Simultaneous fluorescence and conductance studies of planar bilayer membranes containing a highly active and fluorescent analog of gramicidin A

J. Mol. Biol. (1975) 99, 75-92

Simultaneous Fluorescence and Conductance Studies of Planar Bflayer Membranes Containing a Highly Active and Fluorescent Analog of Gramicidin A W. R. VEATCH, R. M~ATHIES,~ . EISENBERGt AND L. STRyI~.R

Delgartment of Molecular Biophysics and Biochemistry Yale University, New Haven, Conn. 06520, U.S.A. (Received 15 April 1975) Gramicidin A is a linear p o l y p e p t i d e antibiotic t h a t facilitates t h e passive di_Cfl~ion of alkali cations a n d hydrogen ions across lipid bflayer membranes b y forming t r a n s m e m b r a n e channels. I t has been proposed t h a t t h e conducting channel is a dimer t h a t is in equilibrium with non-conducting monomers in the membrane. W e have directly tested this model b y carrying out simultaneous fluorescence a n d conductance studies on p l a n a r bilayer m e m b r a n e s containing d a n s y l gramicidin C, a highly fluorescent a n d active analog o f gramicidin A. Dansyl gramicidin C in bilayer membranes has a n excitation m a x i m u m a t 355 nm, an emission m a x i m u m a t 530 nm, and a q u a n t u m yield of 0.4. The magnitude of t h e single-channel conductance of dansyl gramicidin C is 60% of t h a t of gramici. din A. The kinetics a n d equilibria of channel formation are nearly the same for dansyl gramicidin C a n d gramicidin A. Glycerolester membranes having h y d r o . carbon thicknesses ranging from 26 A to 4 7 / ~ were used to enable us to control the proportion of monomeric d a n s y l gramicidin C. F o u r conclusions can be d r a w n from these experiments. (1) A p l o t of t h e logarithm of t h e m e m b r a n e conductance versus the logarithm of t h e m e m b r a n e fluorescence h a d a slope of 2.0±0.3, over a concentration range for which nearly all the gramicidln was monomeric. Hence, the active channel is a dimer of the non-conducting species. (2) I n a m e m b r a n e in which n e a r l y all of the gramicidin was dimeric, t h e n u m b e r of channels was a p p r o x i m a t e l y equal to t h e n u m b e r of dimers. Thus, m o s t rllmers are active channels a n d so i t should be feasible to c a r r y o u t spectroscopic studies of t h e conformation of t h e t r a n s m e m b r a n e channel. (3) The association constant for dimerization is more t h a n 1000-fold larger in a 26 A t h a n in a 47/~ glycerolester membrane. The dimerization constant in a 48 A p h o s p h a t i d y l choline m e m b r a n e was 200 times larger t h a n in a 4 7 / ~ glycerolester membrane, showing t h a t i t depends on t h e t y p e of lipid as wed as on the thickness of the h y d r o c a r b o n core. (4) W e were readily able to detect 10 -14 mol c m -2 of dansyl gramicidin C in a bilayer m e m b r a n e , which corresponds to 60 fluorescence molecules p e r pzn2. The fluorescent techniques described here should be sufficiently sensitive for fluorescence studies of reconstituted gates a n d receptors in p l a n a r bilayer membranes. 1. Introduction G r a m i e i d i n A, a l i n e a r p o l y p e p t i d e a n t i b i o t i c , r e n d e r s biological m e m b r a n e s a n d s y n t h e t i c l i p i d b i l a y e r m e m b r a n e s p e r m e a b l e t o a l k a l i c a t i o n s a n d p r o t o n s (Chappell & Crofts, 1965; H a r r i s & P r e s s m a n , 1967; Mueller & R u d i n , 1967; H l a d k y & H a y d o n , Present address: Health Sciences Center SUNY Stony Brook, N.Y. 11794, U.S.A. 75

76

W. R. VEATCH ~ '

AL.

1970). The amino acid sequence of valine gramicidin A (Sarges & Witkop, 1965) is Formyl.L.Val-Gly-L-Ala-D-Leu-L-A]a-D-Val-L-Val-D-Val. 1 2 $ 4 5 6 7 8 L-Trp-D-Leu-L-Trp-D-Lou-L-Trp-D-Leu-L-Trp-NHCH2CH2oH. 9 10 11 12 13 14 15 The distinctive features of this amino acid sequence are the alternation of D and L amino acids, the presence of hydrophobic side chains, and the absence of any charged groups. Gramieidin C is a naturally occurring variant in which L-tryptophan at position 11 is replaced by L-tyrosine. Gramicidin A induces cation permeability by forming transmembrane channels rather than by acting as a diffusional carrier (Haydon & Hladky, 1972; Krasne et at., 1971). Synthetic bflayer membranes containing very small amounts of gramicidin A exhibit discrete changes in conductance that arise from the formation and breakdown of individual gramicidin channels (Hladky & Haydon, 1970). The conductance of a single channel depends on the concentration and type of ion but not on the thickness or viscosity of the membrane (Hladky & Haydon, 1972). It has been observed that the membrane conductance is approximately proportional to the square of the amount of gramicidin A added to the aqueous phase, which suggested that the conducting channel is a dimer (Tosteson et al., 1968; Goodall, 1970). However, this inference is not unequivocal because gramicidin A has such a low solubility in water that it irreversibly adsorbs to the membrane, and so the actual amount incorporated in the membrane is uncertain. The molecularity of the conducting channel has been further studied by a voltage-jump approach (Bamberg & L~uger, 1973), which is based on the finding that the number of channels increases as the membrane is thinned by an applied voltage (Hladky & Haydon, 1972). The results of these voltage-jump experiments are consistent with the hypothesis of an equilibrium in the membrane between a non-cenductlng monomer and a conducting dimer of gramicidin A (Bamberg & L~uger, 1973), but their data do not strongly exclude the possibility that the conducting species is a trimer (Zingsheim & Neher, 1974). Similar results have also been obtained from an autocorrelation analysis of the conductance fluctuations (Kolb et al., 1975; Zingsheim & Neher, 1974). We have carried out fluorescence and conductance studies of lipid bilayer membranes containing dansyl gramieidin C (Veatch, Gross & Blout, manuscript in preparation), a highly fluorescent and fully active analog of gramicidin A. The fluorescence intensity of membranes containing this analog provided a direct measure of its absolute surface density. The number of channels was ascertained from simultaneous measurements of the membrane conductance. These experiments were designed to answer three questions. (1) Is the conducting channel a dimer? (2) Are all dimers channels? (3) How does the dlmerization constant depend on membrane structure? 2. S t r a t e g y f o r M a k i n g S i m u l t a n e o u s F l u o r e s c e n c e a n d C o n d u c t a n c e Measurements

Consider an equilibrium in the membrane between a non-conducting monomer, G1, and a conducting dimer, 02.

