1 Pharmakologisches Institut, Universität Wien, A-1090 Vienna, Austria; and 2 Instituto de la Grasa, Consejo Superior de Investigaciones Cientificas, E-41012 Seville, Spain
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ABSTRACT |
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We tested the effects of membrane phospholipids on the function of high-conductance, Ca2+-activated K+ channels from the basolateral cell membrane of rabbit distal colon epithelium by reconstituting these channels into planar bilayers consisting of different 1:1 mixtures of phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylserine (PS), and phosphatidylinositol (PI). At low ambient K+ concentrations single-channel conductance is higher in PE/PS and PE/PI bilayers than in PE/PC bilayers. At high K+ concentrations this difference in channel conductance is abolished. Introducing the negatively charged SDS into PE/PC bilayers increases channel conductance, whereas the positively charged dodecyltrimethylammonium has the opposite effect. All these findings are consistent with modulation of channel current by the charge of the lipid membrane surrounding the channel. But the K+ that permeates the channel senses only a small fraction of the full membrane surface potential of the charged phospholipid bilayers, equivalent to separation of the conduction pathway from the charged phospholipid head groups by 20 Å. This distance appears to insulate the channel entrance from the bilayer surface potential, suggesting large dimensions of the channel-forming protein. In addition, in PE/PC and PE/PI bilayers, but not in PE/PS bilayers, the open-state probability of the channel decreases with time ("channel rundown"), indicating that phospholipid properties other than surface charge are required to maintain channel fluctuations.
rabbit colon epithelium; surface charge; cell membrane composition
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INTRODUCTION |
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ION CHANNELS IN BIOLOGICAL membranes are embedded in a lipid double layer consisting of phospholipids, glycolipids, and cholesterol, the phospholipids being the most abundant. The membrane of each cell organelle contains not only specific proteins but also a unique lipid composition. Having different lipid molecules in its membranes most likely is of functional significance for the cell (34).
The present report addresses the effects of membrane phospholipid
composition on the activity of high-conductance
K+ channels from the basolateral
cell membrane of rabbit colon epithelium. These channels are activated
by intracellular Ca2+ and membrane
depolarization. They are highly selective for
K+ over
Na+ and
Cl; are inhibited by
Ba2+ and the scorpion toxin,
charybdotoxin (17, 32); and appear to be involved in basolateral
K+ recycling in the course of
transepithelial Na+ absorption
(33). Using the technique of ion channel reconstitution in planar lipid
bilayers, we studied single-channel activity directly in bilayers of
well-defined lipid composition. In this regard, artificial membranes
provide a system less ambiguous than that provided by the patch-clamp
method. Evidence that membrane phospholipids affect the conductance and
gating of colonic high-conductance, Ca2+-activated
K+
(BKCa) channels is presented.
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MATERIALS AND METHODS |
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Plasma membrane preparation.
Basolateral plasma membrane vesicles (BLMV) were prepared from surface,
i.e., Na+-absorbing, epithelial
cells of rabbit distal colon as described by Wiener et al. (39).
Briefly, after homogenization of mucosal scrapings, the subcellular
particles were fractionated by sucrose density gradients and Ficoll-400
barrier centrifugation. The BLMV preparation was highly enriched in
Na+-K+-ATPase
activity, a recognized enzyme marker for basolateral membranes. The
isolated BLMV were suspended in 250 mM sucrose-10 mM HEPES-Tris, pH
7.2, divided into small aliquots, stored at 80°C, and thawed immediately prior to use.
Reconstitution of ion channels in planar lipid bilayers. The method for production of planar lipid bilayers was essentially that of Schindler (27). A small droplet (~0.5 µl) of the phospholipid solution was taken up into a 10-µl glass capillary pipette. After the inside of the glass capillary had been coated with the lipid by moving the piston of the pipette up and down several times, a bilayer was formed by applying an air bubble from the capillary pipette to the aperture (diameter 240 µm) of a Teflon septum (thickness 12 µm) mounted vertically between the two halves of a Teflon chamber. Three types of phospholipid solutions (25 mg of each component/ml decane) were used alternatively for bilayer formation: 1) phosphatidylethanolamine (PE) and phosphatidylcholine (PC), 2) PE and phosphatidylserine (PS), and 3) PE and phosphatidylinositol (PI).
