DTIC ADA231655: Channel Protein Engineering: A Novel Approach Towards the Molecular Dissection Determinants in Ligand-Regulated Channels

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It. TITLE (Include Security Classification)
Channel Protein Engineering: A Novel Approach Towards the Molecular Dissection
Determinants in Ligand-regulated Channels
12. PERSONAL AUTHOR(S)
Montal, Mauriclo
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FIELD GROUP SUB-GROUP protein structure, orotein design, acetvlcholine receDt:-r,
L3 ionic channels, biological membranes, signal transducri::..
19 ABSTRACT (Continue on reverse if necessary and identify by block number)
Our goal is to establish the molecular determinants that account for the pore
properties of voltage-gated and ligand-gated channel proteins. We are modeling
hypothesized pore-forming structures of sodium channels, calcium channels, potassium
channels and acetylcholine receptors. We succeeded in designing a calcium channel
protein according to our model. Accordingly, a protein was synthesized by solid-phase
methods. The synthetic channel protein forms channels in lipid bilayers that emulate
the pore properties of the authentic channel. The key role of specific residues
in determining the activity of these channel proteins is currently being assessed.
These studies are guided by the results on "computer mutations."
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Dr. Igor Vodyanoy ,(202) 696-4056 ONR
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ca - ('D. R&T Code: 4414911
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.. DATE: 23 January 1991
PROGRESS REPORT ON CONTRACT N00014-89-J- 1469
PRINCIPAL INVESTIGATOR: Dr. Mauricio Montal
CONTRACTOR: University of California, San Diego
CONTRACT TITLE: Channel Protein Engineering: A Novel Approach Towards the Molecular
Dissection Determinants in Ligand-regulated Channels
START DATE: 1 February 1989
RESEARCH OBJECTIVE: To identify the structures forming the channel lining of ligand-gated
channels; to characterize the single channel properties of the pore-forming structure; and to model
the structure using molecular mechanics and dynamics.
PROGRESS (Year 2)
1. Molecular modeling of the pore forming structures of voltage-gated and ligand-
gated channel proteins.
We pursued the modeling of the pore forming structures of the sodium channel and the
acetylcholine receptor (Proteins 8:226-236, 1990, enclosed reprint). Further, we calculated the
energy profiles for cations and found them to be consistent with the permeation properties
observed experimentally. We introduced the use of "computer mutations" whereby specific
residues are substituted and thereby various molecular contributions to the binding sites and energy
barriers are deduced. Notably, for the acetylcholine receptor channel, an innermost site is found to
be attributable to -OH groups of serine residues, consistent with our previous modeling and more
recent experiments. However, a most external site is found to arise from a ring of highly
conserved phenylalanine residues, not usually regarded as providing favorable interactions with
ions. This site may provide favorable interactions with amphipathic channel blockers such as local
anesthetics. Calculations are currently being extended to include a molecule of QX-222, a
quarternary ammonium derivative of the local anesthetic lidocaine, within the lumen of the pore.
Specific predictions for possible experimental mutations are made which can serve to test both the
proposed structure, as well as the computational inferences we have drawn from it (Progr. Cell
Res. 1:195-211, 1990, enclosed reprint).
We are in the process of calculating the pore structure of the rat brain acetylcholine receptor cac, a
neuronal receptor with presumed tetrameric structure. The M2 transmembrane segment may form
the bundle that lines the pore: it shows extensive homology to the M2 segment of the muscle
receptor, it has the same two potential binding sites exposed to the lumen of the pore, namely a
serine and a phenylalanine spaced eight-residues apart and is also amphipathic. Modeling is also
carried out on a tetrameric potassium channel (of the delayed rectifier type) and on a tetrameric
calcium channel (of the dihydropyridine receptor family). The results on the calcium channel are
more extensive and very provocative. We modeled the inner bundle that lines the calcium channel
as a tetramer of S3 segments contributed by the four homolgous repeats. The bundle is funnel-
shaped with the narrowest end pointing to the N- terminus. The exterior of the bundle is
hydrophobic and, presumably, is in contact -v.5 the apoiai membrane interior. The lumen of the
pore is lined with polar/neutral residues and two clusters of acidic residues spaced ten residues
-1-
Mauricio Montal
R&T Code: 4414911
apart. The pore diameter at its narrowest extent is 7.4 A and occurs at the level of a conserved
aspartic acid. Thus, this motif satisfies the structural and energetic requirements for the function of
the inner bundle that forms the pore of calcium channel proteins. We are currently investigating the
binding of QX-222 and how the structurally related dihydropyridine enantiomers of BayK 8644 [
(+) antagonist and (-) agonist] may cause opposite actions. Binding sites for drugs are
- -
evaluated using the docking subroutine in the hNSIGHT program package (Biosym Technoiogies,
CA.), monitoring electrostatic and Van der Waals interactions, simultaneously. We identified a
potential binding site for the 1, 4-dihydropyridine located in the middle of the bundle and formed
by a serine residue of one helix and stabilized by a cysteine in an adjacent helix. We are evaluating
the model by "computer mutations" and "experimental mutations". As described below, we
succeeded in designing a synthetic calcium channel protein according to this model. Therefore.
the current excitement is based on the availability of the structure model and of the synthetic
pore protein, which should prove valuable in assessing the functional role of key residues and
should facilitate the conceptual design of drugs t,:at alter the pore by blocking it from the aqueous
pathways or via the hydrophobic access to the protein from the bilayer interior.