(1)

FLUORESCENT

ANALOG

OF GRAMICIDIN

[a,.] = K [ a , ] ~ k~

K = k-~'

A

77

(~)

(a)

where K is the equilibrium constant for dimerization. The total surface density of gramicidin in the membrane [Go] (mol cm-2), is given by

[Go] = [G1] + 2 [Gs].

(4)

The density of conducting dimer depends on the total surface density of gramicidin according to the equation [Go] 1 -- (1 --f- 8K [Go])lm [G~] = T -18K

(5)

This dimer model can be tested by measuring [G2] as a function of [Go]. Two limiting conditions are of particular interest. When nearly all of the gramieidin is monomeric, [G2] ~ K [Go]2 (when g [Go] << 1).

(6)

Under these conditions, a plot of log [G2] versus log [Go] will have a slope of 2, ff the dimer model is correct. When nearly all of the gramicidin is dimeric,

[a~]

[Go] (when K [Go] >> 1).

_

T

(7)

For this limiting case, the surface density of channels is equal to one haft of the total surface density of gramicidin, provided that all dimers are active channels. In our experiments, [Go] was determined d~ectly by measuring the fluorescence intensity of the membrane, and [G2] by measuring the membrane conductance. Not all combinations of values of'[Go] and [G2] are experimentally accessible. [(70] must be sufficiently high so that the fluorescence of dausyl gramicidin C in the membrane exceeds the background fluorescence due to the membrane lipids. In practice, this occurred when [Go] was greater than about 10-1~ tool c m - 5 . In contrast, [Gs] had to be less than about 4 x 10 - ~4 mol cm- 2, so that the membrane conductance could be measured accurately; at higher values of [Gs], the resistance of the solution is much greater than that of the membrane. The experimental significance of these limits on the range of observable values of [(~0] and [(75] can be appreciated by considering Figure 1, which shows plots of log [Gs] versus log [(70] calculated for different dimerization constants. For a single kind of membrane, it is not feasible to make simultaneous fluorescence and conductance measurements over a range of [Go] yielding mostly monomer ([(7o] << IlK) to mostly dimer ([Go] >> ILK). However, these limiting cases, which are most decisive in testing the dimer model (equations (6) and (7)), are experimentally accessible ff membranes having very different values of K are used. This was achieved by using a series of glycerolester membranes differing in the thickness of their hydrocarbon core (Fettiplace et al., 1971; Hladky & Haydon, 1972) (Table 1). The duration of single channels of gramicidin A in these glycerolester membranes (Hladky & Haydon, 1972) suggested that they have a wide range of dimerization constants, which is in fact the case. We will refer to these membranes by the thickness of their hydrocarbon core (e.g., 26 A glyeerolester membrane).

78

W . R. V E A T C H

ET

AL.

-11

-12

-15

IE -14 cJ

"5 E -15

oo, _J

-16

-17

-18

-17

:-16

-15 -14 -13 Log GO (tool cm-2)

-t2

-II

FIG. l. Theoretical dimerization curves and feasibility region for simultaneous fluorescence and conductance measurements. The curves are plots of the dimer surface density [G~], as a function of ~he total surface density [Go] for values of the dimerization constant (K) ranging from 108 to 1017 mo1-1 em 2, according to equation (5).

TABLE 1

Hydrocarbon thickness of bilayer membranesformed from glycerol monoester-alicanemixtures Glycerol monoester

All~ne

Hydrocarbon thickness (A)

Glycerol- 1-palmitoleate Glycerol- 1-oleate Glycerol- 1-oleate Glycerol- 1-oleate

n-hexadecane n-hexadecane ~-tetradecane n-deeane

26 31 40 47

From Hladky & Haydon (1972).

3. Materials and M e t h o d s (a) Formation of planar lipid bilayer membranes The sample cell for simultaneou~ fluorescence a n d conductance measurements is shown i n the inset of Fig. 2(a). The septum was made of black Teflon (Cadillac Plastics) a n d the walls of white Teflon. The cell a n d quartz windows were clamped together i n a brass housing. Prior ~o each use, the cell was dismantled a n d sequentially sonicated i n chloroform, methanol, distilled water, a n d chromic acid, a n d then rinsed for 30 m~n with deionized water. The cell was t h e n rinsed briefly with methanol a n d chloroform

F L U O R E S C E N T A N A L O G OF G R A M I C I D I N A

79

Photomult~

i',iei i.r.filter

..,,,,j~ f /

1

r=z-5 ~ X V /

.

I

~ ~ ~ ~ r ~ . = . CelI [Xenon 1 Diaphragm /,~ Monochrbmatar ~,/~,Microscope (a)

To emission detectionsv

~todiode

Internal apertureemission \ X~ Ik\,\\.\\\\'k\\\\ L.

It\\\\\

/ rWindow \.\" [ \ \ \ \ \ \ \ \ \ \ X~k~/

.

".41

Septum,

/,-.-mL_ ~'~'-- Membrane ~x.\\\\~-~\.\\\ \\\\\\\NI

Internal excitation aperture cm

Excitationor alignmentbe

External excitation aperture

Visual observation of membrane

(b) 1~o. 2. Schematic d~gram of the membrane fluorimeter. (a) Top view of the physical layout of the standard membrane fluorhneter with a xenon lamp and a monochromator as the excitation source. (b) Cross.section of a modifieation of the standard membrane fluorlmeter in which an argon-ion laser was used with apertures inside the cell to el~m;~te the possibility of detecting torus fluorescence. The emission optics external to the cell were unchanged.

