(Received for publication, November 4, 1994; and in revised form, December 13, 1994)
From the
The electrophysiologic sequelae of arachidonic acid release
mediated by the major phospholipase A (PLA
) in
electrically active tissues (i.e. the 40-kDa
Ca
-independent PLA
) were assessed in Sf9
cells expressing the human recombinant delayed rectifier K
channel Kv1.1. Intracellular administration of
Ca
-independent PLA
increased the rate of
activation of the macroscopic current (from
= 6.25 ± 0.76 ms to
Ca-independent PLA
(
)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
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 in brain and heart (i.e. Ca
-independent PLA
(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
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.
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 (
), peak current-voltage relationship 5 min after
application of arachidonic acid (
), and steady-state
current-voltage relationship 5 min after application of arachidonic
acid (
). 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.
Figure 2:
Fatty acid selectivity of the
bidirectional modulation of Kv1.1 macroscopic current. A, chloroform extracts of Sf9 cells incubated with
[H]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
, 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
,
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).
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.
Electrophysiologic characterization of the effects of
Ca-independent PLA
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
-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 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
. 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
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
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
, which
may modulate the complex spatiotemporal integration of electrical and
biochemical signals at the synaptic membrane.