. * Vol. 189, No. 2, 1992 December
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS Pages
15, 1992
BILAYBRB
COWAIPIRG
CRLCIUN
S.V.Balasubramanian,
IONOPHORE A23187
S.K.Sikdar
FORM
1038-l
042
CHANNELS
and K.R.K.Easwaran*
Molecular Biophysics Unit, Indian Institute of Science, Bangalore - 560 012, INDIA Keceived
October
19,
1992
For the first time, based on bilayer membrane conductance experiments, it has been shown that A23187, a carboxylic calcium single channel ionophore, incorporated in lipid bilayers gives currents similar to the well known gramicidin channel. The current characteristics indicate the possibility that the transmembrane ion transport by this important calcium ionophore is initially by a carrier mechanism but with time is by a channel or pore mechanism due to the aggregation of the molecule in a lipid matrix. 0 1992 Academic Press, Inc.
A23187, a carboxylic calcium ionophore of molecular weight 523 and with a structure consisting of a keto-pyrrole a spiroketal portion and a benzoxazole ring, is extensively group, used to facilitate movement of calcium ions across biological membranes and cells (l-3). However, the mechanism of calcium transport at the molecular level induced by this important ionophore is not well understood. While A23187 is believed to be a typical carrier (1, 4, 5),there have been some recent reports suggesting the possibility of calcium transport across membranes by this ionophore also by a pore or channel mechanism (6, 7). We report here for the first time, based on bilayer membrane conductance experiments, that A23187 incorporated in lipid bilayers gives single channel current in the picoampere range with ion conducting channels opening and closing randomly. The current characteristics indicate the existence of both carrier and channel mechanisms for calcium ion transport by this ionophore. *
To whom correspondence
0006-291X/92
Copyright All rights
should
be addressed.
$4.00
0
I!392 by Academic Press, of reproduction in any form
Inc. reserved.
1038
Vol.
189,
Idatoriala
No.
2,
1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
and Methods
A23187 and egg yolk lecithin used are from Sigma Chemical Co. lipid bilayers and are used without further purification. Planar were painted across a 0.3mm diameter hole in a styrene copolymer lecithin ( Merck ) dispersion of either cup with a n-decane egg (Sigma, USA) or dipalmitoyl phosphatidyl choline (DPPC) or diphytanoyl phosphatidyl choline (DPhPC) (supplied to us by Dr.O.S.Anderson,Cornell Univ.Med.College,New York) with or without A23187. The bilayers of egg lecithin was prepared using 1% w/v in n-decane containing O.lmg/ml of A23187. The styrene copolymer cup lipid bilayer (cis) rested in an outer chamber (trans) and the partition separated aqueous solution of similar ionic composition (150 mM NaC1,5 mM CaCl ,lOmM HEPES, the SOlUtiOn pH was adjusted A low noise operational amplifier (OPA 102B, to 7.4 with TRIS ). was used. Electrical Burr Brown) with frequency compensation connections to the experimental chambers were through Ag-AgCl wires. Connections to the outer chamber were taken to ground, while the cis side was taken to the non-inverting input of the with operational amplifier. Potential differences were cis room respect to trans. Measurements were carried out at temperature (28-30 OC). The voltage and current signals were either plotted on a pen recorder (Bryans, UK) or stored on digital tape audio tapes using a Biologic DTR 1200 (France) digital audio recorder. transients Formation of a bilayer was judged from fast in the current traces due to membrane capacitance following application of 2mV pulses. For amplitude histogram analysis single channel current data were taken off-line, filtered at lKHz, led through a CED 1401 A/D converter (Cambridge Electronic Design, UK), analysed using a PCAT 286 computer (PCL, India).The histogramswere plotted on a Graphtec MP4100 plotter. The PAT version 6 software (J.Dempster, University of Strathclyde, UK) was used for analysis. Ro8ults
and
Disaussion
Fig. 1A shows the plot of current traces at different transmembrane potentials in the absence of A23187 (fig. 1A a) and in presence of A23187 in egg lecithin lipid bilayer membrane recorded within minutes of the addition of the ionophore to the bilayer membrane (fig. 1A b). The current-voltage curve plotted (fig. 1B) using the data clearly showed an increase in conductance from 1.75~s to 4.2~s in the presence of A23187. This increase may be of a carrier induced enhancement in conductance and the observation is comparable to that of the typical carrier molecules such as valinomycin. However, it is interesting to note that at high membrane potentials (such as -1OOmV) some rectangular channel type current profile are also seen. Fig. 2A shows time records of current at different transmembrane potentials half hour after the painting of the lipid bilayer containing A23187. Discrete rectangular channel type current traces are seen with the frequency of channel openings increasing with inorease in membrane potential. Multichannel currents are observed with channel opening 1039
Vol.