80

W. R. V E A T C H E T A L .

(speetrograde, Aldrich) a n d air dried. The sealing rims were coated with silicone grease (Bow Coming) to p r e v e n t leakage a r o u n d the windows. VA.I~ne grA.mleidin A, gr~.micidin A, gramicidin B and gramieidin C, purified b y countercurrent distribution, were the generous gift of D r E h r h a r d Gross of t h e N a t i o n a l I n s t i t u t e s of Health. D a n s y l gramicidin C, p r e p a r e d according to Veatch ct at. (manuscript in preparation), was a t least 90~/o pure. All salts used in this s t u d y were t h e u l t r a p u r e grade from A l p h a Inorganic. The membrane-forming solution was prepared b y mixing a solution of t h e lipid in chloroform w i t h a solution of gramicidin in methanol. This m i x t u r e was e v a p o r a t e d to dryness under nitrogen, a n d t h e n dissolved in a n alkane: n-decane (spectrograde, Aldrich), n-tetradecane, or n-hexadeeane (spectroquality, Matheson, Coleman & Bell). The final concentration of t h e glycerohnonoesters (Sigma) was 20 m g / m l a n d t h a t of dioleoyl p h o s p h a t i d y l choline (Supelco) was I0 rng/mi. The p h o s p h a t i d y l choline solutions were h e a t e d to 70°C for 1 rain. There was some precipitation of dansyl gr~mlcidin C when its concentration in t h e membrane-forming solution approached 10 .4 M; this could be minimized b y adding more chloroform to t h e g r a m i c i d i n - l i p i d m i x t u r e prior to evaporation. Thick membranes were formed using a Teflon syringe needle (gauge 10), flushing t h e hole with fresh lipid for each membrane. The membranes were thinned with t h e voltage clamped to zero, except for p h o s p h a t i d y l choline membranes, which required voltages of 50 to 80 InV. The electrical and fluorescence properties of different membranes were reproducible to within a factor of two, and drift with time was minimal when t h e aqueous solutions were n o t stirred. (b) I"luoresc~nce meavurements on planar bilayer membranes The s t a n d a r d m e m b r a n e fluorimeter is schematically shown in Fig. 2(a). A 150 W xenon arc lamp (Osram XBO150) in a W i l d housing a n d trigger module was imaged on the entrance slit of a monochromator. The excitation wavelength was set to 356 n m a n d a Coming 7-54 filter was used to eliminate s t r a y light. The exit slit of the m o n o e h r o m a t o r was imaged on the excitation diaphragm, which in t u r n was imaged on the membrane. The fluorescence from the m e m b r a n e was collected, filtered through a Coming 3-72 filter, imaged on the emission diaphragm, a n d t h e n on the cathode of a photomultiplier (EMI6256S). The signal from the photomultiplier was amplified b y a K e i t h l e y electrometer (615G) a n d recorded on a dual channel strip chart recorder (Houston Instruments). The time constant was 3 s. The optical system was aligned b y viewing 500 n m light t h a t was reflected from t h e membrane, either before or after t h i ~ i n g . The image of the excitation d i a p h r a g m was viewed through the microscope. A n i m p o r t a n t feature of the alignment procedure was t h e scattering of light off the surface of the emission diaphragm. This scattered light, which traces a p a t h through the system in a direction opposite to t h a t of the fluorescence, was imaged on t h e membrane. The result was a superposition of the images of t h e emission diaphragm and of t h e excitation d i a p h r a g m in t h e plane of the m e m b r a n e when viewed t h r o u g h t h e microscope. I t was simple then to a d j u s t the position of t h e cell so t h a t t h e excitation a n d emission areas were equal in size, completely superposed, a n d centered on t h e fiat membrane. A 0-004 cm 2 circular excitation area was used and, unless otherwise noted, t h e m e m b r a n e a r e a was 0-2 c m 2. The s e p t u m was 0-7 m m t h i c k in t h e region of t h e hole. The membranes were excited only during t h e 30-s measuring periods to a v o i d decomposition. The m e m b r a n e was broken after a b o u t I0 rain b y a p p l y i n g a 500 m V pulse a n d t h e fluorescence was t h e n measured to o b t a i n a b a c k g r o u n d value. F o r a given comb i n a t i o n of lipid a n d alkaue, membranes having increasing concentrations o f d a n s y l gramieidin C were formed without moving t h e diaphragms or t h e cell. F i n e alignment o f t h e flatness of t h e m e m b r a n e was carried out b y withdrawing small volumes o f solution from the a p p r o p r i a t e side of t h e membrane. The fluorescence intensity above b a c k g r o u n d of the first m e m b r a n e of a series, which d i d n o t contain d a n s y l grainicidin C, was s u b t r a c t e d from t h a t of subsequent membranes, which contained the fluorescent antibiotic. F o r a different combination of lipid a n d alkane, t h e cell was disassembled a n d completely cleaned. F o r each series, t h e fluorescence of a 47 A glycerolester m e m b r a n e in t h e presence o f 1.3 × 1 0 - e M-ammonium 8-anilino-l-naphthalenesulfonic acid (Eastman) in 0-1 M-NaCI