2. Synthesis of tetrameric synthetic channel proteins and demonstration of
channel blockade by a local anesthetic.
We have extended our synporin strategy (Proc. Natl. Acad. Sci. USA 87:6929-6933, 1990.
enclosed reprint) to design and synthesize pore proteins based on sequence information and
oligomeric number. This concerted approach encompassing the technologies of molecular
modeling, synthetic chemistry and single channel recordings in lipid bilayers is proving to be
valuable in identifying and further examining the functional pore entities of an entire channel
protein (FASEB J. 4:2623-2635, 1990, enclosed reprint).
Thus far, we have designed and synthesized four homotetrameric proteins which imitate the
sequence of a highly conserved segment predicted to line the pore of channel proteins.
A. For the nicotinic acetylcholine receptor from Torpedo californicaa muscle-like receptor. the
sequence is:
EKMSTAISVLLAQAVFLLLTSQR
B,C. For the nicotinic acetylcholine receptor from brain, a neuronal acetylcholine receptor. the
sequence for the aX3 or u4 subunit is:
EKVTLCISVLLSLTVFLLLITE
and for the 132 subunit is:
EKVTLAISVLLALTVFLLLIS K
Where A is replacing the authentic cysteine in this position.
D. For the dihydropyridine-sensitive L-type calcium channels, the sequence is:
DPWNVFDFLIVIGSIIDVILSE
This sequence is identical in L-tvpe calcium channels from skeletal and cardiac muscle, brain,
aorta and lung (Soc. Neurosci. 16(2): 957a, 1990, abstract enclosed).
As control proteins for the first three cholinergic receptor channels, we synthesized a similar
tethered tetramer with M1 peptides; for the calcium channel, the control protein is a tethered
tetramer of the S5 segment of repeat IV. None of the control proteins form channels in lipid
bilayers, in accord with expectations.
-2-
Mauricio Montal
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The synthetic proteins emulate the pore properties of the authentic channels, including single
channel conductance, cation selectivity, transitions between closed and open states in the
millisecond time range and sensitivity to channel blockers. In membranes composed of
phosphatidylcholine and phosphatidylethanolamine, the single channel conductance in symmetric
0.5M KC1 or 0.5M NaCl of the muscle-like channel protein, is 20 pS and 15 pS, respectively. For
the neuronal-like channel protein, the corresponding values are 28 pS and 14 pS respectively. The
apparent Kd for channel blocking by QX-222 in 0.5M KC1 is = 10pM. Thus, the M2 synthetic
channel proteins exhibit the sensitiity tc ocal anesthetic channel blockers ouifi ici ic
authentic cholinergic receptor. For the calcium channel protein, the apparent selectivity ratio
inferred from single channel conductance ratios is: Ba2+>Ca2+>Sr2+>Na+>K+>>C1-, in
agreement with measurements obtained on authentic calcium channels. To assess the integrity of
the designed pore protein we examined its pharmacology. Remarkably, the synthetic protein
exhibits the pharmacological specifity that is characteristic of authentic calcium channels and
at similar effective concentrations: Nifedipine, verapamil, QX-222, Cd2+ and Ca2+, all block
ion conduction through the synthetic pore protein. A critical test of the model involved the action
of the enantiomeric forms of the 1, 4- dihydropyridine BavK 8644. We showed that the (-) BayK
8644 acts as agonist and increases the frequency of openings and prolongs the channel opentime,
whereas the (+) enantiomer acts as a powerful blocker and drastically reduces the number of
openings and their open lifetime. Hence, the stereospecific activity of 1,4-dihydropyridines on the
synthetic channel matches that exerted on authentic channels.
WORKPLAN (Year 3): We are in the process of examing the key role of specific residues in
determining the activity of these channel proteins and their sensitivity to local anesthetic blockers
by designing and synthesizing analogues where such residues are substituted. These studies are
guided by the results of the "computer mutations" previously described.
PUBLICATIONS AND REPORTS:
I. Two (2) copies of reprints of research supported by the award are enclosed. These include:
Montal, M. Molecular engineering of channel proteins. In: Membrane Technology in Clinical
Pathology, Biochemistry and Pharmacology. Verna, R., ed., Serono Symposia Publications, Vol.
64:9-22, Raven Press, New York, (1989).
Montal, M., M .S. Montal and J. Tomich. Synporins: Synthetic proteins that emulate the pore
structure of biological ionic channels. Proc. Nati. Acad. Sci. USA, 87: 6929-6933 (1990).
Montal, M. Molecular anatomy and molecular design of channel proteins. FASEB J., 4:2623-
2635 (1990).
