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Ion channel transduction in Langmuir-Blodgett lipid membranes
Thin Solid Films, 210/211 (1992) 707-709
707
Ion channel transduction in Langmuir-Blodgett lipid membranes C. A. Palmer, T. L. Fare, D. A. Stenger and C. G. Silvestre Center for Bio/Moleeular Engineering, Code 6090, Naval Research Laboratory, Washington, DC 20375 (U.S.A.)
D. H. Cribbs Biology Department, St. Mary's College of Maryland, St Mary's City, MD 20686 (U.S.A.)
P. P. Bey, Jr. and K. M. Rusin Geo-Centers lnc, 10903 Indian Head Hwy, Ft. Washington, MD 20744 (U.S.A.)
Abstract Multilayers of phospholipid films with VDAC (voltage dependent anion channels) or acetylcholine receptors (AChR) have been deposited onto platinum electrodes using the Langmuir Blodgett technique. Electrical properties of both suspended and platinum electrode-supported phospholipid bilayers containing VDAC were studied. For the VDAC-coated electrodes, the VDAC conductance decreased under appropriate biasing conditions due to the voltage sensitive nature of the protein. The response was asymmetric with respect to the polarity of the offset voltage, possibly the result of steric hindrance of the protein conformational changes by the electrode surface. Physical properties of AChR-lipid films were investigated at the air-water interface as a function of surface pressure. AChR-lipid films were transferred onto electrodes and the conductance of the electrodes was found to increase upon injection of carbamylcholine. Alternative methods for protein transfer onto the electrode have been considered. Stability and sensitivity of these films will be discussed with respect to biosensor development.
I. Introduction Receptor-based proteins are used by the h u m a n b o d y to regulate physiology. A chemical stimulant (ligand) will bind to a receptor protein to activate a cellular response. F o r example, interaction o f carbamylcholine or acetylcholine with the acetylcholine receptor ( A C h R ) protein allows an ion current to flow through a high impedance phospholipid bilayer causing a muscle or nerve to depolarize. If such a protein could be stabilized in a lipid bilayer on an electrode, a sensor for a variety o f neurotransmitters could be made [1]. Since these proteins are to be deposited on an electrode for sensor development, there are problems with polarization due to D C charging. To avoid these charging effects, an A C voltage is used to m o n i t o r the m e m b r a n e current; this minimizes electrode polarization and yields higher noise immunity using lock-in techniques [2]. In this paper, we report the use o f voltage-dependent anion channel ( V D A C ) - p h o s p h o lipid films to develop these A C techniques and A C h R lipid films to study ligand-gated channels on electrodes.
NaH2PO4 buffered to p H 7.0 in doubly distilled, deionized water. The electrodes used in all experiments were 200 lam diameter platinum wires, flamed prior to immersion in the subphase. Lipid-protein films were formed at r o o m temperature in either a J o y c e - L o e b l IV or N I M A teflon-coated m o n o l a y e r trough (Fig. 1). 2.2. V D A C
2. Experimental details
Liposome preparations o f V D A C protein were obtained from yeast by a prescribed m e t h o d [3]. The activity o f the protein was measured by monitoring ion channel currents during fusion o f these vesicles to black lipid m e m b r a n e [4]. To form monolayers o f this preparation for transfer, aliquots o f this preparation were diluted by 20-fold in aqueous subphase aliquots. Various a m o u n t s o f this solution were applied at a rate o f approximately 50 ~tl/min to a clean, 10 m m diameter glass rod which penetrated the air-water interface; an additional 2 0 0 - 2 5 0 ~tl o f subphase solution was used to rinse off the remaining vesicles on the rod [5]. Controlled a m o u n t s o f dissolved D P P E : D L P E (dipalmitoyl phosphatidyl ethanolamine : dilauroyl phosphatidylethanolamine) were then spread onto the interface and the films were compressed to between 35 and 45 m N / m at a rate o f 0.05-0.10 cm2/s.