F L U O R E S C E N T A N A L O G OF G R A M I C I D I N A

81

was also measured so t h a t the observed dansyl gramicidin C m e m b r a n e fluorescence could be related to this standard. A laser excitation modification of the standard m e m b r a n e fluorimeter was used to rigorously exclude the p o s s i b ~ t y of detecting some torus fluorescence. The best previous a t t e m p t to exclude torus fluorescence using conventional illumination utilized a wide emission aperture inside the cell (Alamuti & L~uger, 1970). A diagram of a cross.section through the center of the m e m b r a n e hole is shown in Fig. 2(b). The a t t e n u a t e d beam from a n argon-lon laser (Spectra-Physics, Model 171, with ultraviolet light mirrors) was separated into its two components using a diffraction grating a n d the 351.1-nm line was directed through the cell, as indicated. A silicone photodiode (International Recth%r) monitored the energy incident on the m e m b r a n e during the fluorescence measurement to correct for fluctuations. Movable apertures inside the cell eliminated stray light due to scattering from the windows of the cell. The black Teflon apertures were a b o u t 0.3 m m thick near the holes. The external a n d internal excitation apertures were both a b o u t 0-3 m m thick near the holes. The area of the circular external a n d internal excitation apertures were both a b o u t 3 × 10 -3 cm 2, a n d the two circular holes in the i n t e r n a l emission aperture were each a b o u t 1 × 10 -2 cm 2. The area of the m e m b r a n e hole was 0-2 c m 2. The visible beam from a h e l i u m - n e o n laser (Spectra-Physics 155) was made coaxial with the 351.1-nm beam at the diffraction grating to align the apertures, which were moved horizontally into place after each m e m b r a n e was formed. The alignment procedure for the laser modification differed from t h a t for the s t a n d a r d apparatus because the laser beam was n o t imaged on the membrane. The flatness of the m e m b r a n e was checked with a n additional light source, a n d alignment was achieved b y centering the alignment b e a m i n the right hole of the internal emission aperture. The lower limit on the n u m b e r of dansyl gramicidin C molecules t h a t can be detected is determined b y the background fluorescence of the bare membrane, presumably due to small a m o u n t s of chemically degraded lipids. For the glycerolmoneesters, the bare memb r a n e fluorescence corresponded to a b o u t 2 × 10 -15 reel cm -2 of dansyl gramicidin C ( q u a n t u m yield 0.45). The bare phesphatidyl choline m e m b r a n e fluoresced several times more t h a n those of the glycerolmonoesters. I n the fluorescence studies reported here, the surface density of dansyl gramicidLu C was greater t h a n about 10 -14 mol em -2 (Fig. 1). (c) Gonductance measurement~ on planar bilayer membranes The apparatus used for controlling the voltage across the m e m b r a n e and measuring the current has been described b y Eisenberg (1972). Strip Ag/AgC1 electrodes about 3 cm 2 i n area were used. For the voltage-jump experiments, a 200 m V pulse with 0.01 ms rise time lasting 5 s was applied to the membrane. The current was measured with a 104 to l0 s feedback resistor. The time-course of the voltage a n d current signals was recorded on a dual channel storage oscilloscope (Tektronics 546) with C12 camera. F o r the simultaneous conductance a n d fluorescence measurements voltage pulses of 10 m V lasting 2 s were automatically applied every 10 s a n d the resulting current fed into one channel of a dual channel strip chart recorcer (Houston Instruments). For highly conducting membranes, the resistance of the electrodes a n d solution in the absence of a m e m b r a n e was subtracted from the total resistance to obtain the m e m b r a n e resistance. F o r the laser excitation modification, where the apertures served as a further barrier to current flow, a differential voltmeter recorded the voltage through a n additional set of electrodes so t h a t polarization of the current carrying electrodes did n o t affect the results. Clearly, there is a n upper limit to the accurately measurable m e m b r a n e conductance. F o r a 0-2 c m 2 m e m b r a n e without internal apertures, the upper limit corresponds to channel densities of 5 x I0 -14 mol c m -2 (Fig. 1).

4. Results (a) Dansyl gramicidin G i8 a good analog of gramicidin A T h e c o n d u c t a n c e i n d u c e d b y g r a m i e i d i n A i n p l a n a r lipid bflayer m e m b r a n e s arises from t h e f o r m a t i o n of discrete c h a n n e l s ( H l a d k y & H a y d o n , 1970,1972). T h e m a g n i t u d e a n d trlneties of t h e s p o n t a n e o u s f l u c t u a t i o n s of t h e c o n d u c t a n c e of a 6

82

W . R . VEATCH E T ~4L.

membrane containing a low concentra~on of dansyl gramicidin C are like those of one with valine gramicidin A (Fig. 3). For both gramicidln~ the magnitudes of 95~/o of the channel fluctuations are within 30~/o of the most probable value, and 80~/o of them are within 6~/o of the most probable value. This most probable value of the single channel conductance was 40~/o less for dansyl gramicidin C t h a n for gramieidin A. A highly variable a m o u n t of smaller channels (having an average frequency of less than 5~/o) was observed for dansyl gramicidin C and somewhat less~often for gramicidin A. I n this paper we are concerned solely with the mean channel conductance, which closely a p p r o ~ m a t e s the most common value given in Table 2. Dansyl t5

..... I

I

I

........["'

--_o x

o {;I

L-

g o

5

O0

I

I0

I

20

I

30 Time ts)

I

I

40

50

I

I

40

50

60

(a)

15

I

I

I0

20

I

iO--

.0

50 Time ( s )

60

(b)

I~IG.3. Comparison of dansyl gramioidin C with valine gramioidin A. Single-channel conductance data. These records were obtained with 100 mY applied to a 40 A glycerolester membrane having an area of 7 × 10-8 cm2 in 1 M-KCLThe feedback resistor of the voltage clamp was 108 E and the effeotive time-constant of the system was 250 ms. The single-channel conductance of valine gramiddin A (a) is 4X 10-I1 ~ - I ; the value for dansyl Erau~eidin C (b) is 2-4x 10-11 ~-I.

FLUORESCENT

ANALOG

OF GRAMIOIDIN

A

83

TABL~ 2

OomTariso~ of the single-channe~conductance of dansy~ gramicidin G and ratine gramicidin A in dO ~ ¢lycerole~$erbilayer membranes Conductance ( D - 1) of Dansyl gramicidin C Valine gramicidin A

Ionic environment

1 M-KC1 1 m-NaCl

2.4 x 10 - ~ 1.5 x 10 - xx 4-OX lO -~2

0-I M-NaCI

4-0 x 10 -11

2-4 x 10 - xx 6-Ox 10 -12

gramicidin C also resembles valine gramicidin A at high conductance levels. Under these conditions, discrete channels are no longer resolved. The conductances of membranes formed from solutions containing equal concentrations of either dansyl gramicidin C or reline gramicidin A are compared in Figure 4. In general, the conductance induced by dansyl gramicidin C was two to threefold less than that induced by gramicidin A. This is partly attributable to the smaller unit conductance of the fluorescent analog and the rest is due to a decrease in the partition between membrane and torus, because the dimerization constant of the analog is not less than that of gramicidin A (vide infra). The kinetic characteristics of dansyl gramicidin C and valine gramicidin A were compared by the voltage-jump method of Bamberg & L~uger (1973). The results are given in Table 3. Dansyl gramicidin C and valine gramicidin A have nearly