Oiki, S., V. E. Madison and M. Montal. Bundles of amphipathic transmembrane a-helices as a
structural motif for ion-conducting channel proteins: Studies on sodium channels and acetylcholine
receptors. In: PROTEINS: Structure, function and Genetics., 8:226-236 (1990).
Montal, M. Molecular anatomy and molecular design of channel proteins. July 20-August 3,
1990. Tenth International Biophysics Congress, Vancouver, Canada. S9.4, p.24 (1990).
Grove, A., J. M. Tomich and M. Montal. Design, synthesis and single channel characterization of
d dihydropyridine-sensitive calcium channel protein. Soc. Neurosci. 16(2):95-a. (1990).
-3-
Mauricio Montal
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2. A progress report was submitted on June 15, 1990.
TRAINING: Two trainees are involved in the project.
Women or minorities -0
Non-citizens -2 (India, Spain)
DISTRIBUTION LIST:
Dr. Guenter Albrecht-Buehler Dr. D. W. Deamer
Dept. of Cell Biology Department of Zoology
Northwestern Univ Medical School University of California
303 E. Chicago Ave. Davis, CA 95616
Chicago, IL 60611
Dr. Edward A. Dratz
Dr. Max Berkowitz Department of Chemistry
Dept of Chemistry Montana State University
University of North Carolina Bozeman, MT 59717
Chapel Hill, NC 27599-3290
Dr. Harvey M. Fishman
Dr. Martin Blank Dept of Physiciology & Biophysics
Dept of Physiology University of Texas Medical Branch
Columbia University College of Galveston, TX 77550
Physicians and Surgeons
630 W. 168th St. Dr. Sol M. Gruner
New York, NY 10032 Department of Physics
Jadwin Hall
Dr. William E. Brownell Princeton University
Dept of Otalaryngology-HNS P 0 Box 708
Johns Hopkins University Princeton, NJ 08544
School of Medicine
720 Rutland Avenue Dr. M. Gutman
Baltimore, MD 21205 Tel Aviv University
Department of Biochemistry
Dr. Marco Colombini Laser Lab for Fast Reactions in Biology
Department of Zoology Ramat Aviv, 69978
University of Maryland Tel Aviv, Israel
College Park, MD 20742
Dr. Huey W. Huang
Dr. Michael A. Cusanovich Department of Physics
Dept of Biochemistry Rice University
University of Arizona Houston, TX 77251
Tuscon, AZ 85721
-4-
Mauricio Montal
R&T Code: 4414911
Distribution List cont:
Dr. Israel R. Miller Dr. James C. Weaver
Dept. of Membrane Research Div. Health Sciences & Technology
Weizrnann Institute of Science Room 20A-128
Rehovot 76100 Massachusetts Institute of Technology
Israel Cambridge, MA 02742
Dr. V. Adrian Parsegian Dr. Michael J. Wilcox
Lab of Chemical Biology Department of Anatomy
NIADDK, Room 9N-307 University of New Mexico
Building 10 School of Medicine
Bethesda, MD 20892 Basic Medical Science Building
North Campus
Dr. David S. Perlin Albuquerque, NM 87131
Department of Biochemistry
Public Health Research Institute
455 First Avenue
New York, NY 10016
Dr. H. Gilbert Smith
EG & G Mason Research Institute
57 Union St.
Worcester, MA 01608
Dr. Michael E. Starzak
Department of Chemistry
State University of New York
Binghamton, NY 13901
Dr. Tian Y. Tsong
Department of Biochemistry
University of Minnesota
College of Biological Science
1479 Gormer Avenue
St. Paul, MN 55108
Dr. Peter Vanysek
Department of Chemistry
Northern Illinois University
De Kalb, IL 60115
Dr. Howard Wachtel
Department of Electrical & Computer Engineering
University of Colorado
Campus Box 425
Boulder, CO 80309
-5-
auricio Montal
Proc. Natl. Acad. Sci. USA R&T Code: 4414911
Vol. 87, pp. 6929-6933. September 1990
Biophysics
Synporins-synthetic proteins that emulate the pore structure of
biological ionic channels
MAURICIO MONTAL§ . MYRTA S. MONTAL0, AND JOHN M. ToIlCHOI
,Deparienis of Biology and Physics. Universit" of California. San Diego. La Jolla. CA 92093-0319; and %MedicalG enetics Division. Children' Hospital.