189, No. 2, 1992
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
(a) c. 40 43 -100
/ &
b) 100 '-
I OSPA
Z$p 0-m -20/m -40 /
-100 /
shifts in Fiq. 1. A. Plots of current traces following indicated to the transmembrane potential from 0 to potentials and left of the traces (in mV), in control without A23187 (a) Note with A23187 (b) in egg lecithin lipid bilayer membrane. rectangular channel type current events at higher potentials in membranes B. Current-voltage relations in egg lecithin (b) . without A23187 (control, 0) and with A23187 (0). Each data point represents the mean f S.D. value obtained from 4 different linear experiments. The solid line through the two data sets are and regression lines which give current reversal of OmV (control) (+ A23187), with membrane conductances of 1.75 pS and 4.2 pS, respectively. B 0.6
0.b I---
AC.
induced currents following 2. A. A23187 single channel (in incorporation in egg lecithin bilayer at membrane potentials mV) indicated to the left of the traces. channel current - voltage curves for A23187 B. Single Each.point is a mean of incorporated into the egg lecithin bilayer. vertical bars values obtained from 4 different experiments; The linear regression line through the data points represent S.D. single channel gives current reversal potential of OmV with conductance of 6.43~5.
Fig.
1040
* * Vol.
189, No. 2, 1992
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
2.99
2.39
2
8
'.7g 1.19
,597
0
PA
Fig. eqq
3. lecithin
anathe
centered
Current amplitude distribution at 1OOmV. The unit conductance second open channel conductance level around l.OpA.
of A23187 channel in level is seen at 0.5~A is also seen which is
and closing completely at random. It was also observed that more channels are seen at higher concentration of the ionophore. Experiments done with buffer solution free of calcium chloride showed no stepwise conductance clearly indicating the calcium ion selectivity of A23187. Fig. 2B shows current-voltage curve for A23187 incorporated into egg yolk lecithin lipid bilayer using the data fig. 2A. The value of single channel conductance obtained was 6.43~s. The amplitude histogram of the channel current record is given in fig. 3. The analysis of the histogram clearly indicate that for 1% w/v egglecithin bilayer in n-decane electrolyte solution and the temperature at which the experiment is carried out, the single channel conductance falls within the narrow range of conductance with near gaussian shape. The distribution width primarily depend on the noise level of the experiment. In addition to the single channel conductance profile around 0.5pA current amplitude, a broad approximately gaussian type distribution can also be generated around a current amplitude of nearly 1pA and this is possibly due to the opening of more than one channel simultaneouly. The experiments were also carried out using DPPC and DPhPC as membrane forming solution and the results obtained were similar to that for egg lecithin. However, the single channel 1041
* * Vol.
189,
No.
2,
1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
conductance depended on the nature of the lipid used. The magnitude of conductance obtained followed the order egglecithin < DPPC < DPhPC. Analysis of the data from figures 1 and 2 reveal that the ionophore A23187, while exhibiting a carrier conductivity across planar bilayer activity membrane initially, also induces time. charecterized by discrete rectangular channel openings with The opening and closing of the channels are at random and possibly related to the structural fluctuation of a possible aggregated structure. pore A aggregated dimeric structure for A23187 in phospholipid vesicles has already been proposed based on spectroscopic techniques (6). The observation of channel activity by this ionophore also explains the large turnover number for calcium by A23187, in biological membranes, as compared to other carrier ionophores of similar structure (8). Acknowlodgnents The research was supported by grants Biotechnology, India and in part by the Foundation, Sweden.
from Erna
the Department of & Victor Hasselblad
Reforenaes 1.
2. 3. 4. 5. 6. 7. 8.
(1985) In 'Metal ions in Painter, G.R., and Pressman,B.C. pp-229-289 biological systems', (H.Sigel, Ed), vol. 19, Marcel1 Dekker Inc. New York. Reed, R.W.,and Lardy, H.A. (1972) J.Biol.Chem. 247, 6970-6977. Pressman, B.C., and Fahim, M. (1982) Ann.Rev.Pharmacol. Toxicol. 22, 465-490. Puskin,J.S., Visties,M.I., and Wene,M.T. (1981) Arch. Biochem. Biophys. 206, 164-172. Kolber, M.A., and Haynes,D.H. (1981) Biophys. J. 36,369-390. Balasubramanian, S.V., and Easwaran, K.R.K. (1989) Biochem. Biophys.Res.Communn. 158, 891-897. Easwaran, K.R.K., and Balasubramanian, S.V. (1990) In 'Water in Biomolecular Systems'.(D.Vasilescu et.al, Ed) and Ions ~~-221-228. BirkhBuser Verlag, Basel. Caswell,A.H., and Pressman, B.C. (1972) Biochem. Biophys. Res. Communn. 49, 292-298.