I

/

I

/

/

/ / /•

~,-6 / /

/

/

/

/

I

/ 0

/

/ / / /o •

•

I

I

/

/

o

/o /

/

o/ /

/

/

/

o/ / / /

I

I

-8 -7 -6 -9 Log nominolconcentration(M)

FIG. 4. Comparison of dansyl gramicidin C w i t h reline gramlcidin k . Dependence of the conductance on the concentration in the membrane-forming solution. These measurements were made on 40 A glycerolester membranes in 1 •-KC1 a b o u t 6 rain after t h e y were t.hi~ned b y continuously applying a voltage of 100 i n V . The membrane area was about 7 x 10 -3 cm 2. The broken l i n e h a s a slope of 2.0. ( O ) Experimental results for reline gramicidin A and ( O ) those for r ] a ~ y l gramioidln C.

84

W . R. V E A T C H

ET

AL.

TABLE 3

Oompariso~ of the binaics and ~i~ibria of dimerizatio~ of dansyZ gramicidin G and gramicidi~ A in phospho2idyl choline bilayer membranes Parameter /¢b (s -1) /~ (s-1 reel-1 c m s) K (mol-1 em 2) K]Ko~

Ko (mol - 1 em 2)

Dansyl gramioidin C 0-6 2.5 × 1024 4.5 × 1014

Valine gramieidin A 0.7 2.5 X 101. 3.5 × 1014

N 4

,., 6

1 × 10I~

6 × 1018

These values were determined from voltage-jump experiments carried out in 1 ~r-NaC1. The size of the voltage jump was 200 mV. Uncertain due t o t h e large scatter of points for various membranes.

the same forward (/or) and reverse (kb) rate constants for the formation of channels at 200 mV. There was a large scatter of values for the equilibrium parameter ~ at low conductance, which is required to estimate the ratio of the dimerization constant at 200 mV (K) to the d~merization constant at zero voltage (Ko). There appears to be, at most, a factor of two difference between the Ke values for dansyl gramicidin C and gramicidin A. (b) Fluorescenc~~roperties of dansyl gramicidin G in phos't~hatidyl choline vesicles Phosphatidyl choline vesicles containing one molecule of dansyl gramicidin C per 50 molecules of phospholipid were prepared using the method of Batzri & Kern (1973) to ascertain the fluorescence properties of this dausyl derivative in a lipid bflayer membrane. The chromatographic elution profile of these vesicles on Sepharose 4]3 (Pharmacia) was virtually the same as for vesicles without dansyl gramicidin C, showing that their size distribution was unaltered by the presence of the fluorescent antibiotic. Furthermore, these vesicles retained trapped [14C]glucose to the same extent as vesicles devoid of dausyl gramicidin C. These findings suggest that dansyl gramicidin C does not grossly perturb the lipid bilayer. The corrected excitation and emission spectra of phosphatidyl choline vesicles containing dansyl gramicidin C are shown in Figure 5. The excitation peak at 355 mn and shoulder at 260 nm correspond to peaks in the absorption spectrum of the dansyl group, whereas the excitation peaks at 285 nm and. 290 nm are due to energy transfer from tryptophan. The emission spectrum has a peak at 530 nm~ The quantum yield of the dansyl fluorescence was 0.45±0.1, taking the magnesium salt of 8-an~]ino-1naphthalenesulfonic acid in ethanol as a standard of quantum yield 0.37. The nanosecond emission kinetics did not correspond to a single excited state lifetime. The mean lifetime was about 12 ns. Experiments are underway to determine why more than one excited state lifetime was observed. (e) Simultaneo~ conductance and fluorescence measurements of

dansyl gramicidin C in planar bilayer membranes The conductance and fluorescence intensity of planar bflayer membranes conta]nlng dausyl gramieidin C were measured simultaneously to test directly the elimer model.

F L U O R E S C E N T ANALOG OF G R A M I C I D I N A I ....

I

I

I

I

I

I

:500

320

:340

360

380

400

85

p °~

D rr"

260

280

420

Wavelength (nm) (o)

I

I

I

I

I

I

I

I

I

I

I

>,, ._ m

(=

,o n~

440

480

520

560 600 Wavelength (nm) (b)

640

660

Fzo. 5. Corrected fluorescence excitation and emission spectra of dRn~ylgramicidin C in vesicles The vesicles contained a 1 : 50 mol ratio of dau~yl gramicidin C to dioleoyl phosphatidyl choline in 0.16 M-KCI (plus 4% ethanol). For the excitation spectrum in (a) the emission wavelength was 530 nm and for the emission spectrum in (b) the excitation wavelength was 350 nm. The slitwidths were 4 nm and 2 nm, respectively. A red-sensitive photomultiplier tube (EMI 9658R) was used to obtain these spectra. As mentioned previously, such simultaneous measurements are feasible only if the fluorescence intensity is sumciently high and the membrane conductance is sufficiently low (Fig. 1). We found t h a t simultaneous fluorescence and conductance measurements could be made over a tenfold range in the concentration of dansyl gramicidin C in a thick 47 A glycerolester membrane. A plot of the logarithm of the conductance v e r s u s the logarithm of the fluorescence intensity (Fig. 6) has a slope of 2.0±0.3. For a certain a m o u n t of dansyl gramieidin C fluorescence, the 47 A glycerolester membrane has about 10a lower conductance t h a n the 26 A glyceroles~r membrane. Since gramicidin has the same single-channel conductance on all of the gIycerolester membranes, this means t h a t fewer than one gramicidin molecule in a thousand is

86

W. R. VEATCH . ~ T A L . qD

tD

) 0

-3

o

•

a "o

§

_1

o

o/

•

°/

I

2 Log fluorescence

FIO. 6. Conductance as a function of fluorescence for dansyl gramicidin C in 47 A glycerolester membranes. The laser excitation modification was used to obtain three independent sets of points. These fluorescence values were t h e n normalized using m e m b r a n e - b o u n d 8-anilino-l-naphthalenesulfonie acid as a fluorescence standard. The least-square slopes for these three sets of d a t a are 2-04-0.2 (C)), 2.14-0.2 (D), and 1.7+0.2 (O).

