More than a century ago, studies of tadpole narcosis led to the Meyer–Overton rule describing a positive Keywords Adair theory; kinetic modelling; lipid ⁄ protein interface; nicotinic ac
Trang 1Kinetic analysis of tadpole narcosis
Joachim Altschuh1, Sebastian Walcher2and Heinrich Sandermann, Jr3,4
1 Institute of Biomathematics and Biometry, GSF – National Research Center for Environment and Health, Neuherberg, Germany
2 Lehrstuhl A fu¨r Mathematik, RWTH Aachen, Germany
3 Institute of Biochemical Plant Pathology, GSF – National Research Center for Environment and Health, Neuherberg, Germany
4 ecotox.freiburg, Germany
High-resolution X-ray diffraction structures of integral
membrane proteins have revealed structurally diverse
annular, nonannular, single leaflet and laterally
con-nected lipid-binding sites [1–4] Each of the multiple
lipid-binding sites of integral membrane proteins may
be, to a certain extent, structurally unique, as best
documented for the tightly bound phospholipid,
cardio-lipin [1–4] In spite of their analytical power,
spectro-scopic methods have so far failed to resolve the
multiple protein-bound lipids Both ESR [5,6] and
fluorescence spectroscopy [2,7] use overall relative
macroscopic lipid-binding constants The similarity of
these binding constants among different membrane
proteins has led to the conclusion that binding to the
annular lipid shell shows relatively little structural
specificity [2] The overall microscopic lipid-binding
constants of four integral membrane enzymes also were quite uniform [8] By contrast, there is evidence for special lipid-binding sites having high structural specificity, or dominating the lipid dependence of func-tional proteins For example, the mitochondrial b-hyd-roxybutyrate dehydrogenase needs the specific lipid, phosphatidylcholine, in a background of other phos-pholipids [9,10], and a specific requirement for cardio-lipin in a background of other lipids is documented for cytochrome oxidase [11] and processes of oxidative phosphorylation [12] In order to kinetically resolve protein-bound lipids, an extended Adair-type analysis
of tadpole narcosis was performed employing micro-scopic lipid-binding constants
More than a century ago, studies of tadpole narcosis led to the Meyer–Overton rule describing a positive
Keywords
Adair theory; kinetic modelling; lipid ⁄ protein
interface; nicotinic acetylcholine receptor;
tadpole narcosis
Correspondence
H Sandermann, GSF – National Research
Center for Environment and Health, Institute
of Biochemical Plant Pathology, Ingolsta¨dter
Landstraße 1, D-85764 Neuherberg,
Germany
Fax: +49 89 3187 3383
Tel: +49 89 3187 2285
E-mail: sandermann@gsf.de
(Received 16 December 2004, revised
4 March 2005, accepted 10 March 2005)
doi:10.1111/j.1742-4658.2005.04657.x
High-resolution X-ray diffraction structures of integral membrane proteins have revealed various binding modes of lipids, but current spectroscopic studies still use uniform macroscopic binding constants to describe lipid binding The Adair approach employing microscopic lipid-binding con-stants has previously been taken to explain the enhancement of agonist binding to the nicotinic acetylcholine receptor by general anaesthetics in terms of the competitive displacement of essential lipid activator molecules [Walcher S, Altschuh J & Sandermann H (2001) J Biol Chem 276, 42191–42195] This approach was extended to tadpole narcosis induced by alcohols A single class, or two different classes of lipid activator binding sites, are considered Microscopic lipid and inhibitor binding constants are derived and allow a close fit to dose–response curves of tadpole narcosis
on the basis of a preferential displacement of more loosely bound essential lipid activator molecules This study illustrates the potential of the Adair approach to resolve protein-bound lipid populations
Abbreviations
nAChR, nicotinic acetylcholine receptor; SSD, sum of squared deviations.
