Membrane phospholipid composition affects function
of potassium channels from rabbit colon epithelium
KLAUS TURNHEIM,1 JOHANNES GRUBER,1 CHRISTOPH WACHTER,1
AND VALENTINA RUIZ-GUTIÉRREZ2
1Pharmakologisches Institut, Universität Wien, A-1090 Vienna, Austria; and 2Instituto de la Grasa,
Consejo Superior de Investigaciones Cientificas, E-41012 Seville, Spain
rabbit colon epithelium; surface charge; cell membrane composition
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 highconductance K⫹ channels from the basolateral cell
membrane of rabbit colon epithelium. These channels
are activated by intracellular Ca2⫹ and membrane
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solely to indicate this fact.
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.
MATERIALS AND METHODS
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, Darm-
0363-6143/99 $5.00 Copyright r 1999 the American Physiological Society
C83
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Turnheim, Klaus, Johannes Gruber, Christoph
Wachter, and Valentina Ruiz-Gutiérrez. Membrane phospholipid composition affects function of potassium channels
from rabbit colon epithelium. Am. J. Physiol. 277 (Cell
Physiol. 46): C83–C90, 1999.—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.
C84
PHOSPHOLIPIDS AND K⫹ CHANNEL ACTIVITY
RESULTS
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).
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
Fig. 1. Top: example of the current through a rabbit colon highconductance, Ca2⫹-activated K⫹ (BKCa ) channel reconstituted in a
phosphatidylethanolamine (PE)/phosphatidylcholine (PC) bilayer.
Shown is channel activity immediately after channel fusion with
bilayer and 30 min later. Holding voltage (Vm ) ⫽ ⫺10 mV; initial KCl
concentrations: 150 (cis) and 5 mM (trans); free Ca2⫹ concentration ⬇28 µM. o and c, open (conductive) and closed (nonconductive)
states, respectively, of channel. Bottom: changes of voltage dependence of open-state probability, Po, of BKCa channels reconstituted in
PE/PC bilayers vs. time. Data are means ⫾ SE for 13–19 channels.
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
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.
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stadt, 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.
PHOSPHOLIPIDS AND K⫹ CHANNEL ACTIVITY
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
Fig. 3. Top: example of current through a rabbit colon BKCa channel
reconstituted in a PE/phosphatidylserine (PS) bilayer. Conditions
were as for Fig. 1. Bottom: changes of voltage dependence of Po of
BKCa channels reconstituted in PE/PS bilayers with time. Data are
means ⫾ SE of 8–11 channels.
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
Fig. 4. Examples of the current-voltage (Ic-Vm ) relations of colonic
BKCa channels reconstituted in PE/PC, PE/PS, or PE/PI bilayers,
initially with 150 mM KCl in cis solution and 5 mM KCl in trans
solution.
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Fig. 2. Top: example of current through a rabbit colon BKCa channel
reconstituted in a PE/phosphatidylinositol (PI) bilayer. Conditions
were as for Fig. 1. Bottom: changes of voltage dependence of Po of
BKCa channels reconstituted in PE/PI bilayers with time. Data are
means ⫾ SE for 4 channels.
C85
PHOSPHOLIPIDS AND K⫹ CHANNEL ACTIVITY
C86
Table 1. Gc and Erev of BKCa channels from the
basolateral cell membrane of rabbit distal colon
epithelium reconstituted in planar lipid bilayers
of different phospholipid compositions
Bilayer
Composition
Gc , pS
Erev , mV
n
PE/PC
PE/PS
PE/PI
173 ⫾ 23
224 ⫾ 33*
237 ⫾ 22*
⫺56.0 ⫾ 10.8
⫺56.1 ⫾ 12.5
⫺51.9 ⫾ 10.2
69
82
24
Data are means ⫾ SD. * Statistically different from phosphatidylethanolamine (PE)/phosphatidylcholine (PC) value (2P ⬍ 0.001).
