Membrane phospholipid composition affects function of potassium channels from rabbit colon epithelium

Klaus Turnheim1, Johannes Gruber1, Christoph Wachter1, and Valentina Ruiz-Gutiérrez2

1 Pharmakologisches Institut, Universität Wien, A-1090 Vienna, Austria; and 2 Instituto de la Grasa, Consejo Superior de Investigaciones Cientificas, E-41012 Seville, Spain


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    MATERIALS AND METHODS
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INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-alpha -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.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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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Fig. 1.   Top: example of the current through a rabbit colon high-conductance, 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 approx 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.



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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.



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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.

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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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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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

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, (&Kdot;), 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 (&Kdot;) 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 (&Kdot;) 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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Fig. 5.   Single-channel conductance, Gc, as a function of logarithmic mean K+ activity, (&Kdot;), 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-(&Kdot;) relation for uncharged PE/PC bilayers underlying simple saturation kinetics. Dashed curves represent calculated Gc-(&Kdot;) 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.


                              
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Table 2.   Gc(max) and K for BKCa channels reconstituted in planar bilayers of different phospholipid compositions

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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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.

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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Table 3.   Lipid composition and distribution of the major phospholipids of basolateral membrane vesicles of rabbit distal colon epithelium


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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, psi 'o, can be calculated according to Gouy-Chapman theory from the ratio of the Gc values for the different bilayers
<FR><NU><IT>G</IT><SUB>c(PE/PS)</SUB></NU><DE><IT>G</IT><SUB>c(PE/PC)</SUB></DE></FR> or <FR><NU><IT>G</IT><SUB>c(PE/PI)</SUB></NU><DE><IT>G</IT><SUB>c(PE/PC)</SUB></DE></FR> = exp(−<IT>F</IT>&psgr;′<SUB>o</SUB>/<IT>RT</IT>) (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+)'o, can be obtained from the K+ activity in the bulk solution, (K+)b, and psi 'o from the Boltzmann relation
(K<SUP>+</SUP>)′<SUB>o</SUB> = (K<SUP>+</SUP>)<SUB>b</SUB> exp(−<IT>F</IT>&psgr;′<SUB>o</SUB>/<IT>RT</IT>) (2a)
For PE/PS bilayers, the activity of K+ 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, psi 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
(K<SUP>+</SUP>)<SUB><IT>x</IT></SUB> = (K<SUP>+</SUP>)<SUB>b</SUB> exp(−<IT>F</IT>&psgr;<SUB><IT>x</IT></SUB>/<IT>RT</IT>) (2b)
in which (K+)x and psi x are the K+ activity and the electrostatic potential at distance x, respectively. The dependence of psi x on x is given by
&psgr;<SUB><IT>x</IT></SUB> = <FR><NU>2<IT>RT</IT></NU><DE><IT>F</IT></DE></FR> ln<FENCE><FR><NU>1 + &agr;exp(−&kgr;<IT>x</IT>)</NU><DE>1 − &agr;exp(−&kgr;<IT>x</IT>)</DE></FR></FENCE> (3a)
in which
&agr; = <FR><NU>exp(<IT>F</IT>&psgr;<SUB>o</SUB>/2<IT>RT</IT>) − 1</NU><DE>exp(<IT>F</IT>&psgr;<SUB>o</SUB>/2<IT>RT</IT>) + 1</DE></FR> (3b)
and kappa  is the reciprocal of the Debye length, the thickness of the ionic double layer near the charged surface. The kappa  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 alpha -subunit and a 31-kDa beta -subunit, whereas the functioning alpha -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-(&Kdot;) 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 (&Kdot;) 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
<IT>G</IT><SUB>c</SUB> = <FR><NU><IT>PF</IT><SUP> 2</SUP></NU><DE><IT>RT</IT></DE></FR> (<A><AC>K</AC><AC>˙</AC></A>) (4)
(28), where P represents the permeability coefficient of K+. Clearly, saturation of Gc with increasing (&Kdot;) 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 dependence of Gc is lower in PE/PS and PE/PI bilayers than in PE/PC membranes.

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.