2.1. Materials
2.3. A C h R
The subphase used for m o n o l a y e r formation was an aqueous solution o f 1 . 0 m M CaCI2 and 1 . 0 m M
Vesicle preparations o f A C h R were prepared with varying concentrations o f D O P C (dioleoyl phos-
0040-6090/92/$5.00
© 1992 - - Elsevier Sequoia. All rights reserved
708
('. A. Pahner el a/. / hm channel lransduction in LB lipid membranes
,
LOCK-IN AMP.
I -- yoc0oLso
..~
: : . ~
-
FUNCTION GEN,
etectroc~e I
jl I
etectrode
Fig. 1. Experimental set-up showing the impedance measurement of coated electrodes.
phatidylcholine), cholesterol, and DPPA (dipalmitoyl phosphatidic acid) [6]. Mixtures of DPPE, cholesterol, and DPPA dissolved in 4:1 chloroform/ethanol at a concentration of 0.5 mg/ml were spread to submonolayer coverage at the air-water interface. After waiting 15 min, the vesicle preparation of AChR was then spread at the air-water interface with the lipids. After waiting an additional 15 min, the mixed monolayer was compressed at a rate of 0.05-0.10cm2/s to a surface pressure of 35-40 mN/m.
2.4. Deposition and characterization Lipid-protein films were deposited onto hydrophilic platinum electrodes at a transfer speed of 2 mm/min, while holding the surface pressure constant. Starting with the electrode submerged, a bilayer was transferred by withdrawal and submersion, with transfer ratios close to 1.0. The bilayer admittance was monitored with either an E G & G PAR 5208 or 5210 dual phase lock-in amplifier (LIA). A frequency of 0.5 Hz and amplitude of 100mV was used for the AC voltage, Vac, in all experiments. A function generator provided both the external synchronization to control the LIA reference phase and the DC offset voltage, Volt, used to bias the membrane potential [2, 7]. Coverage was determined by using an impedance technique developed for lipid-coated electrodes [2]. After coating, the total conductance of the electrode was determined. The total conductance of the electrode, Gp, is given by the linear sum of G m the coated-electrode conductance, Ge the uncoated-electrode conductance, and Gc the channel conductance. The total capacitance Cp is given by the sum of Cm the coated-electrode and Ce the uncoated-electrode capacitance [2, 7]. The results are normalized to (Gp/Gpo), obtained by dividing the
total conductance by the maximum conductance, Gp,,, of the film.
3. Results and discussion
3.1. VDAC While VDAC-containing BLMs show approximately profiles, the VDAC-lipid coated bilayer exhibited an asymmetric response [7]. Negative voltages with respect to the electrode caused a decrease of (Gp/Gpo) of VDAC films by about 15%. Little or no decrease in the (Gp/Gpo) values was observed for positive offset voltages in the 0 - 4 0 mV range; little or no change in capacitance of VDAC (or AChR) films was observed. The observed asymmetric response of the supported VDAC conductance may be due to: (1) steric hindrance of the protein by the electode; (2) the attraction of the positively charged residues in the a-helix NHz-terminus for the negatively charged electrode which would electrostatically inhibit protein conformations [3]; or (3), since the VDAC protein is a dimer with each monomer of the protein in opposite orientation, the asymmetry could arise because only one of the monomer's #-sheets is sterically hindered by the electrode when a DC voltage is applied [8]. symmetric (Gp/Gpo) -V
3.2. A ChR For AChR-lipid films, the subphase ionic concentration was increased to 10-40 mM Na + after film deposition to increase the signal from the channels. Controlled aliquots of carbamylcholine were then added to the subphase to induce channel response on the coated electrodes. It was found that the film conductance increased for agonist concentrations on the order of 1 mM (Fig. 2). 100 MILLION ACHR CHANNELS ABOUT 10000 ACTIVE DPPA:DPPE:CHOL 1:1:2 Im V r
CCh
15 minutes
Fig. 2. Change in the in-phase (Im V) and quadrature phase (Re signal from the experimental set-up in Fig. 1 for the response of AChR-coated electrode to an injection of carbamylcholine (CCh) a function of time; nS, nanoSiemens. Note the long period response during which the channels remain desensitized.