involved in channels under these conditions at any given time. Since K[G0] << 1, the observed slope of 2.0 proves that the channel is a dimer of the non-conducting species. Is every gramieidin dimer a conducting channel? The absolute surface density of gramicidin must be known to answer this question. Our task of converting the observed fluorescence intensity to the absolute surface density of gramicidin has been greatly facilitated by the work of Zingsheim & Haydon (1973). They determined the surface density of 8-anilino-1 -naphthalenesuffonic acid adsorbed to a 47/~ glycerolester planar bilayer membrane as a function of the concentration in the ambient solution. We used a 47 A glycerolester membrane in 0-1 ~-NaC1 containing 1.3× 10 -6 M8-anilino-l-naphthalenesulfonic acid, which gives a surface density of 7.1×10 -z8 tool cm -2 (Zingsheim & Haydon, 1973), as a membrane fluorescence standard. The surface density of dansyl gramicidin C could then be calculated from the ratio of the fluorescence intensity of dansyl gramicidin C to that of the 8-ani]ino-1naphthalenesuffonic acid membrane standard. The expression relating these quantities is $'~

~dcdQdH~

2'--~ = ¢;ataQaH a '

(8)

in which -~d is the fluorescence intensity of dansyl gramicidin C in the membrane, a a is the surface density of this species, ~a is its extinction coefficient at 350 nm, Qa is its quantum yield of fluorescence, and Ha is the relative efficiency of detection of the dansyI fluorescence. The corresponding quantities for the 8-anilino-l-naphthalenesulphonic acid membrane standard are denoted by the subscript a. We used the following values: Ea = 4000 cm -z M-z, Qa = 0.45 (estimated from fluorescence measurements of phosphatidyl choline vesicles), Ha ---- 1.0, ~= = 4000 cm -1 I~-1,

F L U O R E S C E N T ANALOG OF GRAMICIDIN A

87

Qa -~ 0.2 (Zingsheim & Haydon, 1973), ~ ---- 1.3, and aa = 7"1 × 10 - i s reel cm -2 (Zingsheim & Haydon, 1973). The surface density of dansyl gramicidin C is then given by Fd aa = ~aa × 6'2 × 10 -is mol cm -2. (9) A 26 A glycerolestsr membrane was used to determine whether every gramicidin dimer is a conducting channel. This membrane was chosen because most gramicidin molecules are dimeric (K[Go] >> 1), whereas t h e y are mostly monomeric (K[G0] ~ 1) in the 47 A glycerolester membrane, at the concentrations required for simultaneous fluorescence and conductance measurements. The experimental results are given in Table 4, which compares surface densities calculated from fluorescence measurements (ad) with those derived from simultaneous conductance measurements (a'd). The surface density a'd is calculated from the conductance b y the expression Membrane-specific conductance a'd -~ 2 × Single-channel conductance '

(10)

in which the factor of 2 takes into account the dimeric nature of the channel. This expression assumes t h a t all dimers are channels, and so the ratio a'd/a a should be equal to 1. The observed ratios range from 0.6 to 2.0 and have an average value of 1.4 (Table 4). We estimate t h a t the surface density derived from fluorescence measurements are correct within a factor of two, and t h a t the value from conductance measurements has a smaller error. Thus, these results strongly suggest t h a t most, if not all, gramicidin dimers are conducting channels. I f only half of the gramicidi~ waz

in the channels, then one would exl~ect a'a/a ~ = 0"5. The 2~resence era significant fractiou of non.conducting gramicidin in solvent lenses in the membrane would also result in a low a'~/a~ ratio, contrary to our result. The conductance and fluorescence of dansyl gramicidin C were simultaneously measured in three other planar bilayer membranes: 31 A and 40 A glycerolester membranes and one of phosphatidyl choline. The number of channels in these TABLE 4

Gomparicon of the sstrface densities of dansyl gramicidin 0 in 26 tt glyeerolester membranes calculated from simultaneous fluorescence arid conductance meazstrements Membrane ~ro~

(ores) 0-06t 0.2~

NaCI

Time after

tenon (~)

th~n~ng (~')

0.1 1.0 0.1

10 10 2 I0 2 10 10

1.0 0.2~

0-1

CMou]atedsurface density (reel ore- s) from from O/d/O'd conductance fluorescence (~'~) (~d) 5-5x 4.8x 1.8× 2-3 × 1.3× 2.7× 2.7×

I0 -lt I 0 -~4 10 - ~ 10 -~4 10 -~4 10 -~4 10 - ~

t Fluorescence measured by standard membrane fluorimetor. Fluorescence measured b y laser excitation modification.

2.8× 4"0× 2.0× 1-8 X 2.0× 1.5× 1-5×

10 -14 10 -1~ 10 -1~ 10 -14 10 -1~ 10 -14 10 -14

2.0 1-2 0.9 1.3 0-6 1.8 1.8

88

W.R.

VEATCH ET AL.

m e m b r a n e s is s h o w n as a f u n c t i o n o f t h e surface d e n s i t y o f d a n s y l g r a m i c i d m C in Figure 7. T h e previously described results for the 26 A a n d 47 ,~ glycerolester m e m branes are also included in this plot. I t is interesting to note t h a t the n u m b e r o f channels for a given surface density o f dansyl gramicidin C depends m a r k e d l y on the thickness a n d composition o f t h e p l a n a r bilayer membranes. I n particular, t h e 26 A glyeerolester m e m b r a n e has a b o u t 1000-fold more channels t h a n t h e 47 A glycerolester membrane.

