation of arachidonic acid in electrically active membranes.




Figure 3: Electrospray mass spectroscopy of Sf9 cellular phospholipids. Electrospray mass spectroscopy was performed on chloroform extracts of Sf9 cells grown in standard Sf9 cell culture media (Control) and chloroform extracts of Sf9 cells grown in standard media supplemented with 5 µM arachidonic acid (+AA). Traces are shown for each phospholipid class: phosphatidylethanolamine (PE), phosphatidylinositol (PI), and phosphatidylcholine (PC). * denotes arachidonic acid-containing phospholipids within each class. Phosphatidylethanolamine molecular species are: m/z 689, 14:0-18:1; m/z 717, 16:0-18:1; m/z 743, 18:1-18:1 and 18:0-18:2; m/z 767, 18:0-20:4. Phosphatidylinositol molecular species are: m/z 836, 16:0-18:1; m/z 864, 18:0-18:1; m/z 886, 18:0-20:4. Phosphatidylcholine molecular species are: m/z 781, 16:0-18:2; m/z 783, 16:0-18:1; m/z 809, 18:1-18:1; m/z 831, 18:1-20:4; m/z 833, 18:0-20:4.




DISCUSSION

Electrophysiologic characterization of the effects of Ca-independent PLA(2) on the properties of cloned ion channels expressed in Sf9 cells possesses several inherent advantages, including: 1) released arachidonic acid is not oxidatively metabolized into eicosanoid metabolites; 2) K channel expression occurs in the presence of a null electrophysiologic background (i.e. Sf9 cells do not contain intrinsic voltage-activated channels); and 3) the lipid environment surrounding the ion channel can be easily manipulated by appropriate modification of culture medium. The results of the present study establish that PLA(2)-mediated arachidonic acid release results in the potent, specific, and reversible bidirectional modulation of transmembrane ion flux mediated by the delayed rectifier K channel, Kv1.1.

One prominent feature of electrically active membranes is their substantial enrichment in arachidonic acid. Herein, we demonstrate that the action of the major PLA(2) activity present in excitable cells results in the direct modulation of K channel macroscopic currents mediated through the release of arachidonic acid and its subsequent rapid lateral diffusion in the plane of the membrane, thereby facilitating its direct interaction with ion channel proteins. Thus, a potent modulator of ion channel function (i.e. non-esterified arachidonic acid) resides latent in the appropriate electrically active subcellular membrane compartment, awaiting activation by esterolytic cleavage of the parent phospholipid at the sn-2 position catalyzed by PLA(2). This stands in contrast to traditional paradigms of arachidonic acid-mediated cellular activation, which first require its translocation to specific intracellular compartments followed by subsequent oxidation and a second translocation step of the oxidized eicosanoid metabolite across the plasma membrane where it can finally interact with an exofacial plasma membrane receptor. Through the direct and proximal interaction of the released arachidonic acid with a potent biologic effector (e.g. a K channel and perhaps other plasma membrane proteins), the rapid, specific, and efficient delivery of a lipid second messenger can be effected.

Prior studies have either stressed the importance of oxygenated metabolites of arachidonic acid as modulators of K channel function (7, 8, 10) or alternatively have concluded that many different molecular species of fatty acids modulate channel function irrespective of the number or the regiospecificity of the olefinic linkages present (11, 12, 13, 17) . For example, Honore et al.(17) demonstrated that application of either arachidonic acid or docosahexaenoic acid to Chinese hamster ovary cells expressing Kv1.5 resulted in identical electrophysiologic alterations in macroscopic current. The effect of these fatty acids as described by Honore et al.(17) on Kv1.5 is similar to the effect we observe for arachidonic acid on Kv1.1. However, the application of docosahexaenoic acid does not alter Kv1.1 function. One, but not the only possible explanation underlying these dissimilarities, is that there are differences in the interaction of arachidonic acid with K channel proteins comprised of distinct (yet homologous) primary sequences.

The results in this simplified reconstituted system underscore the importance of arachidonic acid, and not other fatty acids or oxidized metabolites, as the biologic effector of the Kv1.1 channel. Comparison of the structure-activity relationship of arachidonic acid to that of its cognate methyl ester on K channel function demonstrates that ablation of the negative charge on the carboxylate terminus completely attenuates the modulatory effects of arachidonic acid on K channel activation and inactivation. The acceleration of activation mediated by eicosa-5,8,11-trienoic acid, but not eicosa-8,11,14-trienoic acid, in conjunction with the fact that neither eicosatrienoic acid induces inactivation further underscores the chemical specificity of the observed interactions. Collectively, these results demonstrate the obligatory structural requirements of both the extent and extant of olefinic linkages in the hydrophobic region and the carboxylate functionality in facilitating the bidirectional functional modulation of Kv1.1. The simplest, but not the only, explanation is the direct interaction of the arachidonic acid (either in situ generated or exogenously supplied) with Kv1.1 protein itself. Yet, we cannot rule out the possibility that arachidonic acid acts indirectly through one or more K chan nel regulatory proteins.

The functional characteristics of the delayed rectifier K channel are one of the primary determinants of many cells' membrane excitability(26, 27) . Since in situ generation of arachidonic acid by Ca-independent PLA(2) induces substantive changes in activation and inactivation time constants of the delayed rectifier Kv1.1 K channel and recent studies localize Kv1.1 to axons and synaptic terminals(28) , the potential sequelae of PLA(2) hydrolysis of electrically active membranes includes alterations in action potential characteristics, calcium transients, neurotransmitter release, and refractory period. The marked increase in the rate of neuronal K channel macroscopic current activation represents a heretofore undescribed consequence of activation of Ca-independent PLA(2), which may modulate the complex spatiotemporal integration of electrical and biochemical signals at the synaptic membrane.


FOOTNOTES

*
This research was supported by National Institutes of Health Grant 41250. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Division of Bioorganic Chemistry and Molecular Pharmacology, Washington University School of Medicine, 660 South Euclid, Box 8020, St. Louis, MO 63110. Tel.: 314-362-2690; Fax: 314-362-1402.

(^1)
The abbreviations used are: PLA(2), phospholipase A(2); BEL, (E)-6(bromomethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran-2-one; 5-HETE = 5-hydroxyeicosa-6,8,11,14-tetraenoic acid; HuKI, human brain potassium channel Kv1.1; Me(2)SO, dimethyl sulfoxide; Sf9, Spodoptera frugiperda insect cell line; , time constant of activation; , time constant of inactivation.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.