Toward an understanding of structure and function ... - Semantic Scholar

Toward

an understanding

function

of structure

and

of ion channels1

BRUCE

K. KRUEGER

Department

of Physiology,

University

of Maryland

School of Medicine,

Baltimore,

Maryland

21201,

USA

tion) in terms of specific voltage-dependent ABSTRACT

The second half of the 1980s is certain to be considered a turning point in the study of ion channels. Within the last few years, monumental advances of molecular biology, single-channel

rect molecular

characterization

in the application

recording, and dihave been brought to

bear on the problem of relating the molecular structure of the ion channel proteins to their function

membrane. studied

Structure-function

at a level of detail

relationships

that was unimagined

in the cell can now be a decade

with the techniques of molecular biology appear to have dominated the literature in this field; however, innovative strategies of structural characterization and electrical measurements of functioning channels in native and artificial membranes continue to break new ground. This paper is a selective review of current progress in understanding structureago.

Recently,

advances

made

function relationshipsin ion channels. The

relative use-

changes

in

membrane conductance for Na and K ions. Although they suggested that the ions might move across the squid axon membrane through discrete ion-selective pores, the concept that specific transmembrane proteins behaving as

channels remained

conferred those properties as speculation for nearly

on the membrane 2 decades. During

that period, research in the developing field of membrane biophysics concentrated on characterizing the kinetics of these conductance changes with the increasingly sophisticated voltage clamp technique and investigating the permeation of cations across nerve or muscle membranes with radioactive tracers. Meanwhile, intensive electrophysiological studies revealed that there are several distinct types of K channels, as well as voltage-gated channels selective for Ca or chloride and channels regulated by a variety of neurotransmitters, notably the nicotinic acetylcholine receptor (AChR) at the neuromuscular junction.

fulness of determining amino acid sequences of channel proteins together with the resulting deductions about 3-dimensional structure and function will be evaluated with respect to the potential importance of studying the channel molecules more directly by biochemical, immunological, and electrophysiological methods. A full understanding of the details of channel structure and its relationship to function may be realized in the near future as a resultof the interdisciplinary application of

A major breakthrough occurred in the 1960s with the discovery and characterization of tetrodotoxin (TTX), the highly potent and selective inhibitor of the nerve Na conductance. This substance was used as a pharmacological tool not only to simplify the nerve membrane conductance pattern by eliminating the Na conductance, but also to provide direct evidence for the first time that the Na and K conductance pathways are probably separate and discrete entities. Moreover, the high affinity of toxin binding (K1 109_108 M) made it unlikely that the

biophysical,biochemical, and molecular biologicaltechniques.KRUEGER, B. K. Toward an understanding of

voltage-dependent Na conductance mechanisms such as rearrangements

structure and function 1906-1914; 1989. Key Words: ing

ION Past

ion channel

.

tetrodotoxin

voltage clamp technique

CHANNEL and

FASEB j

of ion channels.

.

.

single-channel

/ subunits

.

3:

record-

Na channel

STRUCTURE-FUNCTION

present

Interest in ion channels2 (1) was originally the work of Hodgkin, Huxley, and Katz

stimulated by (2, 3) some 37 years ago. These investigators were the first to describe the process of nerve excitation (action potential genera1906

was caused by other

in membrane

phos-

‘From the Symposium “Ion Channel Reconstitution and Single Channel Recording in Molecular Pharmacology” presented at the Annual Fall Meeting of the American Society for Pharmacology and Experimental Therapeutics, August 17-21, 1986, Baltimore, Maryland. Speakers were R. L. Barchi (University of Pennsylvania), D. K. Bartschat (University of Maryland), R. Coronado (Baylor University), R. Huganir (Rockefeller University), B. K. Krueger (University of Maryland), and R. L. Rosenberg (Yale University). 2Space limitations preclude reviewing this active field of research exhaustively and citing all relevant publications. In many cases I have cited recent reviews that contain more complete listings of references to the original literature. An excellent background source on ion channels is Ionic Channels of Excitable Membranes by Hille (1). This monograph provides a historical overview of the field as well as detailed coverage of the methods and theory of research on channels through 1983.