The lipid bilayer separated the cis solution from the trans solution (1.5 ml each). The cis and trans solutions contained initially 150 and 5 mM KCl, respectively, both in 10 mM HEPES-Tris, pH 7.2, and 250 µM CaCl2. Ion channels were incorporated into the bilayer by flushing 9 µl of the BLMV suspension (containing ~0.5-1 µg protein) directly toward the bilayer from the cis side. In some experiments the free Ca2+ concentration in the bathing solutions was lowered to ~28 µM by the addition of a buffered (pH 7.2) K+-EGTA solution. Rapid mixing of added compounds was insured by using magnetic stirrers in both chamber halves. The solutions on the cis and trans sides of the bilayer were connected to a patch-clamp amplifier (EPC-7; List, Darmstadt, Germany) via 0.5 M KCl-agar bridges in series with Ag-AgCl electrodes. To shield from electromagnetic and mechanical interferences, the bilayer chamber and the head stage of the patch-clamp amplifier were placed in a Faraday cage mounted on an antivibration table. Vm, the "holding" voltage across the bilayer, is expressed as the electrical potential of the cytosolic (i.e., Ca2+-sensitive) side of the channel with respect to the extracellular side (ground). The electrical current across the bilayer was passed through an eight-pole low-pass Bessel filter and visualized on a storage oscilloscope.Acquisition and analysis of channel data. The unfiltered single-channel current was stored via a pulse code modulator on a commercial videocassette recorder (VCR). For analysis of channel activity with the program pCLAMP (Axon Instruments, Foster City, CA) on a personal computer, the data recorded on the VCR were filtered at 500 Hz and digitized at a frequency of 2,000 Hz by an analog-to-digital converter. At each holding voltage, channel activity for 30-40 s was analyzed. The channel current, Ic, at a given voltage was obtained from the mean of the current amplitude histogram. The open-state probability, Po, was defined as the fraction of total time the channel was conductive or open with the open-closed discriminator set at half the amplitude of the open current level.
Membrane lipid analysis. Total lipids were extracted from BLMV by the method of Folch and coworkers (8) and quantified gravimetrically with an electrobalance. The lipid classes were separated on thin, silica-coated quartz rods (Chromarod S) equipped with a TLC flame ionization detector (Iatroscan, Technical Marketing Associates, Mississauga, ON, Canada) as described previously (35).
Materials.
PE (bovine brain), PC
(L--diphytanoyl-lecithin), PS
(bovine brain), and PI (bovine liver) for bilayer work were obtained in
chloroform from Avanti Polar Lipids (Alabaster, AL) and stored at
80°C. The lipids for daily use were kept at
20°C. Decane was from Aldrich (Vienna, Austria), and KCl
Specpure was from Alfa Products, Johnson Matthey
(Vienna, Austria). SDS, dodecyltrimethylammonium (DDTMA)
bromide, and the lipid and phospholipid standards for lipid
analysis were purchased from Sigma Chemical. All other chemicals were
obtained from local suppliers.
Statistics. Results are given as means ± SD or SE. n represents the number of experiments. The statistical significance of a difference between means was calculated by the t-test. Linear and nonlinear regression analyses were performed by the least-squares method.
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RESULTS |
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Figures
1-3 (top) illustrate
activity tracings of single BKCa
channels from the basolateral membranes of rabbit colonocytes reconstituted in either PE/PC, PE/PI, or PE/PS bilayers under identical
ionic conditions (initial KCl concns: 150 and 5 mM for cis and
trans solutions, respectively; free
Ca2+ concn 28 µM). In PE/PC
bilayers, channel activity was characterized by a "rundown"
phenomenon, i.e., channel activity decreased with time so that current
fluctuations were almost absent 30 min after channel fusion with the
bilayer (Fig. 1). In PE/PI bilayers, channel rundown was slower (Fig.
2), but channel activity was stable in PE/PS bilayers (Fig. 3).