1042
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS Pages
15, 1992
BILAYBRB
COWAIPIRG
CRLCIUN
S.V.Balasubramanian,
IONOPHORE A23187
S.K.Sikdar
FORM
1038-l
042
CHANNELS
and K.R.K.Easwaran*
Molecular Biophysics Unit, Indian Institute of Science, Bangalore - 560 012, INDIA Keceived
October
19,
1992
For the first time, based on bilayer membrane conductance experiments, it has been shown that A23187, a carboxylic calcium single channel ionophore, incorporated in lipid bilayers gives currents similar to the well known gramicidin channel. The current characteristics indicate the possibility that the transmembrane ion transport by this important calcium ionophore is initially by a carrier mechanism but with time is by a channel or pore mechanism due to the aggregation of the molecule in a lipid matrix. 0 1992 Academic Press, Inc.
A23187, a carboxylic calcium ionophore of molecular weight 523 and with a structure consisting of a keto-pyrrole a spiroketal portion and a benzoxazole ring, is extensively group, used to facilitate movement of calcium ions across biological membranes and cells (l-3). However, the mechanism of calcium transport at the molecular level induced by this important ionophore is not well understood. While A23187 is believed to be a typical carrier (1, 4, 5),there have been some recent reports suggesting the possibility of calcium transport across membranes by this ionophore also by a pore or channel mechanism (6, 7). We report here for the first time, based on bilayer membrane conductance experiments, that A23187 incorporated in lipid bilayers gives single channel current in the picoampere range with ion conducting channels opening and closing randomly. The current characteristics indicate the existence of both carrier and channel mechanisms for calcium ion transport by this ionophore. *
To whom correspondence
0006-291X/92
Copyright All rights
should
be addressed.
$4.00
0
I!392 by Academic Press, of reproduction in any form
Inc. reserved.
1038
Vol.
189,
Idatoriala
No.
2,
1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
and Methods
A23187 and egg yolk lecithin used are from Sigma Chemical Co. lipid bilayers and are used without further purification. Planar were painted across a 0.3mm diameter hole in a styrene copolymer lecithin ( Merck ) dispersion of either cup with a n-decane egg (Sigma, USA) or dipalmitoyl phosphatidyl choline (DPPC) or diphytanoyl phosphatidyl choline (DPhPC) (supplied to us by Dr.O.S.Anderson,Cornell Univ.Med.College,New York) with or without A23187. The bilayers of egg lecithin was prepared using 1% w/v in n-decane containing O.lmg/ml of A23187. The styrene copolymer cup lipid bilayer (cis) rested in an outer chamber (trans) and the partition separated aqueous solution of similar ionic composition (150 mM NaC1,5 mM CaCl ,lOmM HEPES, the SOlUtiOn pH was adjusted A low noise operational amplifier (OPA 102B, to 7.4 with TRIS ). was used. Electrical Burr Brown) with frequency compensation connections to the experimental chambers were through Ag-AgCl wires. Connections to the outer chamber were taken to ground, while the cis side was taken to the non-inverting input of the with operational amplifier. Potential differences were cis room respect to trans. Measurements were carried out at temperature (28-30 OC). The voltage and current signals were either plotted on a pen recorder (Bryans, UK) or stored on digital tape audio tapes using a Biologic DTR 1200 (France) digital audio recorder. transients Formation of a bilayer was judged from fast in the current traces due to membrane capacitance following application of 2mV pulses. For amplitude histogram analysis single channel current data were taken off-line, filtered at lKHz, led through a CED 1401 A/D converter (Cambridge Electronic Design, UK), analysed using a PCAT 286 computer (PCL, India).The histogramswere plotted on a Graphtec MP4100 plotter. The PAT version 6 software (J.Dempster, University of Strathclyde, UK) was used for analysis. Ro8ults
and
Disaussion
Fig. 1A shows the plot of current traces at different transmembrane potentials in the absence of A23187 (fig. 1A a) and in presence of A23187 in egg lecithin lipid bilayer membrane recorded within minutes of the addition of the ionophore to the bilayer membrane (fig. 1A b). The current-voltage curve plotted (fig. 1B) using the data clearly showed an increase in conductance from 1.75~s to 4.2~s in the presence of A23187. This increase may be of a carrier induced enhancement in conductance and the observation is comparable to that of the typical carrier molecules such as valinomycin. However, it is interesting to note that at high membrane potentials (such as -1OOmV) some rectangular channel type current profile are also seen. Fig. 2A shows time records of current at different transmembrane potentials half hour after the painting of the lipid bilayer containing A23187. Discrete rectangular channel type current traces are seen with the frequency of channel openings increasing with inorease in membrane potential. Multichannel currents are observed with channel opening 1039
Vol.