0892-6638/89/0003-1906/$01.50. © FASEB

pholipid structure. Only a protein of substantial mass was likely to provide a structure sufficiently complex to bind a small toxin molecule with such high affinity and form an aqueous pore across the membrane. The synthesis of radiolabeled derivatives of TTX (and the functionally similar Na channel blocker saxitoxin, or STX) provided the means to actually count the number of channels on a cell membrane; later, these labeled toxins would provide an assay for purifying the Na channel molecule. At about the same time, it was discovered that a snake polypeptide toxin from the banded krait - a-bungarotoxin bound tightly to the nicotinic AChR and, by blocking its activation, paved the way for an analogous biochemical and functional characterization of the AChR and its associated channel. One of the most significant breakthroughs in the study of ion channels came in the middle 1970s with the development of the patch clamp technique (4, 5). With this method, the measurement of transmembrane ionic currents across a small patch of cell membrane is facilitated by the formation of a gigaohm seal between the electrode tip and the membrane. Unitary, stepwise current fluctuations reflecting the opening and closing of single-channel molecules were observed, providing dramatic evidence that ion channels are discrete macromolecules in the cell membrane. At about the same time another singlechannel recording technique, based on reconstitution of ion channels in artificial planar bilayers (6), began to be applied to the study of naturally occurring channels from plasmalemmal

and intracellular membranes.

The

latter

has been widely used to study the conductance properties of membranes not easily accessible to other means of recording and to study previously characterized channels in well-defined membrane phospholipids and electrolyte solutions. In the last half of this decade (7-9), structural studies of ion channels accelerated rapidly and the AChR and Na channel were purified. The AChR was found to be a pentamer with four different subunits in the ratio ct2/3-y; the Na channel was found to consist of a single large a subunit associated (apparently only in mammalian Na channels) with one or two smaller f3 subunits. Progress with other channel types has been less dramatic because of the lack of specific high-affinity ligands, although the finding that drugs of the dihydropyridine class are potent modulators of certain types of Ca channels enabled the purification of at least one type of Ca channel (9-11). Purified toxin binding sites are indeed capable of functioning normally as channels, as revealed by the reconstitution of purified AChR, Na channel and Ca channel proteins in phospholipid vesicles and bilayers, and by measurement of channel function by tracer flux, planar bilayer, or patch clamp techniques (5, 6). Purified channel proteins have also been used to produce polyclonal and monoclonal antibodies, which has enabled the topographical mapping of the channels’ structures. method

Future

of ion channel

research

During the last 5 years, the dominant force in the study of structure-function relationships in ion channels has been STRUCTURE

AND

FUNCTION

OF ION CHANNELS

the application of gene cloning methods to determine the primary amino acid sequence of channel proteins (12). Many investigators in the field expect to see within the next 10 years a fairly complete description of the structure of several channel proteins at the atomic level and also to understand intramolecular motions of the proteins as the channels respond to such physiological stimuli as changes in membrane potential or binding of modulatory ligands. These expectations often lead to lively discussions about the relative contributions of molecular biology structural biochemistry and immunochemistry, and electrophysiology and membrane biophysics in achieving these goals. Some insight into these issues developed during a symposium entitled “Ion Channel Reconstitution and Single Channel Recording in Molecular Pharmacology” presented by the Society for Pharmacology and Experimental Therapeutics in August of 1986. Although the emphasis was on current electrophysiological and biochemical approaches to studying ion channels, the symposium was organized and presented against a background of frequent reports of advances based on cloning of channel genes and cDNA manipulation. Drawing on new results reported at the symposium as well as on more recent publications, this review will examine the potential contributions of biophysics, biochemistry, and molecular biology to achieving the goal of understanding ion channel function at the molecular level. BIOPHYSICS

OF

ION

CHANNELS

The functional properties of ion channels are generally characterized by using a biophysical approach, that is, by measuring ionic currents across membranes containing the channels under voltage clamp conditions. The interesting biophysical properties of ion channels can be divided into two categories: 1) the properties of ion movement through the open channel (permeation), and 2) the probability that the channel will be open (gating). Permeation includes the ability to select ions that will pass easily through the channel while retarding or rejecting others (selectivity). This may result simply from the exclusion of ions too large to pass through the narrowest region of the channel pore or from specific interactions

between

permeant

ions and charged

or polar

regions

of

the channel structure within the pore. Gating is presumed to be a mechanism for shutting off the movement of permeant ions through the channel pore that is coupled, via

a conformational change in the protein structure, to an extracellular receptor (e.g., the AChR), an intracellular modulatory influence (e.g., G protein or phosphorylation), or a change in the membrane potential (e.g., voltage-gated Na, K, or Ca channels). Voltage-dependent gating presumably requires intrachannel charge movement or dipole reorientation in response to changes in the transmembrane electric field. In early biophysical studies, the membrane of intact cells was voltage clamped to produce a macroscopic current resulting from the activity of a large number of channels; the net macroscopic current is determined by both permeation and gating of the ensemble of functional channels. The patch clamp (5) and planar bilayer (6) techniques allow the ionic currents 1907

through single-channel molecules to be studied, so that permeation (as revealed by single-channel current) can be unambiguously distinguished from gating (manifested as the probability of occurrence of unitary opening and closing events). Generally, limited information about channel structure can be inferred from biophysical measurements alone.