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Mean values of the voltage dependence of Po for channels reconstituted in PE/PC, PE/PI, or PE/PS bilayers are shown in Figs. 1-3 (bottom). As reported earlier (32), the colonic BKCa channel is voltage gated and Po increases with membrane depolarization. In PE/PC bilayers, Po at a given voltage decreased with time, the half-life being 6-8 min. In PE/PI bilayers, the half-life of channel rundown, 20-25 min, was longer than that in PE/PC bilayers, whereas the Po-Vm relation in PE/PS bilayers was practically unchanged with time.
One of the differences between the phospholipids used for bilayer production is that the zwitterionic PE and PC carry no net charge,1 whereas PI and PS have a net negative charge. Hence with PE/PI and PE/PS bilayers there is a negative electrostatic surface potential in the aqueous phase adjacent to the membrane, which causes local accumulation of cations. This attraction of positively charged particles is expected to be especially prominent for divalent cations such as Ca2+. Po of colonic BKCa channels is markedly dependent on the Ca2+ concentration (17, 32), but the sensitivity to Ca2+ varies considerably between channels. When we compare the Po-Vm relations immediately after channel fusion, the Po values for a given Vm in PE/PI and PE/PS bilayers are higher than that in PE/PC bilayers (Figs. 1-3). This finding is consistent with local attraction of Ca2+ to the surface of the charged bilayer, but the possibility that some channel rundown had already occurred in the PE/PC bilayers within the time span of the several minutes necessary to generate the initial Po-Vm relation cannot be excluded.
On closer inspection of the tracings of channel activity shown in Figs.
1-3, it was noted that the current amplitude was somewhat higher
in PE/PI and PE/PS bilayers than in PE/PC bilayers (these recordings
were all obtained at a
Vm of 10
mV). This phenomenon is more clearly apparent from the
Ic-Vm
relations of BKCa channels inserted in either PE/PC, PE/PI, or PE/PS bilayers (Fig.
4). For all three types of bilayers, the
relation between
Ic and
Vm was linear or
ohmic in the voltage range from the reversal potential, Erev, to +40 mV.
But the single-channel conductance,
Gc, obtained from
the slope of the
Ic-Vm
relation, in PE/PI and PE/PS bilayers was higher than that in PE/PC
bilayers. A summary of the values of
Gc and
Erev for all
BKCa channels reconstituted in the
three types of bilayers is given in Table
1. Mean
Gc values for
PE/PS and PE/PI membranes were significantly higher than that for PE/PC membranes, whereas
Erev values were
not
different.2
The difference between
Gc values for
PE/PS and PE/PI bilayers was not statistically significant.
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According to surface charge theory, the size of the electrostatic
potential at the membrane-solution interface is dependent on the
electrolyte concentration in the bulk aqueous solution (19, 21, 22).
Therefore, if the difference in channel conductance is a result of the
surface charge of the phospholipid bilayers, this difference in
conductance should be abolished at high
K+ concentrations. Figure
5 shows the relation between
Gc and the logarithmic mean K+ activity on
the two sides of the channel, (), given by
[(K+)c - (K+)t]/ln[(K+)c/(K+)t],
where
(K+)c
and
(K+)t
are the K+ activities in the
cis and
trans solutions, respectively.
Starting from initial KCl concentrations of 150 (cis) and 5 mM
(trans), the
K+ concentrations on the
cis or
trans side of the bilayer were
increased in a stepwise manner by the addition of appropriate amounts
of a 2 M KCl stock solution. In the three types of bilayers,
Gc depended on
(
) in a hyperbolic manner that can be
described by saturation kinetics. When the experimental data of Fig. 5
were fitted by the Michaelis-Menten equation, it became clear that the
values of maximum
Gc at infinite
(
) in the three bilayers were not significantly
different (Table 2). But the apparent
half-saturation constant, i.e., the value at which
Gc is half
maximal, was significantly lower in PE/PS and PE/PI bilayers than in
PE/PC bilayers. These findings are in agreement with the notion that
the high Gc
values at low K+ activities for
PE/PS and PE/PI bilayers compared with those for PE/PC bilayers is a
result of the electrostatic attraction of the permeating cations in
charged membranes.