-~4

IE (J o

E oJ

-J5

== c o

g' ..J

-16

-14

-1:5

Log total qromicidin, (mol cm-2)

FIG. 7. Channel surface density as a function of total gramieidin surface density for planar bilayer membranes of different composition. The lines are theoretical dimerization eurves like those in Fig. 1. The fluorescence has been converted to total gramicidin surface density according to equation (9), and specific conductance has been converted to channel surface density according to equation (10). The single-channel conductance of the glycerolester membranes in 0-1 ~-NaCI was taken to be 4 × 10-12 ~-1. For the phosphatidyl choline membrane in I M-NaC1, the singlechannel conduetanoe was estimated to be 7 × 10-12 ~ - 1 The laser excitation modification was employed for the 40 A and 47 A glycerolester membranes and the standard membrane fluorimeter for the others. (O) 47 A glyeerolester membrane; ({D) 40 A glycerolester membrane; ([-]) 31 A glycerolester membrane; ( • ) 26 A glyeerolester membrane; ([]) phosphatidyl choline membrane. (d) Dependence of the surface density of gramicidin A on its

concentration in the membrane-forming solution W e can n o w determine whether t h e a m o u n t of gramieidin in a m e m b r a n e is directly proportional to its nominal concentration, t h a t is, to its concentration in t h e m e m brane-forming solution. T h e 47 A glycerolester m e m b r a n e s were f o r m e d f r o m solutions

FLUORESCENT

ANALOG OF GRAMICIDIN

A

89

i n w h i c h t h e c o n c e n t r a t i o n o f g r a m i c i d i n A r a n g e d f r o m a b o u t 1 0 - 7 M t o 10 - 4 M. T h e surface d e n s i t y o f g r a m i c i d i n A, d e t e r m i n e d f r o m t h e v a l u e o f t h e m e m b r a n e c o n d u c t a n c e , is p l o t t e d as a f u n c t i o n o f t h e n o m i n a l c o n c e n t r a t i o n in F i g u r e 8. T h e e x p e r i m e n t a l p o i n t s e x h i b i t c u r v a t u r e a n d fall b e l o w t h e c a l c u l a t e d line h a v i n g a s l o p e o f 1. T h e p a r t i t i o n c o e f f c i e n t for g r a m i c i d i n A b e t w e e n t h e 47 A g l y c e r o l e s t e r m e m b r a n e a n d t h e t o r u s w a s a b o u t t e n f o l d less a t a n o m i n a l c o n c e n t r a t i o n o f 10 - 4 M t h a n a t 1 0 - 7 M. Clearly, t h e s u r f a c e d e n s i t y o f g r a m i c i d i n A i n a g l y c e r o ] e s t e r m e m b r a n e is n o t d i r e c t l y p r o p o r t i o n a l t o i t s n o m i n a l c o n c e n t r a t i o n . I n p h o s p h a t i d y l choline m e m b r a n e s , t h e p a r t i t i o n coefficient is m o r e c o n s t a n t o v e r a c o m p a r a b l e range of nominal concentration. I

1

-12

I

I

SLOPE"I~~ c

-15 -14

/o

o, -16

I -7

I

I

I

-6 -5 -4 Log nominol concentrolion (M)

FIG. 8. Dependence of the surface density of gramicidin A in 47 A glycerolester membranes on its nominal concentration. The lowe¢ concentration samples were obtained by serial dilution of membrane-forming solutions having a high nominal concentration of gramicidin A into membraneforming solutions lacking gramicidin A. The membrane area was 0-04 cm ~, the salt concentration was 0.1 M-NaC1, and brief 10-mV pulses were used to monitor the conductance, which varied little over the first 15 rain after thinning. The total gramioidin A surface density was calculated from the measured specific conductance and a dimerization constant of 10ix reel- x em 2. The line having a slope of 1 corresponds to the ideal case in which the partition coefficient is independent of nominal concentration. The experimental points (C)) fall below this line. TABLE 5

Dimerization constant8 for dan~yl graraicidin C estimated from 8imultaneo~ fluorescence and conducgance meae~ren~ent8 Membrane Glycerolcster

Hydrocarbon thickness (A)

Dimerization constant (mol-1 cm 2)

47t 40t 31~

3× 101~

26~ Phosphatidyl choline

48~

t From Hladky & Haydon (1972). From Bamberg & LtLuger (1973).

1 x 1011

~ 1014 __~101" 2 x 10~s

90

W. R. VEATCH B T A Z .

We also investigated the effect of membrane thickness on the partition of gramieidin between the membrane and the torus. Glycerolester membranes ranging in thickness from 26 A to 47 A were formed from membrane-forming solutions containing 10 -5 Mdansyl gramicidin C. The fluorescence intensities of these membranes differed by less than a factor of three, indicating that the partition coefficient does not markedly depend on membrane thickness. This finding suggests that the partition coefficient is relatively independent of the ratio of monomer to dimer. 5. Discussion The structure of the gramicidin transmembrane channel is of interest as a model for the passive ion-pathways of ceil membranes. The fluorescent chromophore of dansyl gramicidin C provides a useful probe of that structure. The conductance properties of dansyl gramicidin C are similar to those of gramidicin A. The strong fluorescence of th~ active analog enabled us to measure its absolute surface density in planar bilayer membranes. In previous studies, the concentration of gramicidin A in the bilayer membrane was not directly measured. The number of active channels corresponding to a particular surface density of gramicidln was ascertained from simultaneous measurements of the membrane conductance and fluorescence. Three conclusions can be drawn from these experiments. (1) The slope of 2-0±0.3 in the double logarithmic plot of the conductance versu8 the fluorescence intensity, over a concentration range for which nearly all of the gramicidin is non-conducting (Fig. 6), proves that the channel is a dimer of the nonconducting species, as first suggested by Tosteson et al. (1968). (2) In the 26 A glycerolester membrane, nearly all of the dansyl gramicidin G was dimeric. The number of channels in this membrane was approximately equal to the number of dimers (Table 3). Hence, most i f not all dimers are channels. Thus, it should be possible to use spectroscopic techniques to characterize the conformation of the transmembrane channel. (3) The dimerization constant of dansyl gramicidin C increases 30.fold in going from a 47 A to a 40/~ glycerolester membrane (Table 4). The dimerization constant in a 48 A phosphatidyl choline membrane is 200 times larger than in a 47 A glycerolester membrane. Thus, the dimerization constant depends markedly both on the type of lipid and on the thickness of the hydrocarbon core. Hladky & Haydon (1972) found that the mean duration of a single channel increases monotonieaUy the thinner the membrane. They attributed this effect to a variation in the extent of local thinn~ug or dimpling of the membrane in the vicinity of the conducting channel. Our data are consistent with their proposal. The greater than 1000-fold decrease in the dimerization constant in going from a 26 A to a 47 A glycerolester membrane corresponds to an increase in the free energy of dimerization of at least 4.5 kcal/mol. This free energy difference might represent the cost of deforming a small region of the 47 A membrane to match the length of the gramieidin channel. The d~merization constant of 2 × 1018 mol-: cm9 from fluorescence measurements for dansyl gramicidin C in dioleolyl phosphatidyl choline is a factor of five less than the value we obtained using the voltage-jump technique of Bamberg & L~uger (1973) for dansyl gramicidin C on the same membrane (Table 3). This agreement is fairly good, since the higher value would correspond to only a 40~o decrease in the total surface density from the absolute fluorescence. It should be noted that our fluorescence