V) an as of
c. A. Palmer et al. ] Ion channel transduction in LB lipid membranes
Calculations based on film conductance and a-bungarotoxin binding assays showed that about 104 out o f 108 channels on the electrode were active. Clearly, the most important consideration in these experiments is maintaining an environment compatible with protein functionality. W o r k on improving the protein-lipid preparations is underway. We have shown in this w o r k that A C voltages m a y be used to measure the m o d u l a t e d conductance o f a voltaged or ligand-gated ion channel without polarizing the ion channels. A l t h o u g h the films were stable for several hours, stability o f the proteins for long-term use is of concern. Attempts are being made to maintain the protein in a native environment to improve aging. O u r experiments suggest that admittance measurements of protein-lipid films on electrodes might be a suitable a p p r o a c h for making ion channel-based biosensors.
709
Acknowledgment Research was conducted while one o f the authors ( C A P ) held an O N T Postdoctoral Fellowship.
References 1 F. E. 2 T. 3 E. 4 5 6 7 8
S. Ligler, T. L. Fare, K. Seib, J. Smuda, A. Singh, P. Ahl, M. Ayers, A. Dalziel and P. Yager, Med. Instrum., 22 (1988) 247. L. Fare, Langmuir, 6(1990) 1172. Blachly-Dyson, S. Peng, M. Colombini and M. Forte, Science, 247 (1990) 1233. M. Montal and P. Mueller, Proc. Natl. Acad. Sci. USA, 69 (1972) 3561. R. Verger and F. Pattus, Chem. Phys. Lipids, 16 (1976) 285. T. M. Fong and M. G. McNamee, Biochemistry, 25 (1986) 830. D. A. Stenger, D. H. Cribbs and T. L. Fare, Biosensors, 6 (1991) 423. M. Colombini, J. Membr. Biol., 111 (1989) 103.
707
Ion channel transduction in Langmuir-Blodgett lipid membranes C. A. Palmer, T. L. Fare, D. A. Stenger and C. G. Silvestre Center for Bio/Moleeular Engineering, Code 6090, Naval Research Laboratory, Washington, DC 20375 (U.S.A.)
D. H. Cribbs Biology Department, St. Mary's College of Maryland, St Mary's City, MD 20686 (U.S.A.)
P. P. Bey, Jr. and K. M. Rusin Geo-Centers lnc, 10903 Indian Head Hwy, Ft. Washington, MD 20744 (U.S.A.)
Abstract Multilayers of phospholipid films with VDAC (voltage dependent anion channels) or acetylcholine receptors (AChR) have been deposited onto platinum electrodes using the Langmuir Blodgett technique. Electrical properties of both suspended and platinum electrode-supported phospholipid bilayers containing VDAC were studied. For the VDAC-coated electrodes, the VDAC conductance decreased under appropriate biasing conditions due to the voltage sensitive nature of the protein. The response was asymmetric with respect to the polarity of the offset voltage, possibly the result of steric hindrance of the protein conformational changes by the electrode surface. Physical properties of AChR-lipid films were investigated at the air-water interface as a function of surface pressure. AChR-lipid films were transferred onto electrodes and the conductance of the electrodes was found to increase upon injection of carbamylcholine. Alternative methods for protein transfer onto the electrode have been considered. Stability and sensitivity of these films will be discussed with respect to biosensor development.