Trang 2correlation between the lipophilicity of chemicals and
their anaesthetic potency [13,14] The underlying
molecular mechanism has remained controversial
General anaesthetics were proposed to act primarily by
perturbing the membrane lipid phase [13,14], or by
attacking hydrophobic protein pockets [15,16] The
latter hypothesis is favoured by site-directed amino
acid exchanges [17–19] and by photoaffinity labelling
[19,20] However, many of the findings supporting lipid
or protein target sites can also be interpreted in terms
of the lipid⁄ protein interface as a third candidate
target site We have previously developed a kinetic
framework for anaesthetics acting by competitive
displacement of essential lipid activators from the
lipid⁄ protein interface [21–23] This mechanism has
been tested with synaptosomal Ca2+-ATPase [24] and
the Torpedo nicotinic acetylcholine receptor (nAChR)
[23] Subsequent to our report [23], a competitive
dis-placement of lipids bound to the nAChR has also been
reported for free fatty acids [25] and local anaesthetics
[26] The nAChR is the by far best-characterized
mem-ber of the superfamily of ligand-gated ion channel
proteins [27], which is currently thought to harbour
the true target site(s) of general anaesthesia [15–20]
The lipid⁄ protein interface of the nAChR and its
microscopic binding and inhibition constants are used
here as a prototype that allows a close fit to dose–
response data for tadpole narcosis
Results
Experimental system
This study was based on the fact that the reported Hill
coefficients of tadpole narcosis are in a diagnostic
win-dow that is typical for the activation [28] and
inhibi-tion [21] of lipid-dependent funcinhibi-tional membrane
proteins, namely Hill coefficients in the range 2.5–4.0
Tadpole narcosis has been characterized by Hill
coeffi-cients of around 4.0 when 14 different aliphatic
alco-hols were tested [29] and of around 3.4 with four
different cycloalcohols [30] The Hill coefficients of the
previously analyzed enhanced agonist binding to the
nAChR were between 2.3 and 4.0 [23,31], and were
thus in the same diagnostic window The multiple-site
kinetic formalism for lipid-dependent proteins has
therefore previously been applied to the nAChR [23]
and is now applied to tadpole narcosis General
anaes-thetics are well known to also interact with the
lipid-free pore region of the nAChR, but this inhibitory
effect has much lower kinetic cooperativity (Hill
coeffi-cients 1.0–1.3) [32,33] In order to employ the
pre-viously characterized lipid⁄ protein interface of the
nAChR as a prototype target site of tadpole narcosis, the microscopic inhibition constants of narcotic chemi-cals need to be known The KI-value of 1-hexanol has previously been determined [23] The additional KI val-ues needed for the present study were derived from other previously determined KI values as described in Experimental procedures and summarized in Table 1
Kinetic modelling
A basic task of our kinetic modelling was to explain the ‘leftward shift’ of the dose–response curves of tad-pole narcosis relative to those of nAChR agonist bind-ing enhancement It is known that the members of ligand-gated channel superfamilies [18–20], and mem-brane proteins generally [1–4], have sequence-specific rough surfaces that are likely to lead to nonuniform binding constants for the multiple essential lipid acti-vators Therefore, it seemed appropriate to develop a general Adair-type formalism for the case of different binding sites (see Appendix) The more loosely bound lipid ligands have higher microscopic lipid dissociation binding constants, KL, and will more easily undergo displacement by general anaesthetics The KL-value previously derived for the nAChR [23] is therefore allowed to increase in order to simulate the unknown narcotic receptor(s) of tadpoles, all other parameters characterizing the nAChR lipid⁄ protein interface remaining constant The initial analysis assumes a uni-form increase in the microscopic dissociation constant,
KL, of all 40 lipid activator sites of the nAChR This
is illustrated for 1-hexanol in Fig 1 An approximation
to the data points for tadpole narcosis and the ‘left-ward shift’ is achieved by increasing KL 8.2-fold to
387 nm This overall increase is within the 10-fold range of average macroscopic lipid-binding constants
of various membrane proteins as documented by ESR [5] and fluorescence spectroscopy [2] A 10-fold range
of overall macroscopic binding constants for different lipids has recently also been documented by ESR-spectroscopy for the nAChR [26] In the case of b-hydroxybutyrate dehydrogenase there was a 15-fold difference in macroscopic binding constants between the specific lipid activator, phophatidylcholine, and the nonactivating lipid, phosphatidylethanolamine [10] The curve fits to the data points for tadpole narcosis
by the additional alcohols of Table 1 are shown in Fig 2 along with the theoretical curves for the enhancement of agonist binding to the nAChR The latter curves were calculated with the KI values of Table 1 and Eqn (1) of Walcher et al [23]
As discussed above it is unlikely that all lipid-bind-ing sites of a sensitive target protein differ by the same
Trang 3factor from closely related nontarget members of the same superfamily We therefore applied our general-ized Adair-type formalism to differentiate between two lipid-binding classes (Appendix) A fixed number, m,
of the total of 40 lipid-binding sites of the receptor is allowed to have increased KL values For example, if