FLUORESCENT ANALOG O F GRAMICIDIN A

91

technique only provides a lower boundary for high dimerization constants, whereas the voltage-jump technique provides only an upper boundary for low dimerization constants. The design of the membrane fluorimeter merits some comment. In general, there are two major hindrances to ma~ing precise measurements of the fluorescence intensity of a planar bilayer membrane. Raman scattering and fluorescence arising from the solution can give a much larger signal than the fluorescent species in the membrane. The torns surrounding the bilayer membrane also poses a problem, because its volume may be l0 s times larger than that of the bilayer membrane. These background signals can be greatly reduced by exciting and collecting the emission from a small area of the membrane. I t is also important that the angle between the excitation and emission beams be between about 60 ° and 120% These constraints make it difficult to align different membranes for quantitative comparisons of the fluorescence intensity. This alignment problem was solved by using the devices and procedures described in Materials and Methods. A most useful criterion for assessing whether any torus fluorescence is being detected is to use a membrane-bound fluorescence standard that is present at a negligible concentration in the torus. Such a standard is provided by the fluorescence of a 47 A glycerolester membrane in equilibrium with a solution of 8-anillno-l-naphthalenesuffonic acid (Zingsheim & Haydon, 1973). We found that the ratio of dansyl gramicidin C fluorescence to that of 8-anilino-1naphthalenesuffonic acid was approximately the same for 0.06-cm 2 and 0.2-cm2 membranes (Table 4). Also, the ratio observed with the standard membrane fluorimeter was about the same as with the laser excitation modification, showing that very little fluorescence was detected from the torus. The laser excitation modification yields consistent results independent of the optical quality of the cell windows, which can deteriorate with time in experiments involving many membranes such as those in Figure 6. We were readily able to detect 10 -1~ tool cm -2 of dansyl gramicidin C in a bflayer membrane. There are about l0 s lipids per gramicidin under these conditions. This surface density corresponds to 60 fluorescent molecules per ~m ~. I t is interesting to compare this detection limit with the surface density of receptors in biological membranes, which are known to range from about 1/~m 2 (e.g. the insulin receptor in the plasma membrane of adipose cells; Cuatrecasas, 1971) to 2 × 104/~m 2 (e.g. the acetylcholine receptor in postsynaptic membranes; Porter ~ al., 1973). Thus, the fluorescence techniques described here should be sufficiently sensitive for fluorescence studies of the conformation and dynamics of reconstituted assemblies in planar bflayer membranes. We thank Dr Erhard Gross for providing the countercurrent-purified gramicidins. One of us (W. V.) was supported by the Membrane Pathology training grant (GM-00167). One of us (R. M.) is a Helen Hay Whitney Fellow and the third author (M. E.) was an IB1VI Fellow. We thank Mr Gerald Johnson for expert technical assistance. This work was supported by grants from the National Institute of General Medical Sciences (GM-16708 and Gi~L21716). REFERENCES Alamuti, N. & L~uger, P. (1970). Biochim. B~phys. Ac~, 211, 362-364. Bamberg, E. & L~uger, P. (1973). J. Membr. BioL 11, 177-194. Batzri, S. & Korn, E. D. (1973). Biochim. Biophys. Ac~, 298, 1015-1019.

92

W . 11. V E A T C H

ET

AL.

Chappell, J. B. & Crofts,A. R. (1965). Biochem. J. 95, 393-402. Cuatrvcasas, P. (1971). Proo. N a t Acid. ~ . , U.S.A. 68, 1264-1268. Eisenberg, M. (1972).Ph.D. Thesis, CaliforniaInstituteof Technology. Fettiplace, R., Andrews, D. M. & Haydon, D. A. (1971). g. Membr. BIOL 5, 277-296. Goodall, M. C. (1970). Biochim. Biophys. Act~, 219, 471-478. Harris, E. J. & Pressman, B. C. (1967)./Vat, re (London), 216, 918-920. Haydon, D. A. & Hladky, S. B. (1972). Quart Rev. Biophys. 5, 187-282. Hladky, S. B. & Haydon, D. A. (1970). iVa~ure ( ~ ) , 225, 451-453. Hladky, S. B. & Haydon, D. A. (1972). Bioch/m. Biophye. ~4ctu, 274, 294-312. Kolb, H. A., L~uger, P. & Bamberg, E. (1975). J. Membr. Biol. 20, 133-154. Krasne, S., Eisenman, G. & Szabo, G. (1971). ~c/once, 174, 412-415. Mueller, P. & Rudin, D. O. (1967). Biochem. Biophys. Res. Commun. 26, 398-404. Porter, C. W., Chiu, T. H., Wieckowski, J. & Barnard, E. A. (1973). iVature New BIOL 241, 3-7. Sarges, R. & Witkop, B. (1965). J. Ame~'. Chem. Soo. 87, 2011-2020. Tosteson, D. C., Andreoli, T. E., Tieffenberg, M. & Cook, P. (1968). J. Gen. Physiol. 51, 373S-384S. Zingsheim, H. P. & Haydon, D. A. (1973). Biochim. Biophys. Acta, 298, 755-768. Zingsheim, H. P. & Neher, E. (1974). Biophys. Chem. 2, 197-207.