    ACKNOWLEDGEMENTS

The chamber used for the reconstitution experiments was kindly provided by Dr. H. Schindler, Univ. of Linz, Linz, Austria.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Apell, H. J., E. Bamberg, and P. Läuger. Effects of surface charge on the conductance of the gramicidin channel. Biochim. Biophys. Acta 552: 369-378, 1979[Medline].

2.   Bell, J. E., and C. Miller. Effects of phospholipid surface charge on ion conduction in the K+ channel of sarcoplasmic reticulum. Biophys. J. 45: 279-287, 1984[Abstract].

3.   Bienvenüe, A., and J. Sainte Marie. Modulation of protein function by lipids. Curr. Top. Membr. 40: 319-354, 1994.

4.   Carruthers, A., and D. L. Melchior. How bilayer lipids affect membrane protein activity. Trends Biochem. Sci. 11: 331-335, 1986.

5.   Chang, H. M., R. Reitstetter, and R. Gruener. Lipid-ion channel interactions: increasing phospholipid headgroup size but not ordering acyl chains alters reconstituted channel behavior. J. Membr. Biol. 145: 13-19, 1995[Medline].

6.   Coronado, R., and H. Affolter. Insulation of the conduction pathway of muscle transverse tubule calcium channels from the surface charge of bilayer phospholipid. J. Gen. Physiol. 87: 933-953, 1986[Abstract].

7.   Doyle, D. A., J. M. Cabral, R. A. Pfuetzner, A. Kuo, J. M. Gulbis, S. L. Cohen, B. T. Chait, and R. MacKinnon. The structure of the potassium channel: molecular basis of K+ conduction and selectivity. Science 280: 69-77, 1998[Abstract/Free Full Text].

8.   Folch, J., M. Lees, and G. H. Sloane-Stanley. A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 226: 497-509, 1957[Free Full Text].

9.   Garcia, M. L., H.-G. Knaus, P. Munujos, R. S. Slaughter, and G. J. Kaczorowski. Charybdotoxin and its effects on potassium channels. Am. J. Physiol. 269 (Cell Physiol. 38): C1-C10, 1995[Abstract/Free Full Text].

10.   Giangiacomo, K. M., M. Garcia-Calvo, H.-G. Knaus, T. J. Mullmann, M. L. Garcia, and O. McManus. Functional reconstitution of the large-conductance, calcium-activated potassium channel purified from bovine aortic smooth muscle. Biochemistry 34: 15849-15862, 1995[Medline].

11.   Green, W. N., L. B. Weiss, and O. S. Andersen. Batrachotoxin-modified sodium channels in planar lipid bilayers. Ion permeation and block. J. Gen. Physiol. 89: 841-872, 1987[Abstract].

12.   Hauser, H., and G. Lipka. Lipid dynamics in brush border membrane. Curr. Top. Membr. 40: 167-195, 1994.

13.   Hille, B. Ion Channels in Excitable Membranes (2nd ed.). Sunderland, MA: Sinauer, 1992.

14.   Ji, S., J. N. Weiss, and G. A. Langer. Modulation of voltage-dependent sodium and potassium currents by charged amphiphiles in cardiac ventricular myocytes. Effects via modification of surface potential. J. Gen. Physiol. 101: 355-375, 1993[Abstract].

15.   Kaczorowski, G. J., H.-G. Knaus, R. J. Leonard, O. B. McManus, and M. L. Garcia. High-conductance calcium-activated potassium channels; structure, pharmacology, and function. J. Bioenerg. Biomembr. 28: 255-267, 1996[Medline].

16.   Kielland, J. Individual activity coefficients of ions in aqueous solutions. J. Am. Chem. Soc. 59: 1675-1678, 1937.

17.   Klærke, D. A., H. Wiener, T. Zeuthen, and P. L. Jørgensen. Ca2+ activation and pH dependence of a maxi K+ channel from rabbit distal colon epithelium. J. Membr. Biol. 136: 9-21, 1993[Medline].

18.   Kleinfeld, A. M. Current views of membrane structure. Curr. Top. Membr. Transp. 29: 1-27, 1987.

19.   Latorre, R., P. Labarca, and D. Naranjo. Surface charge effects on ion conduction in ion channels. Methods Enzymol. 207: 471-501, 1992[Medline].

20.   Lee, G. M., and K. Jacobsen. Lateral mobility of lipids in membranes. Curr. Top. Membr. 40: 111-142, 1994.

21.   McLaughlin, S. Electrostatic potentials at membrane-solution interfaces. Curr. Top. Membr. Transp. 9: 71-144, 1977.