I. Introduction Receptor-based proteins are used by the h u m a n b o d y to regulate physiology. A chemical stimulant (ligand) will bind to a receptor protein to activate a cellular response. F o r example, interaction o f carbamylcholine or acetylcholine with the acetylcholine receptor ( A C h R ) protein allows an ion current to flow through a high impedance phospholipid bilayer causing a muscle or nerve to depolarize. If such a protein could be stabilized in a lipid bilayer on an electrode, a sensor for a variety o f neurotransmitters could be made [1]. Since these proteins are to be deposited on an electrode for sensor development, there are problems with polarization due to D C charging. To avoid these charging effects, an A C voltage is used to m o n i t o r the m e m b r a n e current; this minimizes electrode polarization and yields higher noise immunity using lock-in techniques [2]. In this paper, we report the use o f voltage-dependent anion channel ( V D A C ) - p h o s p h o lipid films to develop these A C techniques and A C h R lipid films to study ligand-gated channels on electrodes.
NaH2PO4 buffered to p H 7.0 in doubly distilled, deionized water. The electrodes used in all experiments were 200 lam diameter platinum wires, flamed prior to immersion in the subphase. Lipid-protein films were formed at r o o m temperature in either a J o y c e - L o e b l IV or N I M A teflon-coated m o n o l a y e r trough (Fig. 1). 2.2. V D A C
2. Experimental details
Liposome preparations o f V D A C protein were obtained from yeast by a prescribed m e t h o d [3]. The activity o f the protein was measured by monitoring ion channel currents during fusion o f these vesicles to black lipid m e m b r a n e [4]. To form monolayers o f this preparation for transfer, aliquots o f this preparation were diluted by 20-fold in aqueous subphase aliquots. Various a m o u n t s o f this solution were applied at a rate o f approximately 50 ~tl/min to a clean, 10 m m diameter glass rod which penetrated the air-water interface; an additional 2 0 0 - 2 5 0 ~tl o f subphase solution was used to rinse off the remaining vesicles on the rod [5]. Controlled a m o u n t s o f dissolved D P P E : D L P E (dipalmitoyl phosphatidyl ethanolamine : dilauroyl phosphatidylethanolamine) were then spread onto the interface and the films were compressed to between 35 and 45 m N / m at a rate o f 0.05-0.10 cm2/s.
2.1. Materials
2.3. A C h R
The subphase used for m o n o l a y e r formation was an aqueous solution o f 1 . 0 m M CaCI2 and 1 . 0 m M
Vesicle preparations o f A C h R were prepared with varying concentrations o f D O P C (dioleoyl phos-
0040-6090/92/$5.00
© 1992 - - Elsevier Sequoia. All rights reserved
708
('. A. Pahner el a/. / hm channel lransduction in LB lipid membranes
,
LOCK-IN AMP.
I -- yoc0oLso
..~
: : . ~
-
FUNCTION GEN,
etectroc~e I
jl I
etectrode
Fig. 1. Experimental set-up showing the impedance measurement of coated electrodes.
phatidylcholine), cholesterol, and DPPA (dipalmitoyl phosphatidic acid) [6]. Mixtures of DPPE, cholesterol, and DPPA dissolved in 4:1 chloroform/ethanol at a concentration of 0.5 mg/ml were spread to submonolayer coverage at the air-water interface. After waiting 15 min, the vesicle preparation of AChR was then spread at the air-water interface with the lipids. After waiting an additional 15 min, the mixed monolayer was compressed at a rate of 0.05-0.10cm2/s to a surface pressure of 35-40 mN/m.
2.4. Deposition and characterization Lipid-protein films were deposited onto hydrophilic platinum electrodes at a transfer speed of 2 mm/min, while holding the surface pressure constant. Starting with the electrode submerged, a bilayer was transferred by withdrawal and submersion, with transfer ratios close to 1.0. The bilayer admittance was monitored with either an E G & G PAR 5208 or 5210 dual phase lock-in amplifier (LIA). A frequency of 0.5 Hz and amplitude of 100mV was used for the AC voltage, Vac, in all experiments. A function generator provided both the external synchronization to control the LIA reference phase and the DC offset voltage, Volt, used to bias the membrane potential [2, 7]. Coverage was determined by using an impedance technique developed for lipid-coated electrodes [2]. After coating, the total conductance of the electrode was determined. The total conductance of the electrode, Gp, is given by the linear sum of G m the coated-electrode conductance, Ge the uncoated-electrode conductance, and Gc the channel conductance. The total capacitance Cp is given by the sum of Cm the coated-electrode and Ce the uncoated-electrode capacitance [2, 7]. The results are normalized to (Gp/Gpo), obtained by dividing the
total conductance by the maximum conductance, Gp,,, of the film.