Gc , channel conductance; Erev , reversal potential; BKCa , highconductance, Ca2⫹-activated K⫹; PI, phosphatidylinositol; PS, phosphatidylserine.
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).
Fig. 5. Single-channel conductance, Gc, as a function of logarithmic
mean K⫹ activity, (K̇), on the two sides of BKCa channels reconstituted in PE/PC, PE/PS, or PE/PI bilayers. Free Ca2⫹ concentration ⫽
250 µM. Data are means ⫾ SD of 5–7 experiments. Solid curve,
nonlinear regression analysis of Gc-(K̇) relation for uncharged PE/PC
bilayers underlying simple saturation kinetics. Dashed curves represent calculated Gc-(K̇) relations according to Gouy-Chapman theory
(Eqs. 2b, 3a, and 3b), assuming that channel entrances are removed
from charged surfaces of PE/PS and PE/PI bilayers by indicated
distances.
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).
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 memTable 2. Gc(max) and K for BKCa channels
reconstituted in planar bilayers
of different phospholipid compositions
Bilayer
Composition
Gc(max) , pS
K, mM
n
PE/PC
PE/PS
PE/PI
437 ⫾ 50
431 ⫾ 32
472 ⫾ 28
59.7 ⫾ 19.2
29.8 ⫾ 8.8*
34.9 ⫾ 7.0†
5
7
5
Data are means ⫾ SD. Maximum Gc (Gc(max) ) and logarithmic mean
K⫹ activity at which Gc is half maximal (K ) were calculated by
approximating parameters of saturation kinetics to measure values
in Fig. 5 by using an iterative curve-fitting procedure. Statistically
significant difference from corresponding value for PE/PC bilayers:
* 2P ⬍ 0.01; † 2P ⬍ 0.05.
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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.
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, (K̇), 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
(K̇) 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
(K̇) 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.
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
PHOSPHOLIPIDS AND K⫹ CHANNEL ACTIVITY
C87
be similar (29); in proximal colon epithelium PS and PI
contribute one-third of the total phospholipid (30).
DISCUSSION
branes 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
Table 3. Lipid composition and distribution of the
major phospholipids of basolateral membrane vesicles
of rabbit distal colon epithelium
Lipid or
Phospholipid
%Total
Lipids
Cholesterol
Phospholipids
Diacylglycerols
Free fatty acids
Cholesterol esters
18.3 ⫾ 5.6
74.1 ⫾ 4.6
3.9 ⫾ 1.5
3.2 ⫾ 2.4
0.48 ⫾ 0.08
Phospholipids
PE
PC
PS
PI
Sphingomyelin
Cardiolipin
Lyso-PE
58.7 ⫾ 1.7
6.5 ⫾ 0.7
9.2 ⫾ 0.4
16.1 ⫾ 0.6
8.9 ⫾ 0.4
0.24 ⫾ 0.03
0.54 ⫾ 0.01
Data are means ⫾ SD of 6 separate determinations. %Total is
percentage (wt/wt) of total lipids for lipids and percentage of total
phospholipids for phospholipids. Lyso-PE, 1-acyl-2-hydroxyglycero-PE.
Gc(PE/PS)
Gc(PE/PC)
or
Gc(PE/PI)
Gc(PE/PC)
⫽ exp(⫺F␺8o /RT)
(1)
where F is Faraday’s constant, R is the gas constant,
and T is the absolute temperature (1, 19). From this
equation and the Gc values given in Table 1, the surface
potential attracting K⫹ into the BKCa channels is found
to be ⫺6.6 mV for PE/PS bilayers and ⫺7.7 mV for
PE/PI bilayers. The K⫹ activity at the channel mouth,
(K⫹ )8o, can be obtained from the K⫹ activity in the bulk
solution, (K⫹ )b, and ␺8o from the Boltzmann relation
(K⫹)8o ⫽ (K⫹)b exp(⫺F␺8o /RT)
K⫹
(2a)
For PE/PS bilayers, the activity of
at the entry
pathway of the channel is 1.30-fold higher than that in
the bulk solution; in PE/PI bilayers this accumulation
factor is 1.35.