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In addition, we examined the effect of altering the charge density of
the membrane on
Ic by introducing
the charged lipidlike amphiphiles SDS and DDTMA into neutral PE/PC
bilayers. SDS and DDTMA have an identical 12-carbon backbone, but the
head groups are oppositely charged, negative for SDS and positive for
DDTMA. These amphiphiles are inserted into the bilayer and alter the lipid surface charge. SDS was shown to increase current through Na+ and
Ca2+ channels of cardiac myocytes,
whereas DDTMA had the opposite effect (see Ref. 14). Similarly, in the
present experiments the addition of a 20 µM concentration of the
negatively charged SDS to the cis and
trans solutions increased
Gc of
BKCa channels by 33%, whereas 20 µM DDTMA decreased
Gc by 19% (Fig.
6).
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Finally, the lipid composition of the basolateral membrane vesicle
preparation used for the channel reconstitution experiments was
assessed (Table 3). The total
lipid content of the vesicles was 2.91 ± 0.12 mg/mg protein. The sum
of all phospholipids accounted for three-fourths of the total lipid,
with cholesterol, diacylglycerols, and fatty acids making up the rest.
The cholesterol-to-phospholipid ratio in basolateral membranes of
rabbit distal colon epithelium was 0.25 compared with 0.67 for
basolateral membranes of rabbit proximal colon (30) and 0.72 for these
cell membranes of rabbit small intestine (29), indicating an increase
in fluidity of the basolateral membranes in the aboral direction. Among
the phospholipids, PE was the dominant species in basolateral membranes
from distal colon epithelium. PE, PC, and sphingomyelin, phospholipids
that carry no net charge, accounted for almost three-fourths of the total phospholipids; one-fourth was made up by the negatively charged
PS and PI. In basolateral membranes of rabbit small intestinal epithelium, the relative contents of PS and PI were found to be similar
(29); in proximal colon epithelium PS and PI contribute one-third of
the total phospholipid (30).
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DISCUSSION |
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The conductance and gating behavior of
BKCa channels from the basolateral
membrane of rabbit distal colon epithelium are markedly dependent on
the phospholipids surrounding the channels, as shown by the fusion of
native basolateral membrane vesicles with planar phospholipid bilayers of different composition. Hence the lipids of the
native vesicles appear to be diluted and replaced by the lipids of the
artificial bilayer after vesicle fusion. It is one of the basic tenets
of the fluid "mosaic" model of membrane structure that membrane
lipids undergo lateral diffusion within the plane of the membrane with
a diffusion coefficient of
~108
cm2/s, which is 100-fold faster
than the diffusion rate of proteins (31). In planar bilayers the
diffusion coefficient of lipids may approach
10
7
cm2/s (20).
The conductance of a channel to permeable ions depends on the number of ions near the pore entrance. This number is influenced by the density, location, and sign of the fixed charges in the neighborhood of the ion conductance pathway (19). Previously we reported that cations are attracted into the entrance of BKCa channels of rabbit colon epithelium by negative charges on the channel protein itself (38). The present results provide evidence that in addition Ic is affected by the negative surface charges of the phospholipid membrane.
In neutral bilayers the local K+
activity next to the bilayer surface is equal to the bulk
K+ activity, whereas in negatively
charged membranes the local K+
activity is expected to be higher than that for the bulk solution. The
surface potential affecting the K+
activity at the channel entrance,
'o, can be calculated according to Gouy-Chapman theory from the ratio of the
Gc values for the
different bilayers
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(1) |
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(2a) |
The charge density of a bilayer composed of a 1:1 mixture of neutral
and negatively charged phospholipids is ~1/120
Å2
(see Ref. 21). With this value, the full surface potential, o, of PE/PS and PE/PI bilayers
is predicted from the Gouy equation (21, 22) to be
92 mV for a
salt concentration of 150 mM and a temperature of 22°C. The fact
that the electrostatic potential to which the permeating
K+ is exposed in the channel is
much lower than the actual surface potential of the charged lipid
bilayer indicates that the conduction pathway of the channel is
insulated from the phospholipid head groups, most likely by the protein
wall of the channel.