m¼ 4 lipids are assumed to be more loosely bound, a factor of 270 is required to achieve a fit to the data points for 1-hexanol The results of a more extensive analysis of assuming two lipid-binding classes are sum-marized in Table 1 The corresponding dose–response curves are shown in Fig 1 for 1-hexanol and in Fig 2 for the other tested alcohols
Statistical quality Although the focus of the mathematical approach is on the ‘leftward shift’, illustrated in Figs 1 and 2, a statisti-cal analysis was performed for additional justification The sums of squared deviations (SSD) for a uniform increase in KL were between 0.022 and 0.38 but were higher for small values of m All SSD values are listed
in Table 1 It should be noted that the SSD values of tadpole narcosis are of poorer quality than of agonist binding enhancement [23] However, a survey of the lit-erature on tadpole narcosis showed that the two studies used [29,30] were among the best documented The curve fits of Figs 1 and 2 were obtained by varying only
Fig 1 Fitted dose–response curves for 1-hexanol The principle
(upper) is to mathematically transfer the dose–response curve for
the nAChR [23] to the data set for tadpole anaesthesia [29] This
‘leftward shift’ of the whole-animal response is achieved by
increasing the microscopic lipid dissociation constant, K L , of the
nAChR over all binding sites: m ¼ 40 (solid line), or over part of
the binding sites: m ¼ 10 (dotted line), and m ¼ 4 (dashed line).
The data points for enhancement of agonist binding (s) were taken
from Miller et al [31] and those for tadpole narcosis (d) from
Alifimoff et al [29].
Table 1 Results of regression analyses Values of log KOW, KI, KL¢ and SSD for all alcohols analysed In addition to the fitting procedure des-cribed in the Results, the data were also fitted using the standard two-parameter fits of the Gauss and logistic procedures; the correspond-ing SSD values are shown in the last two columns.
Chemical log KOWa KIb (m M )
No receptors
m with KL¢ c KL¢ c (l M ) SSD d,e SSD d,f SSD d,g
1-Hexanol 2.03 h 0.069 j 40 0.387 0.22 0.21 0.21
1-Octanol 3.00 h 0.011 k 40 0.642 0.38 0.23 0.22
Cyclohexanol 1.23 h 0.25 k 40 0.182 0.022 0.0092 0.011
Cycloheptanol 1.88 i 0.080 k 40 0.214 0.022 0.028 0.029
Cyclooctanol 2.53 i 0.026 k 40 0.244 0.038 0.012 0.011
a
Logarithms of octanol ⁄ water partition coefficient b
Microscopic inhibition constant.cMicroscopic lipid dissociation binding constant of a class of more loosely bound activator molecules binding to a number m of receptor binding sites d Sum of squared deviations e For nonlinear regression with the present kinetic model f Gauss fit g Logistic fit h From Hansch et al [43] i Estimated with ALOGP 2.1 [45] j From Walcher et al [23].kFrom regression analysis, see Experimental procedures.
Trang 4a single parameter, KL Curve fits with comparable
SSD values were obtained when the data sets were
sub-jected to the standard two-parameter fits of the Gauss
and logistic procedures [34] As expected, two variable
parameters generally produced a better fit, but the
mechanism-related model was, at least for m¼ 40, of
comparable quality Overall, statistical quality was
determined by the quality of the data sets used
Discussion
Animal narcosis
Narcosis is assessed by some all-or-none quantal
response of individuals in an animal test population
Theoretically, if all members of the population were
equally responsive, the dose–response curve would be
a sharp step-function with infinite steepness [18,35]
Hill coefficients for anaesthesia of mice, rats, dogs and
humans have been derived and were found to be
between 10 and 22 [15,18,36] Similarly, high slope
val-ues were reported in a recent study with several mouse
strains when either the loss of righting reflex or a tail
clamp assay was employed [37] High Hill coefficients
of around 13 were also reported for narcosis of brine
shrimps exposed in artificial sea water [38] Uniquely
low slope values of narcosis have been reported for tadpoles The two independent studies [29,30] re-ana-lysed here used different tadpole species and experi-mental conditions Loss of the righting reflex was assessed in one study [29], whereas the second study [30] measured the lack of sustained swimming Hill coefficients of around 4.0 [29] and 3.4 [30] were in a diagnostic window for lipid-dependent enzymes and receptors [21–23,28] so that our analysis became possible The quantal responses of tadpoles seem to reflect the titration of some as yet unidentified lipid-embedded target site(s) The much higher slope values
of narcosis obtained with other animal species and man are attributed to population heterogeneity [35] and⁄ or synaptic-block [36] or threshold sensing [18] mechanisms It seems unlikely that the low Hill coeffi-cients for tadpoles can be explained by population heterogeneity alone, because this would imply an extre-mely high heterogeneity for tadpoles in contrast to all other animal species A search for a particular mech-anism therefore seems reasonable
Role of loosely bound lipid activators The above analysis shows that loosely bound essential lipid activator molecules could act as an anaesthetic
Fig 2 Fitted dose–response curves for the straight-chain and cyclic alcohols of Table 1 Only the microscopic lipid-binding constant
K L was allowed to vary, all other parameters describing the lipid ⁄ protein interface [23] remaining constant The curves for the nAChR shown on the right were calculated
as described in the Results The following fitted curves to the data points (d) for tadpole narcosis [29,30] are shown on the left: m ¼ 40; (solid line), m ¼ 10 (dotted line), and m ¼ 4 (dashed line).