Los Angeles. CA 90027
Communicited by Bruno Zirnm. May 3, 1990 (received for review February 27. 1990)
ABSTRACT A class of proteins that mimic the fundamen- with stoichiometry a./y6 (4). In vivo. the AcChoR pentamer
tal pore structure of authentic ionic channels has been de- acts as a ligand-activated cation channel with an effective
signed, synthesized, and characterized. The design is based on pore diameter of 7 A (see ref. 1). A high degree of amino
our earlier result that a 23-mer peptide with the sequence of the acid sequence homology exists among its four subunits, and
M2 segment of the Torpedo californica acetylcholine receptor 5 each exhibits four presumed transmembrane regions de-
subunit-Glu-Lys-Met-Ser-Thr-Ala-le-Ser-VaI-Leu-Leu-Ala- signed as MI, M2. M3, and M4 (5). A synthetic peptide with
Gln-Ala-Val-Phe-Leu-Leu-eu-Thr-Ser-GIn-Arg-forns cat- a sequence that corresponds to M25 forms discrete channels
ion-selective channels in lipid bilayers, presumably by self- in lipid bilayers. The channel conductance and selectivity
assembly of conductive oligomers. Accordingly, a tethered sequence are comparable to those characteristic of the au-
parallel tetramer was synthesized with four M235 peptides thentic AcChoR channel (2).
attached to a carrier template-a 9-amino acid backbone with This molecular-engineering approach has the great virtue
four attachment sites. As expected, the complete 101-residue of simplicity. The design. however, dictates that these pep-
protein does form channels in lipid bilayers reproducing sev- tides self-assemble in the membrane to g.enerate discrete
eral features that are characteristic of authentic acetvlcholine conductive oligomers. expressed as channel units. We show
receptor channels, such as single-channel conductance, cation here that this lack of control over the final product (oligo-
selectivity, transitions between closed and open states in the meric number) can be circumvented by synthesizing a teth-
millisecond time range, and sensiti-ity to local anesthetic ered parallel tetranier with four Torpedo AcChoR M28 nep-
-hanel blockers. An analogue protein, in which the serine tides attached to a carrier template-a 9-amino acid backbone
residue in position 8 is replaced with alanine in each of the four with four attachment sites (6). When incorporated in lipid
M25 23-mer peptides ([AlaS]N126), also forms channels that, bilayers. the complete 101-residue protein forms channels
however, exhibit lower single-channel conductance. By con- that reproduce several features that are characteristic of
trast, a similar tethered tetramer with MIS peptides does not authentic AcChoR channels: single-channel conductance (y).
form channels, in accord with expectations. The general va- cation selectivity, transitions between closed and open states
lidity of this strategy to other channel sequences and oligomer in the millisecond time range, and sensitivity to a local
numbers is anticipated. Thus, synporins-a term coined to anesthetic channel blocker. Thus, synthetic pore proteins-
identify this class of synthetic pore proteins-enrich our ar- synporins-provide a strategy to investigate structure-
mamentarium directed toward the elucidation of structure- function relationships in channel proteins. A preliminary
function relationships, account of this research was presented elsewhere (7).
Ionic channels are transmembrane protein arrays organized MATERIALS AND METHODS
around a central aqueous pore (for review, see ref. 1). A key Materials. 1,2-Diphytanoyl-sn-glycero-3-phosphocholine
issue has been to account for the known permeation prop- (PC). 1-palmitoyl-2-oleyl-sn-glycero-3-phosphocholine
erties of channels. specifically the pore size and ionic selec- (POPC). and 1-palmitoyl-2-oleyl-sn-glycero-3-phosphoeth-
tivity of the open channel, in terms of structural elements anolamine (POPE) were obtained from Avanti. All other
predicted from the deduced amino acid sequences. chemicals were of the highest purity available commercially.
In one approach to this problem. a hypothesis is first Synthesis of Proteins. Synthesis of the four-helix bundles
formulated about the existence of such functional segments. was accomplished by a two-step procedure. A common
The design of a functional peptide that mimics the predicted 9-amino acid template was synthesized using automated
structural element is followed by its chemical synthesis using solid-phase synthetic techniques starting with 0.5 mmol of
solid-phase methods, and the peptide's ability to form ion t-Boc-glycine-PAM resin with a substitution of 0.79 mmol/g
chdni,€s is tested by incorporating it into a synthetic lipid (where t-Boc is N..V-tert-butyloxycarbonyl; Applied Bio-
bilayer. The channel activity of the peptide is characterized systems). Amino acids (2.0 mmol) were added as the pre-
in terms of ion conduction and channel gating. This charac- formed hydroxybenzotriazole (HOBt esters on an ABI
terization is compared with the specific features of the model 431 peptide synthesizer (2, 3). Coupling efficiencies
authentic channel. The comparison. in turn, leads to a were monitored at all steps by using quantitative ninhydrin
redesign of the peptide to match the anticipated character- assays (8). Coupling steps were repeated as required to
istics of the authentic channel. Finally, the identification of a generate coupling efficiences >99% per residue. Accord-
specific residue thought to be critical for the function under ingly, the template NH:-K*K±K*PGK*E*K*G-PAM resin
study can then be tested by substitution (2. 31. was generated, where E' is L-glutamic acid, y-benzyl ester.