By applying the Gouy-Chapman formalism (19, 21, 22), we can calculate the K+ activity at a given distance x from the charged lipid surface as a function of bulk K+ activity
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(2b) |
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(3a) |
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(3b) |
The insulating distance of 20 Å suggests that the
BKCa channel unit has large
dimensions. The BKCa channel of
rabbit colon epithelium has not been cloned; its size, therefore, is
not known. Purified BKCa channels
from bovine tracheal and aortic smooth muscle were reported to consist
of a 60- to 70-kDa pore-forming -subunit and a 31-kDa
-subunit,
whereas the functioning
-subunit has a molecular mass of ~125 kDa
(15). The predicted mass of the
mslo protein, the mouse brain
BKCa channel, is 140 kDa (9). Recently, the molecular structure of the
K+ channel from
Streptomyces lividans
(KcsA channel), the amino acid
sequence of which is similar to that of vertebrate
BKCa channels, was examined by
X-ray crystallographic analysis (7). From these data, the diameter of
the KcsA channel can be estimated
to range between 30 and 50 Å, so the conduction pore would be
15-25 Å away from the bilayer phospholipids, consistent
with the present calculations. But the exact geometry of the rabbit
colon BKCa channel is unclear; for
instance, it is not known if all parts of the channel mouth are at the
same distance from the lipid matrix. These uncertainties may be
responsible for the finding that the Gc-(
)
data do not precisely fit the dashed curves of Fig. 5, which represent
a simple Gouy-Chapman model.
Channels larger than the BKCa channel, for instance, the acetylcholine receptor (molecular mass 280 kDa), appear to be completely insensitive to the surface potential (see Refs. 6 and 23), suggesting that the channel entrance is farther away from the charged phospholipids than is the case for channels that are sensitive to the surface potential. Lipid surface charge also does not influence the conductance of the voltage-gated Na+ channel from canine brain (11). In contrast, the small peptide channel gramicidin A (molecular mass 2 kDa) senses almost the entire lipid surface potential (1).
Negatively charged phospholipids have also been reported to increase the current through the K+ channels of the sarcoplasmic reticulum (2) and those of the plasma membrane of skeletal muscle (23) and vascular smooth muscle (10). In addition, in Ca2+ channels of muscle transverse tubules the Na+ conductance was found to depend on phospholipid surface charge (6). From the Gouy-Chapman theory of the diffuse double layer, applied to the K+ channels of the sarcoplasmic reticulum and skeletal muscle plasma membrane and the Ca2+ channels of muscle transverse tubules, it was calculated that the entry pathways of these channels are located at a distance of 9-20 Å from the lipid bilayer surface (2, 6, 23).
When the () in the bulk solution bathing colonic
BKCa channels is increased, the
rise in Gc
exhibits saturation (see Fig. 5). Assuming simple electrodiffusion
through the channel and the ohmic properties of the channel, the
relation between channel conductance and the
K+ activity on the
cis and
trans sides of the channel is given
theoretically by
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(4) |
The findings of an increase in Gc by introducing the negatively charged SDS into a neutral bilayer, whereas the positively charged DDTMA reduces Gc, additionally support the notion of a surface charge effect of the lipid membrane on the permeating ions, although detergents may also affect channel function by other types of interactions with the bilayer and channel protein (26).
The Erev of the colonic BKCa channels was not affected by the phospholipid composition, in agreement with the assumption that the structures of the conduction pathway and the selectivity filter are not influenced.
Attraction by the negative surface charge of phospholipids is also expected for Ca2+. Ca2+ increases the Po of colonic BKCa channels at a given Vm (17, 32), so Po is expected to be higher in a negatively charged bilayer than in a neutral bilayer at the same Ca2+ activity. In fact, this was observed with BKCa channels from rat skeletal muscle (23). However, in the present experiments with BKCa channels from rabbit colon epithelium it was difficult to assess the influence of phospholipid composition on Po because in PE/PC and PE/PI membranes there was channel rundown. But immediately after channel fusion Po was higher in PE/PI and PE/PS bilayers than in PE/PC bilayers (see Figs. 1-3), a finding that is consistent with local attraction of Ca2+ in charged membranes. If the Ca2+ activation site of the channel senses the same surface potential that attracts K+ into the channel entrance, the free Ca2+ level at the activation site would be 1.7-fold higher than that in the bulk solution.