Trang 5target site as an alternative to lipid-free hydrophobic
protein pockets which are currently favoured in the
lit-erature [18,20] So far, there is only indirect
experimen-tal evidence for such loosely bound lipid activator
molecules X-Ray and electron diffraction studies have
well documented the opposite situation, namely the
existence of particular tightly bound lipid molecules
such as cardiolipin [1–4] The lipid-binding sites of the
nAChR or other ligand-gated channel proteins have
not been resolved into subclasses by direct
measure-ment although the sum of the available evidence is
strongly in favour of heterogeneous lipid-binding sites
[19,26,27] Indirect support comes from the case of a
K+-channel-associated peptide, in which mutation of a
single amino acid has been shown by
ESR-spectro-scopy to change lipid specificity [39]
The lipid⁄ protein interface of the nAChR was used
here as a prototype to explain tadpole narcosis by
assu-ming a preferential displacement of loosely bound lipid
activator molecules This analysis employed Adair-type
kinetics and microscopic lipid and inhibitor binding
constants Recent reviews on high-resolution X-ray and
electron diffraction structures agree with a picture of
high diversity in lipid-binding modes [1–4] The
func-tional importance of individual lipid⁄ protein binding
sites is illustrated by site-directed amino acid
exchan-ges along transmembrane helices [40] Amino acid
exchanges near the interface region, or in the lipid
hydrocarbon region, have been shown to influence the
orientation and function of transmembrane proteins
[19,40] The existence of multiple lipid⁄ protein binding
sites has led to different conclusions On the one hand,
it was stated that a full description of binding to such a
heterogeneous surface is an impossibly difficult task [2]
On the other hand, functional assays of reconstituted
proteins are proposed to be most crucial to yield
infor-mation on the specificity of binding of particular vs
bulk lipids, the number of their binding sites and the
occupancies and the cooperativity of their potential
interactions [1] In these proposed functional assays,
Adair-type kinetics and the use of microscopic binding
constants appear to be one of the tools to achieve the
required resolution Methods to directly determine
microscopic lipid-binding constants still remain to be
developed However, an indirect support of the present
analysis is provided by molecular dynamics simulation
that has recently identified the lipid⁄ protein interface as
anaesthetic target site of the gramicidin ion channel [41]
Experimental procedures
This study is based on two independent data sets for
tad-pole narcosis by various alcohols [29,30] The previously
published characterization of the lipid⁄ protein interface of the nAChR [23] including the total number of n¼ 40 lipid-binding sites of the nAChR [19,42] was adopted, as were the other kinetic constants previously derived [23] This concerns in particular the microscopic lipid dissociation constant, KL, of the nAChR under the first-order assump-tion of identity of lipid-binding sites In terms of ‘two-dimensional’ kinetics, KLamounted to 3.75 lipid molecules per receptor molecule This corresponded to a bulk lipid concentration of 46.9 nm [31] The previously determined
KI value of 1-hexanol (5500 molecules per receptor mole-cule, corresponding to 68.7 lm) [23] is adopted here, while the KIvalues of the other straight-chain and cyclic alcohols studied were calculated from a regression equation of the logarithms of KI values [23] vs the logarithms of octa-nol⁄ water partition coefficients, log KOW [43] The regres-sion is based on data of ethanol, ether, 1-hexanol, isoflurane, and methoxyflurane: log KI¼)0.759Ælog KOW