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
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Fig. 6. Effects of amphiphiles SDS or dodecyltrimethylammonium
(DDTMA) on Gc of BKCa channels reconstituted in PE/PC bilayers
compared to controls (0) before addition of amphiphiles. Free Ca2⫹
concentration ⫽ 250 µM. SDS or DDTMA (20 µM each) was added to
both the cis and trans sides of the bilayer, and the resulting change in
Gc was recorded 10 min later. Data are means ⫾ SD for 6 experiments
for SDS and 5 experiments for DDTMA.
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
⬃10⫺8 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, ␺8o, can be calculated according to
Gouy-Chapman theory from the ratio of the Gc values
for the different bilayers
PHOSPHOLIPIDS AND K⫹ CHANNEL ACTIVITY
C88
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
(K⫹)x ⫽ (K⫹)b exp(⫺F␺x /RT)
(2b)
␺x ⫽
2RT
F
31 ⫺ ␣exp(⫺␬x)4
ln
1 ⫹ ␣exp(⫺␬x)
(3a)
in which
␣⫽
exp(F␺o /2RT) ⫺ 1
exp(F␺o /2RT) ⫹ 1
(3b)
and ␬ is the reciprocal of the Debye length, the thickness of the ionic double layer near the charged surface.
The ␬ is dependent on the bulk salt concentration. The
values of (K⫹ )x at various distances x from the charged
bilayers were used to calculate Gc from the parameters
of the saturation kinetics for PE/PC bilayers given in
Table 2. The results of these calculations are shown by
the dashed curves of Fig. 5. As the distance between the
conduction pathway of the channel and the charged
phospholipids decreases, Gc increases because (K⫹ )x
rises. The enhanced Gc of the channels reconstituted in
PE/PS and PE/PI bilayers corresponds fairly well to a
distance of 20 Å between the conduction pore and the
charged lipid surface.
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-(K̇) data do not
Gc ⫽
PF 2
RT
(K̇)
(4)
(28), where P represents the permeability coefficient of
K⫹. Clearly, saturation of Gc with increasing (K̇) is not
in agreement with Eq. 4, which predicts a linear
relation between Gc that passes through the origin.
Hence the current through the BKCa channel does not
conform to the ‘‘independence principle,’’ which asserts
that ion movement within the channel is not affected by
the presence of other ions (28). In fact, saturation of ion
flux through channels is a frequent observation and
may be a result of interactions of the diffusing particles
with specific sites in the channel or multi-ion occupancy
of the channel (13). Whatever the mechanism of current saturation, the maximum conductances at high K⫹
activities in neutral or charged bilayers are not significantly different (see Table 2). This finding is in agreement with surface charge theory, because the surface
potential is expected to decrease as negative surface
charges are screened by increasing bulk K⫹. The accumulation of K⫹ in the channel entrance is also illustrated by the fact that the apparent half-saturation
constant (K; see Table 2) of the concentration depen-
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in which (K⫹ )x and ␺x are the K⫹ activity and the
electrostatic potential at distance x, respectively. The
dependence of ␺x on x is given by
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 voltagegated 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 (K̇) 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
PHOSPHOLIPIDS AND K⫹ CHANNEL ACTIVITY
lateral 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.
The chamber used for the reconstitution experiments was kindly
provided by Dr. H. Schindler, Univ. of Linz, Linz, Austria.
This study was supported by the Medizinisch-Wissenschaftlicher
Fonds of the Mayor of the City of Vienna, Vienna, Austria.
Address for reprint requests and other correspondence: K. Turnheim, Pharmakologisches Institut, Währinger Strasse 13a, A-1090
Vienna, Austria (E-mail: [email protected]).
Received 27 October 1998; accepted in final form 7 April 1999.
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