The phenomenon of channel rundown (i.e., a decrease in Po with time) of colonic BKCa channels appears not to be a function of surface charge, as was observed both for neutral PE/PC bilayers and for negatively charged PE/PI bilayers but not for charged PE/PS membranes. The function of membrane proteins may be affected not only by unspecific electrostatic factors but also by specific lipid-protein interactions owing to differences in the lengths and structures of the acyl chains and the head group sizes of the lipids (4). In the present study, primarily tissue-derived phospholipids were used for planar bilayer production. These lipids differ not only in head group charge but also in acyl chain length and saturation. Changes in the phospholipid composition can affect the secondary, tertiary, and quaternary structures of membrane proteins (18). The size of the phospholipid head group may affect channel open time, as reported for BKCa channels from rat brain (5). There is also evidence that PS is important for transporter activity, because the Na+/Ca2+ exchanger of dog cardiac sarcolemma was shown to exhibit a high level of activity when reconstituted in PS-containing vesicles, whereas the exchanger activity is low in vesicles composed of PI or phosphatidylglycerol, which also carries a net negative charge (37). Lipid modulation of membrane protein function has also been shown for a number of other transporters (3).
What are the functional properties of the colonic
BKCa channels in their natural
environment, the basolateral plasma membrane of the epithelium? The
native basolateral membrane vesicle preparation was measured to contain
74% phospholipids and 18% cholesterol (percentages of total lipids).
The dominant phospholipid was PE, with almost 60% of total
phospholipids; PS and PI made up ~25% of phospholipids (Table 3).
Hence, the charge density of the native basolateral cell membranes may
be taken to be 1/240
Å2,
one-half the charge density of the 1:1 of PE/PS and PE/PI bilayers used
in the present reconstitution experiments. The full surface potential
of the native basolateral membranes may be estimated to be 60 mV
at a KCl concentration of 150 mM; at a distance of 20 Å, the
thickness of the insulating layer surrounding the
BKCa conduction pathway, the
potential attracting K+ would be
4.6 mV. Therefore, under "physiological" conditions the
activity of K+ at the channel
mouth would only be 1.20-fold higher than that in the bulk solution and
Gc would be 207 pS. However, an asymmetric distribution of phospholipids between the
outer and inner leaflets is a characteristic feature of
cell membranes, as shown for erythrocytes (25) and small intestinal and
renal brush border membranes (12, 36). Typically, the negatively
charged PS and PI are located predominantly in the cytoplasmic layer,
possibly resulting in rectifying properties of ion flux through the
membrane channels in the outward direction under physiological conditions.
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ACKNOWLEDGEMENTS |
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The chamber used for the reconstitution experiments was kindly provided by Dr. H. Schindler, Univ. of Linz, Linz, Austria.
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FOOTNOTES |
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This study was supported by the Medizinisch-Wissenschaftlicher Fonds of the Mayor of the City of Vienna, Vienna, Austria.
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. §1734 solely to indicate this fact.
1 The PE/PC bilayers are assumed to be electrically neutral. Bell and Miller (2) reported a surface charge density of 1/1,250-1/2,500 Å2 for a 4:1 PE/PC bilayer; from these values the surface potential can be estimated to be 1-2 mV at a KCl concentration of 150 mM. This surface potential is considered to be negligible.
2
At the conclusion of these experiments, the
K+ concentrations in the
cis and
trans solutions were measured by flame
photometry. The K+ activity
averaged 111.0 ± 2.0 mM (n = 47) in
the cis solution and 10.3 ± 0.5 mM
(n = 50) in the
trans solution, calculated by using
the activity coefficients given by Kielland (16) and Robinson and
Stokes (24). Hence the Nernst equilibrium potential for K+ is 60.5 mV, which is not
significantly different from the
Erev values
(Table 1), as noted earlier for this channel (32).
Address for reprint requests and other correspondence: K. Turnheim, Pharmakologisches Institut, Währinger Strasse 13a, A-1090 Vienna, Austria (E-mail: klaus.turnheim{at}univie.ac.at).
Received 27 October 1998; accepted in final form 7 April 1999.
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