189, No. 2, 1992
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
(a) c. 40 43 -100
/ &
b) 100 '-
I OSPA
Z$p 0-m -20/m -40 /
-100 /
shifts in Fiq. 1. A. Plots of current traces following indicated to the transmembrane potential from 0 to potentials and left of the traces (in mV), in control without A23187 (a) Note with A23187 (b) in egg lecithin lipid bilayer membrane. rectangular channel type current events at higher potentials in membranes B. Current-voltage relations in egg lecithin (b) . without A23187 (control, 0) and with A23187 (0). Each data point represents the mean f S.D. value obtained from 4 different linear experiments. The solid line through the two data sets are and regression lines which give current reversal of OmV (control) (+ A23187), with membrane conductances of 1.75 pS and 4.2 pS, respectively. B 0.6
0.b I---
AC.
induced currents following 2. A. A23187 single channel (in incorporation in egg lecithin bilayer at membrane potentials mV) indicated to the left of the traces. channel current - voltage curves for A23187 B. Single Each.point is a mean of incorporated into the egg lecithin bilayer. vertical bars values obtained from 4 different experiments; The linear regression line through the data points represent S.D. single channel gives current reversal potential of OmV with conductance of 6.43~5.
Fig.
1040
* * Vol.
189, No. 2, 1992
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
2.99
2.39
2
8
'.7g 1.19
,597
0
PA
Fig. eqq
3. lecithin
anathe
centered
Current amplitude distribution at 1OOmV. The unit conductance second open channel conductance level around l.OpA.
of A23187 channel in level is seen at 0.5~A is also seen which is
and closing completely at random. It was also observed that more channels are seen at higher concentration of the ionophore. Experiments done with buffer solution free of calcium chloride showed no stepwise conductance clearly indicating the calcium ion selectivity of A23187. Fig. 2B shows current-voltage curve for A23187 incorporated into egg yolk lecithin lipid bilayer using the data fig. 2A. The value of single channel conductance obtained was 6.43~s. The amplitude histogram of the channel current record is given in fig. 3. The analysis of the histogram clearly indicate that for 1% w/v egglecithin bilayer in n-decane electrolyte solution and the temperature at which the experiment is carried out, the single channel conductance falls within the narrow range of conductance with near gaussian shape. The distribution width primarily depend on the noise level of the experiment. In addition to the single channel conductance profile around 0.5pA current amplitude, a broad approximately gaussian type distribution can also be generated around a current amplitude of nearly 1pA and this is possibly due to the opening of more than one channel simultaneouly. The experiments were also carried out using DPPC and DPhPC as membrane forming solution and the results obtained were similar to that for egg lecithin. However, the single channel 1041
* * Vol.
189,
No.
2,
1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
conductance depended on the nature of the lipid used. The magnitude of conductance obtained followed the order egglecithin < DPPC < DPhPC. Analysis of the data from figures 1 and 2 reveal that the ionophore A23187, while exhibiting a carrier conductivity across planar bilayer activity membrane initially, also induces time. charecterized by discrete rectangular channel openings with The opening and closing of the channels are at random and possibly related to the structural fluctuation of a possible aggregated structure. pore A aggregated dimeric structure for A23187 in phospholipid vesicles has already been proposed based on spectroscopic techniques (6). The observation of channel activity by this ionophore also explains the large turnover number for calcium by A23187, in biological membranes, as compared to other carrier ionophores of similar structure (8). Acknowlodgnents The research was supported by grants Biotechnology, India and in part by the Foundation, Sweden.
from Erna
the Department of & Victor Hasselblad
Reforenaes 1.
2. 3. 4. 5. 6. 7. 8.
(1985) In 'Metal ions in Painter, G.R., and Pressman,B.C. pp-229-289 biological systems', (H.Sigel, Ed), vol. 19, Marcel1 Dekker Inc. New York. Reed, R.W.,and Lardy, H.A. (1972) J.Biol.Chem. 247, 6970-6977. Pressman, B.C., and Fahim, M. (1982) Ann.Rev.Pharmacol. Toxicol. 22, 465-490. Puskin,J.S., Visties,M.I., and Wene,M.T. (1981) Arch. Biochem. Biophys. 206, 164-172. Kolber, M.A., and Haynes,D.H. (1981) Biophys. J. 36,369-390. Balasubramanian, S.V., and Easwaran, K.R.K. (1989) Biochem. Biophys.Res.Communn. 158, 891-897. Easwaran, K.R.K., and Balasubramanian, S.V. (1990) In 'Water in Biomolecular Systems'.(D.Vasilescu et.al, Ed) and Ions ~~-221-228. BirkhBuser Verlag, Basel. Caswell,A.H., and Pressman, B.C. (1972) Biochem. Biophys. Res. Communn. 49, 292-298.
1042