A test case using this approach has been the nicotinic KI is N'-2-chlorobenzyloxycarbonyl-L-lysine. and K* is N-
acetylcholine receptor (AcChoR). The Torpedo californica
AcChoR is composed of four glycoprotein subunits (a.p.y,S) Abbreviations: y. single-channel conductance: AcChoR. acetylcho-
line receptor: TFE. trifluoroethanolh PC. 1.2-diphytanoyl-sn-glycero-
3-phosphocholine: POPE. 1.palmitoyl-2-oleyl-sn-glycero-3-phos-
The publication costs of this article were defrayed in part by page charge phoethanolamine; POPC, 1-palmitoyl-2-oleyl-sn-glycero-3-phospho-
payment. This article must therefore be hereby marked "adverrisement" choline.
in accordance with 18 U.S.C. 41734 solely to indicate this fact. ITo whom reprint requests should be addressed.
6929
R&T Code: 441!911
Transport Mechanisms and S9 Mdcanlsmes de transport et
Model Systems ____ syst mes mod&les
S3.3 IONOPIIOPES: STRUCTURE AND INTERACTION IN RELATION TO TRANSMEMf3RANE ION-
TRANSPORT. K.R.K. Easwaran. Molecular Biophvsics Unit. Indian Institute of
Science. Bangalore - 560 012, india.
Much of the current understandcln of the molecular mechanism of transmembrane ion-transport
has come essentially due to the discovery of certain transport moiecules. lonophores, which
act as carriers or channels In selectively enhancing the Ionic permeability across natural
and model membranes. There are essentially two aspects of the problem which need a detailed
study to understand the process of carrier or channel mediated ion.-transpcrt across membranes
at the molecular level, namely, a) structure alld conformation of icnophores and their cati-'n
complexes b) the mode of interaction of these lonophores with model and biological membranes.
Our results on the structure and conformation of carrier tonophores and their cation complexes
using NMR and CU techniques in solutIon and X-ray crystallography in the solid state studied
under different conditions clearlv showed that the ion selectivity of the lenophore depends
not only on the nature of the liqands but also on the conformational state of the whole molecule
and that the conformatinn !s very r,'ch dlporr ndant on the s7ze and charge of the catton and
the nature of solvent and anion used . The studles on the Interaction of innophores with
model membranes, in particular phesphoLipids. usinq DSC. ESR. III and 31P NMR gave Information
on the preferred locateon of the ionophores in the lipid bilaver and the mode of inserticn
In the lipid matrix. These results will be presented In reiation to tI~e transmembrane ion
transporting properties of ionophores.
S9.4 MOLECULAR ANATOMY AND .MOLECULAR DESIGN OF CHANNEL PROTEINS. Mauricio Montal.
Departments of Biology and Physics. University of California. San Diego. La Jolla. California 92093-0319.
A central goal in membrane biophysics is to understand how channel proteins work in tens of their underlying protein
structures. Ionic channels are symmetric (or pseudosymmetric) transmembrane protein assemblies organized around a central
aqueous pore. The two key functional elements are the ionic channel - the actual polar pathway that permits the selective
passage of ions across the apolar core of the lipid bilayer and the senor - the structure that detects the stimulus and couples it
to the opening or closing (gating) of the channel. Molecular cloning and sequencing led to the elucidation of the primary
structures of several muli-member gene families. Given this structural information, molecular modeling has been used to
postulate structural models of the protein with emphasis on the identification of structural elements: the channel and the sensor.
To test the models the putative f,-nctional peptides are synthesized by solid phase methods and their abiliry to form ion channels
is assessed by incorporating them into synthetic lipid bilayers. This approach - molecular engineering - will be briefly
described with particular emphasis on the progress we have made in the design and synthesis of peptides and proteins that
emulate the pore structure of two prototype proteins of two major gene families in the brain, namely the voltage sensitive
sodium channel and the nicotinic cholinergic receptor (Supported by NIH, NIMH and ONR).
24
TJIURBDAY AM CALCIU V CHlANNELS IIIrii f~l~
R&T Code: 4414911
FUNNEL-WEB SPIDER TOXIN (PUX)S ELECTIVELY BLOCKS THE SUSTAINED. DISTINCT RAT BRAIN CLASS-C CALCIUM ChLANNELS ARE
BUT NOT THE TRANSIENT, CALCIUM CURRENT IN RETINAL HORIZONTAL CENERATED BY ALTERNATIVE SPLICING. W--gmjij
CELLS J..S0 n Olpff, Of Physioogy a-id M-. Gilbert Ind T-P Snutch. Biotechnology Laboratorv.
Optehalmology. Urti. of Utah SciofO Medicine. Saft Lake. City. Utah. 84 108 Unin. of British Columrbia. Vancouver, B.C.. Canada V6T IW5.
Using the winculi petctscfamp techvilue. we Pe" IderAfed two &"prate Rat brain class C Ca channel cDNA3 (rbC) hybridize on Northern
Ca curreres-onet saosairked PlCS). the oterw trarnsient Pl.-TR)-in cunurert blots to two mRNAf of approx. I and 12 kilobome (kb). The deduced
horizonta cea 04CA) laisatd Pmrmf idut .4l'r bea rritesi Ca cijrAt wre priroy structutre of two o..er-Ig iones CrbC-6I and rbC-30)
ernncd uinng to mM @zdracelAar Ca. whie Na .and K' currents wsrg show%t hat the clam C gene product is 390% ideaitcci to tse rabbit-
pharrrucocogcly suprestfed The large trarmerit Ca cuff"a Is sirtar. but nr cardiac dihydropyridit receptior/Ca channel (designated rbC-1).