)2.67 (N ¼ 5, r2¼ 0.76, SD ¼ 0.44) The higher alcohols studied in tadpole narcosis [29,30] had log KOWvalues out-side the range of applicability of the regression and were therefore not included in the analysis mathematica 4.1 software was employed for all calculations
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Appendix
The Adair approach employing microscopic
lipid-bind-ing constants has previously been taken to explain the
enhanced agonist binding to the nAChR in terms of
the competitive displacement of essential lipid activator
molecules [23]
We generalize and extend this approach by allowing
for different lipid-binding constants in this study Thus
we consider a macromolecule P with a number n of
bind-ing sites for lipid L as well as for inhibitor I Because the
sites may no longer be identical for lipid, we have
micro-scopic lipid dissociation constants KL1, KL2, , KLn
However, we assume that the sites are identical for small
inhibitor molecules that are assigned a uniform
micro-scopic lipid dissociation constant KI
According to the basic model of lipid dependence
[28], there is an integer a < n with the following
prop-erty: a macromolecule is not functional if fewer than
n) a binding sites are occupied by lipid, and fully
functional if at least n) a sites are occupied by lipid
We derive a formula for the fraction r of functional
macromolecules
To begin, we give an expression for the total
concen-trations Consider a fixed binding site (say, of number
j) and denoted by [Pj0] the concentration of
macromol-ecules with empty site j According to the mass-action
law, the concentration of molecules with lipid bound
to site j is given by
Pj0
ơL
KLj and the concentration of molecules with inhibitor
bound to site j is given by
Pj0
ơI
KI Therefore the total concentration is given by
Pj0
1 ợ ơL
KLj
ợơI
KI
Because the sites are independent, the total concentra-tion is equal to the product
P0
ơ 1 ợ ơ L
KL1
ợơ I
KI
1 ợ ơ L
KL2
ợơ I
KI
::: 1ợ ơ L
KLn
ợơ I
KI
with [P0] the concentration of ỔemptyỖ molecules (with
no binding site occupied) This expression may be rear-ranged as
ơP0 Xn kỬ0
Qk Lơ k with
Qk Ử 1ợơI
KI
Sk 1
KL1;
1
KL 2
::: 1
KLn
and Sk denoting the k-th elementary symmetric poly-nomial in n variables, thus
S0đx1;x2; ;xnỬ 1;
S1đx1;x2; ;xnỡ Ử x1ợ x2ợ xn
Snđx1;x2; ;xnỡ Ử x1 x2 xn Generally, Sk (x1, , xn) is formed by taking all possible products of k distinct factors among the
x1, , xn, and then summing these up For more information see Lang [44]
To verify this expression, note
1ợơ I
KI
ợơ L
KLj
KLj L
ơ ợ KLj 1 ợơ I
KI
and use the algebraic identity
L
ơ ợ x1
đ ỡ Lđơ ợ x2ỡ Lđơ ợ xnỡ
Ử Lơ nợXn1
kỬ0
Snkđx1; ;xnỡ Lơ k
The biochemical interpretation of this rearrangement is the following: The term
P0
ơ Qk Lơ k
is equal to the concentration of those macromolecules
to which exactly k lipid molecules are bound Thus the fraction of functional molecules is given by
Trang 8Pn k¼na
Qk½Lk
Qn j¼1
1þK½L
Ljþ½IK
I
because [P0] cancels in numerator and denominator
For practical computations (with a usually quite small
compared with n) another transformation is useful:
Divide numerator and denominator by [L]n, and set
S¼ 1 ⁄ [L] Then
r¼
Pa k¼0
QnkSk
Qn j¼1
1
KLjþ S þ S K½I
I
Here the numerator is just the degree a Taylor poly-nomial of the denominator, which can be deter-mined using built-in functions of symbolic computation systems
If all the KLj¼ KL are equal, we recover the for-mula from Walcher et al [23] (with q¼ 1)
In this study we discuss the scenario when there are two classes of lipid-binding sites, i.e there is a number
m, 0 < m £ n, such that m sites have dissociation con-stant KL0, and the remaining n) m sites have dissoci-ation constant KLfor lipid