3. Results and discussion
3.1. VDAC While VDAC-containing BLMs show approximately profiles, the VDAC-lipid coated bilayer exhibited an asymmetric response [7]. Negative voltages with respect to the electrode caused a decrease of (Gp/Gpo) of VDAC films by about 15%. Little or no decrease in the (Gp/Gpo) values was observed for positive offset voltages in the 0 - 4 0 mV range; little or no change in capacitance of VDAC (or AChR) films was observed. The observed asymmetric response of the supported VDAC conductance may be due to: (1) steric hindrance of the protein by the electode; (2) the attraction of the positively charged residues in the a-helix NHz-terminus for the negatively charged electrode which would electrostatically inhibit protein conformations [3]; or (3), since the VDAC protein is a dimer with each monomer of the protein in opposite orientation, the asymmetry could arise because only one of the monomer's #-sheets is sterically hindered by the electrode when a DC voltage is applied [8]. symmetric (Gp/Gpo) -V
3.2. A ChR For AChR-lipid films, the subphase ionic concentration was increased to 10-40 mM Na + after film deposition to increase the signal from the channels. Controlled aliquots of carbamylcholine were then added to the subphase to induce channel response on the coated electrodes. It was found that the film conductance increased for agonist concentrations on the order of 1 mM (Fig. 2). 100 MILLION ACHR CHANNELS ABOUT 10000 ACTIVE DPPA:DPPE:CHOL 1:1:2 Im V r
CCh
15 minutes
Fig. 2. Change in the in-phase (Im V) and quadrature phase (Re signal from the experimental set-up in Fig. 1 for the response of AChR-coated electrode to an injection of carbamylcholine (CCh) a function of time; nS, nanoSiemens. Note the long period response during which the channels remain desensitized.
V) an as of
c. A. Palmer et al. ] Ion channel transduction in LB lipid membranes
Calculations based on film conductance and a-bungarotoxin binding assays showed that about 104 out o f 108 channels on the electrode were active. Clearly, the most important consideration in these experiments is maintaining an environment compatible with protein functionality. W o r k on improving the protein-lipid preparations is underway. We have shown in this w o r k that A C voltages m a y be used to measure the m o d u l a t e d conductance o f a voltaged or ligand-gated ion channel without polarizing the ion channels. A l t h o u g h the films were stable for several hours, stability o f the proteins for long-term use is of concern. Attempts are being made to maintain the protein in a native environment to improve aging. O u r experiments suggest that admittance measurements of protein-lipid films on electrodes might be a suitable a p p r o a c h for making ion channel-based biosensors.
709
Acknowledgment Research was conducted while one o f the authors ( C A P ) held an O N T Postdoctoral Fellowship.
References 1 F. E. 2 T. 3 E. 4 5 6 7 8
S. Ligler, T. L. Fare, K. Seib, J. Smuda, A. Singh, P. Ahl, M. Ayers, A. Dalziel and P. Yager, Med. Instrum., 22 (1988) 247. L. Fare, Langmuir, 6(1990) 1172. Blachly-Dyson, S. Peng, M. Colombini and M. Forte, Science, 247 (1990) 1233. M. Montal and P. Mueller, Proc. Natl. Acad. Sci. USA, 69 (1972) 3561. R. Verger and F. Pattus, Chem. Phys. Lipids, 16 (1976) 285. T. M. Fong and M. G. McNamee, Biochemistry, 25 (1986) 830. D. A. Stenger, D. H. Cribbs and T. L. Fare, Biosensors, 6 (1991) 423. M. Colombini, J. Membr. Biol., 111 (1989) 103.