Iila o &T -Cuyae descred In ottreptratIons I.TRnactivates abom.e Anslysni or a numnber of other eDNAs reseals dtil1 Clas C coding
-OD mV. Is inactvated at a hjInig potential of -40 mV and s carried less ne4I sequences (designated rbC-IT). Further, In 1400 amino acids
by Be' " thsen Ca. Urdfkr the 1-Currant. IC.TR is r"c preferentily blocked by comoared. rbC-I essd rbC-rf transcripts differ at only two sites. ia
WNt. This is the Mteatti rst it a large trantswir Ca curnre has been s"en, nIte first intane. rbC-fl trascripts have a three amnino'a cid
MCA.o r In a non-nmnrnllan vertebrate pteparation. The suntained Ca curdr~- inseCrtion (ProAleArg) corresponding to the putativec cyti~ptcsnuc loop
Is similar. brA net hieertlal. to the L-Crrrsr described in many pepearatioa between homology dotmains it and 111. The secontd rite. a 28 auttino/
IC.S activateabove -3OrrV. is arger when Be recptcesCandIenaced acid rrgion which includes the third patative trorutnembra" snee given t
by the d~ ropyridle agorr.tAY K 96 (Calbocicenn. Unikie the LCr"r. (S3) of domain IV of rbC-I. is replaced by a different 28 amino ac~
I5 SIs noct preferentall~y blocked by Cd '. nr~ Is A reduced bryi vcirnro ;n hvdrapfrobic stretch. By Southern blof analysii. rbC-1 In~d rbC-11
traction GVIA (a girt fromr Dr. B. P.. Olvaera) FTX (a git from Or A, lrrail. a trrcific Probes are shown to hybridize an identical pattern of rsu
factor Isolated Phornthse venoms of the Isr"e web spider (1-ias at af, P N A S. renomnicD NA fragments. In addition, polytnerase chain reaction
A§:689. t1989).s electively blocks lC.S at very ov, concentlratins n t 300.0 anslyisi of rat Sertomic DNA showes that the rbC-I and r6C-ll'
dlttkon of the cdtmer, traction cotanrning FPU blocked "2 of the sustained traflacriptt are generated from the same genomric DNA fragment.
curant,while a 1tt0,00.000 dilution bioclkad 10-- of ths current This block These results suagest that in addition to the distinct genies which
was mairtained even after weashing with FrX~ree Ringer for up to 20 mn These encode the rac brain class A. B. C and DCis channiels. alternative
results strongly suggest that the sustained and transient Ca currentis aie carried sptlicing provides a further toure of Ca channel divniry in the
through two differeut arid unique tye oPc I-anne't. cnd support the notocnt hat CNS
there Is a wie variety of Ca channels arriong differit spiscies aendI lssue mopes
This workt was supported by N IH. Grant EY05972 to E M L
395.11 395.12
Biochemical Charoetuitorto of a High Affinity (11H]RYonodtnse Receptor DIVERSE as-COPOT 0XIN OVIA BINDDNG PROTEINS IN ELECTXC ORGAN
Prom Rabbit Brain Membrartes. PS . Uc'tino-es and fPCmg AND BOVINE BRAIN - Hom &KLDIM andR -W Tic, DTLao l Moine.
Hocward Hughas Medtcal Irntie ana Ptogram In Neroeclence and Dacit of A Celt. Phisol.. Stanord Univsity Schioool f Miedicinse.S torfced. Calif. 94305.
Physiology ard BlophysicaUs,n iversity oPI ow@C ollege oPM edicine. Iowa City. Binding stes foe the marne trail rosin. marsonlin OVIA (mCgTx).P iaveb ent
tA 5Z242. idmstffd in a vnsy of neunal ditsuer ypm and haoe beenass signed eccisnnely to
The skelealM rneide receptor toe the pla t alklo ryndn has been, noltage-depenident calciun channels. atCsTa has bee shotwnt o hind with high
raasfihcouwumnt o. bHe igIhr ticaaffji nittoy thj3eH Cjr8y2a ierdefne seb liVndleinngn e hoaPs theb ~seaenrc oprctaexrioniyc ahfrfaininit y( Y(aXr(m,-3a gPsxMc)eh ttio amJ.t.Ji rBsCil y2 6f-3e. w9 4n9e1p). x anadie ws i(t0h. 4 lapwn isafofilneistyt rpOinrtgtt,s .i ) I ipna bmr)o tows ia
cleontrte i islaedbrinnwibrires(Ahly.RJ .HM im. ,grealr n-tnr of teceptora t- (2M pinotestnrgp ro-e ) in p"qr~es~qe
Pe11:17r1l.9 9) isoaett l~7b9ra.9in~ lWtthaea t 1a3vIebasnnrh y aasoi odd(Arriee. bRiinniinn gisis ennriicliee iin Or ~ ( tinreIhsuelte sl ehcutrlitcic so rwtah ato tfh Dewit cboipn7d1.ig0 anustrma r ep(rMes5enil sca! ) otef Ie, oBarmnA nRp eosp4 l5fa3i.o 2n4 7o).f
Mmrrsfrmthe hippocajnpi but is signiftcaritty lower in ttemtbrnes, esCgla binding proteinis and that. in alectric orgam oaiwl l ac tualy caiciam
Prm The brain mam and atinsl cord. Appnoxnetal 50% OP(3 Hlr-aicdhne chanrnets. Crosutirking experimnents with the botnobiturncdonal intidoester.