22.   McLaughlin, S. The electrostatic properties of membranes. Annu. Rev. Biophys. Biophys. Chem. 18: 113-136, 1989[Medline].

23.   Moczydlowski, E., O. Alvarez, C. Vergara, and R. Latorre. Effect of phospholipid surface charge on the conductance and gating of a Ca2+-activated K+ channel in planar lipid bilayers. J. Membr. Biol. 83: 273-282, 1985[Medline].

24.   Robinson, R. A., and R. H. Stokes. Electrolyte Solutions (2nd ed.). New York: Academic, 1959.

25.   Roelofsen, B., and J. A. F. Op den Kamp. Plasma membrane phospholipid asymmetry and its maintenance: the human erythrocyte as a model. Curr. Top. Membr. 40: 7-46, 1994.

26.   Sawyer, D. B., R. E. Koeppe II, and O. S. Andersen. Induction of conductance heterogeneity in gramicidin channels. Biochemistry 28: 6571-6583, 1989[Medline].

27.   Schindler, H. Planar lipid-protein membranes; strategies of formation and of detecting dependencies of ion transport functions on membrane conditions. Methods Enzymol. 171: 225-253, 1989[Medline].

28.   Schultz, S. G. Basic Principles of Membrane Transport. Cambridge, UK: Cambridge Univ. Press, 1980.

29.   Schwarz, S. M., H. E. Bostwick, M. D. Danzinger, L. J. Newman, and M. S. Medow. Ontogeny of basolateral membrane lipid composition and fluidity in small intestine. Am. J. Physiol. 257 (Gastrointest. Liver Physiol. 20): G138-G144, 1989[Abstract/Free Full Text].

30.   Schwarz, S. M., A. S. Lambert, and M. S. Medow. Ontogeny of proximal colon basolateral membrane lipid composition and fluidity in the rabbit. Biochim. Biophys. Acta 1107: 70-76, 1992[Medline].

31.   Storch, J., and A. M. Kleinfeld. The lipid structure of biological membranes. Trends Biochem. Sci. 10: 418-421, 1985.

32.   Turnheim, K., J. Costantin, S. Chan, and S. G. Schultz. Reconstitution of a calcium-activated potassium channel in basolateral membranes of rabbit colonocytes into planar lipid bilayers. J. Membr. Biol. 112: 247-254, 1989[Medline].

33.   Turnheim, K., J. Gruber, and H. Plass. Quinidine inhibition of basolateral potassium channels of rabbit colon epithelium (Abstract). Naunyn Schmiedebergs Arch. Pharmacol. 56, Suppl. 1: R23, 1997.

34.   Van't Hof, W., and G. van Meer. Lipid polarity and sorting of epithelial cells. Curr. Top. Membr. 40: 539-563, 1994.

35.   Vázquez, C. M., N. Rovira, V. Ruiz-Gutiérrez, and J. M. Planas. Developmental changes in glucose transport, lipid composition, and fluidity of jejunal BBM. J. Am. Physiol. 273 (Regulatory Integrative Comp. Physiol. 42): R1986-R1093, 1997.

36.   Venien, C., and C. Le Grimellec. Phospholipid asymmetry in renal brush-border membranes. Biochim. Biophys. Acta 942: 159-168, 1988[Medline].

37.   Venuri, R., and K. D. Philipson. Phospholipid composition modulates the Na+-Ca2+ exchange activity of cardiac sarcolemma in reconstituted vesicles. Biochim. Biophys. Acta 937: 258-269, 1987.

38.   Wachter, C., and K. Turnheim. Inhibition of high-conductance, Ca2+-activated potassium channels of rabbit colon epithelium by magnesium. J. Membr. Biol. 150: 275-282, 1996[Medline].

39.   Wiener, H., K. Turnheim, and C. H. van Os. Rabbit distal colon epithelium: I. Isolation and characterization of basolateral plasma membrane vesicles from surface and crypt cells. J. Membr. Biol. 110: 147-162, 1989[Medline].


Am J Physiol Cell Physiol 277(1):C83-C90
0002-9513/99 $5.00 Copyright © 1999 the American Physiological Society




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