labeled receptor is solublitzed from brain mnrbaies us"n CHA'.PS dirtieilyluberainiiou (DMS4)t.e re tat lI"twC&Tx specdifily labelltsw o .ars
ll-ceiutlichdsecontaining t M N.aC9. TI-e brain IJHI"ryanc newcon in bovat brainw ith K, sises of 330 and 220DkD ar detanainedb y SDS-PAGE. aid
ocimigratee i-fough suose gradlents with the hlrsalt recepitor as at large tian Proftins in electric Organ , repodrr wisthM e,v alues of 170. 60. aid 45 ItD.
complex (-30S). SoluAitted rocepstor Is snpecfically 41 inicplad by We hare demonstated tht thet4 3 LDp rotnr of tines-ic color is the 0-subrustofthse
arertceeeppt orp cctoydpr~lea toc sparMoeiLine~1 ,A &So£pi-auareotsp su. rt1e3dI Rysakissoedtihn ret tlraobieelle d rryercvedceiosre O' f the tOta-lin ysuwmtob efrIbeo f m-C~gIT- x b(ainAi ngR )i iat.n d OFWor ittsaecncooeu tsi tf ocraani bgen nifeipcoaatt fufr acfrtoiomn~
blinds to hepe moonw et high af",ty buAd oes tri blind to ,nst gem bot L- 170a id 60 kDa protin, by immaasopraecpttatownit h a Mob againstt he
agglutinin liectn -441yarcidin " receptor is isolated using hevern- oACbR. All three electrie argan mCSTat binding proteins itat similar
"rsh onrtgo ooo lbelxisednlt detcrrualr PlUnsacolroics propentiesin, Chhalf trialc tm esia of '2f-ImrCSTos =n
A proen o -4Wogr0aoyis ercheb y aion ~dNe~ey~arnsdalyn e Wcrdriteun ugtjort in the rangeo f 0. 1- .0 JM mICgTea n the binding of latI-m(gTxs am inhbtdb
Aa pi retenii cohfe 40i0n00p0~ k -Prct~ooa00he caliums entry blochea such asn itirwndipine. cc by ct-isagfornija and other
oPh epacli-aigarbsa eluttac arndau mcroe grirns, and is crosnoective with agnt blown to interse, with the ILAChiR.t oCITs iitiing both in brainaenedl ecsti
artbsodles raisead0 9"k the ailti treacle ryoadene receptor. We proprose rgon is chorcaistically inhibited by higher conceowa cif Call (0.5-1.0 mm)
'hat the -400.000 Do protesnI ist* bains Pormo P the high a"hrt rytarodlie -hee-a" Is brain prepshLIorsI WOsil esMNm that OCIT2 bindl" If testalY
racspitor and ttat I furicaborsiats c Co; rlaise clsartrii brain ndsoptsnrrrc rilulAted or us 3-fold &Il ower Cau concentratios (03-1.0 taM). it is toausiy
hatiulut E~jLQaX Is an lrrveeslgitor oP te Howard Hughes he this 11siratf"-C 'mct acess -- Le rinane sis range is Ca"* blotting
Medical trntr~e. to sitesc onrtrollig Pmeio icd insactivation of calcium dusmek.
3011.13
DESIGN.SYNTMEIS. AND $tNGLECt{ANNELC{ARIACTERMATIONJOF
A DIRYDROPY~RMIDINIUE-SlaIEnNdIS MVE CALCIUM CHANNEL PROTEFIN.
M. MInLL
'tESD. is Ails CA 9209 andC halran Hospital. Los Ancelet. CA 90023.
The s'a oprn tetpoinee of a dlirytkopylathssenstane rajeses asell
krota elscA ckwasunddoestaqoat(Tuube-. c. t96.Nnnvr32J1.
I l3-3 ) . pIenWs y wtatius ~ugesd iouec of four Infrmal repeats
' rdss dqix lefernabitio-tebeal oromnonbrn,. segmts. We deItned
Atinplose(X U XO) (M. Jt ofanl.I "S. 7 8)awi~tanre
ameo iirmlhenumly st oz iamstiaooffoo rttaa 22-mia ppt
*diN seeqveacec coreseponding to tnaanomembrone segmeat IVS3
(tPW'NVPDFUJqOSDVR.M Tills stauiet proto formsc utao-sicis
S~inM,al,ndSua,m72* 1.,9.1 * luS6A OA pS.t ~otvy.
aed IDA* lpS11S50IntMlla Owsriareboeltedby 10'Maitedom.
104 M" MININ go 100 M ofteu local mttie asolga ofitldosit QX-2. -
Adiferme Ind gisseboarceslnig fowl I " ibmqI s-aemodsg
bo IJMYSe u VALLZrha4J'FYAVIOMQ0aGTK doesam forem e
3,11, ambpN G (Gh(42340).NDAICM4N-4438mel P.0778uaiONlR
silchewc e wmm annisegauf too mv in syatsr 5o mm sCa, pH 7.L
SOCIrTY MR61 4111080rn1ct AIRSTACM1 VIIIIMIE 16 it"
Mauricio Montal
R& Code: 4414911
PROTEINS Structure. Function. and Genetics 8 226-236 11990)
Bundles of Amphipathic Transmembrane a -Helices as
a Structural Motif for Ion-Conducting Channcl
Proteins: Studies on Sodium Channels and
*] Acetylcholine Receptors
Shigetoshi Oiki,' Vincent Madison,2 and Mauricio Montal'
'Department of Neurosciences. Roche Institute of .11olecularB iology. Nutley. New .Jersey 07110. 2Roche Research
Center. Hoffmann.La Roche Inc., Nutley. New Jersey 07110, and -'Departm,,nttso f Biology and Physics. University
of California, San Diego, La Jolla. California 92093-0319.
ABSTRACT Channel proteins are trans- INTRODUCTION
membrane symmetric (or pseudosymmetric) Channel proteins are a special class of membrane
oligomers organized around a central ionic proteins that mediate the selective passage of ions
pore. We present here a molecular model of the across the low dielectric constant apolar core of the
pore forming structures of two channel pro- membrane lipid bilayer (for review see 1). Under-
teins with different primary structures and oi- standing their mode of action has progressed with
gomeric size: the voltage-sensitive sodium the advent of recombinant DNA technology that re-
channel and the nicotinic cholinergic receptor. suited in the elucidation of the primary structure of
We report low-energy arrangements of a- several channel proteins.'" A remarkable feature
helical bundles calculated by semiempiricial inferred from the amino acid sequences is the high
potential energy functions and optimization homology conservation among members of this fam-
routines and further refined using molecular ily of proteins. They are composed of homologous
dynamics. The ion-conducting pore is consid- subunits or domains which contein several trans-
ered to be a symmetric or pseudosymmetric membrane segments. Further, segments that could
homooligomer of 3-5 amphipathic a-helices ar- be folded into amphipathic transmembrane a-
ranged such that the polar residues line a cen. helices are identifiable in all channel proteins for
tral hyd'-ophilic oathway and the apolar resi- which sequence information is available. -' This is
dues face the hydrophobic bilayer interior. The significant because amphipathic a-helices could
channel lining exposes either charged (Asp, form a bundle with one side facing an internal hy-
Glu, Arg, Lys) or polar-neutral (Ser, Thr) resi- drophilic pore and the other facing hydrophobic he-
dues. A bundle of four parallel helices con- hiceo rl he membrane cor°I f addition, i-r. c eal-
strain d to C4 symmetry, the helix axis aligned ysis of eiectron micrographs obtained from ordered
with the symmetry axis, and the helices con- arrays ofchannel proteins in their native membrane
strained to idealized dihedral angles, produces environment produced low resolution images (- 18
a structure with a pore of the size inferred for A) that are consistent with radial symmetric or
the sodium channel protein (area - 16 A2). Sim- pseudosymmetric protein assemblies organized
ilarly, a pentarneric array optimized with con. around a central aqueous pore. ' "' These key fea-
straints to maintain Cs symmetry and backbone aon eta qeu oe
torsions chaaceistic syomf melic and ao tures, homology and symmetry, raise the notion that
torsions characteristic of -helices adopts a a unifying motif in the biological design of ion chan-
structure that appears well suited to form the nels is an array of transmembrane a,-helices ar-
lining of the nicotinic cholinergic receptor ranged such that amphipathic helices face a central
(pore area - 46 A2). Thus, bundles of amphi- hydrophilic poe whereas hydrophobic helices sur-
pathic a-helices satisfy the structural, ener-
getic, and dynamic requirements to be the mo-
lecular structural motif underlying the function
of ionic channels.
Received December 18, 1989; revision accepted March 16,
1990.
Key words: protein structure, ionic pores, neu- Address reprint requests to Mauricio Montal, Departments
ral membranes, protein design, en- of Biology and Physics. University of California. San Diego, La
ergy minimization, molecular dy- JoSll.a .O CikAi' s9 p2r0e9se3n-0t 3a1d9d.ress is Department of Physiology, Kyoto
namics University Faculty of Medicine. Kyoto 606. Japan.
0 1990 WILEY-LISS, INC.