nel, by a single gene product consisting of several repeating homologous sequences, each playing roughly the same role as an AChR subunit. Of course, proof was required that the cloned and sequenced genes actually code for functional membrane channels or that the subunit or subunits in question are sufficientto produce channels. This has been accom-

For example, the permeation rates of various ions at different concentrations and in different proportions can

plished by introducing channel-coding into an appropriate expression system

mRNA

or cDNA

that normally lacks the channel being studied(22, 23).Crude mRNA from a be used to develop rate theory models for permeation (in varietyof sources,when injectedintoXenopus oocytes,diwhich permeation is viewed as ions hopping over energy rects the synthesis and subsequent expression of functional barriers across a series of energy minima, which may represent ion binding sites within the channel pore) (1, channels of several types (22, 24, 25). The functional competence of the protein transcripts of channel-coding 13). Similarly, the voltage and time dependence of changenes has been established by similar means: messenger nel gating can be modeled as a series of voltage-dependent RNA synthesized from cDNA clones for a, /3,-y,and #{244} transitions among closed or open states. An example of of the AChR and the a subunit of the ratbrain Na chanhigh-resolution, single-channel patch clamp recording nel directs the functional expression of the correspondto model voltage-dependent Na channel gating can be in the oocyte membrane. By injecting found in a recent paper by Aldrich and Stevens (14). ing channels mRNA eitherfor individual AChR subunits or various combinations of subunits, Mishina et al. (26) found that MOLECULAR BIOLOGY OF ION CHANNELS all four subunits were required for normal channel function, whereas only the a subunit was required for aRecent progress bungarotoxin binding. Injection of mRNA coding for only the a subunit of the rat brain Na channel resulted Before 1982, structuralstudiesof ion channels had been in expression of normal channels, which indicated that restrictedprimarily to a directand laborious method of the /3 subunits associatedwith a in purified STX bindpurificationof the AChR and Na channel proteins(based ing site preparations from rat brain (9) apparently are on high-affinity neurotoxin binding),subsequent analysis not required for expression of functional channels in this by physicochemical and ultrastructural characterization, system (27). However, /3 subunits may modulate Na and, to a limited extent, sequencing of portions of the channel inactivation (28). purifiedchannel proteins.Noda et al.(15)provided the Molecular biological approaches have yielded proposed firstcomplete sequence (a subunit of the AChR) by amino acid sequences for a putative Ca channel (dihydroisolatingan mRNA that recognized a synthetic probe pyridine binding protein) (29), several neurotransmitter that coded for a previously determined portion of the modulated channels (30-32), and voltage-gated Na (33) a subunit sequence. Complementary cDNA was then and K (34) channels from Drosophi& Traditional Drosophila synthesized and sequenced, which permitted the deducgenetics techniques (35) were used to determine the setion of the full amino acid sequence. In rapid succession, quence of the gene at the shaker locus where mutations the remaining three (13,‘y, b) chains were sequenced, rehad previously been linked to K channel deficiencies. substantial

vealing

homologies

in their

amino

acid

se-

(16, 17). Using an analogous strategy, the a subunit of the Na channel in the rat brain was determined (18); subsequent work (19) revealed that there are at least three similar genes in the brain with highly homologous sequences. All these types of Na channels were found to consistof four repeating homologous segments (20). It has been suggested that many, if not all, channels have several common structuralelements at the gross molecular level (20, 21).These include:1) multiplehydrophobic membrane-spanning regions, 2) internal charges quences

that

comprise

the membrane

in the case of voltage-gated

potential-sensing apparatus channels, 3) hydrophilic ele-

intothe native 3-dimensional structure of the channel, form the aqueous transmembrane pore, and 4) sitesfor glycosylationin portions of the sequence facingthe externalsideand regulatoryphosphorylation siteson cytoplasmic segments. These elements may be generated by the associationof multiple distinctsubunits (as in the case of the AChR) coded by multiple genes or, as has been found for the Na chanments

1908

that,

Vol. 3

when

organized

June 1989

What can be learned about channels from molecular biology Channel gene sequencing and manipulation reveal two general types of information. First,the primary amino acid sequence can be analyzed to determine itshydrophobicityprofile,which can lead to predictions of the locations of possiblemembrane spanning regions and extracellular and cytoplasmic regions (21, 36). Potential sites for N- and O-glycosylation serine or threonine) and

nine,

or tyrosine)

the external

and

place

(arginine and phosphorylation

the segments

cytoplasmic

sides

less frequently (serine, threo-

containing

them

on

of the channels,respec-

tively. Analyses of this kind have led to several proposals for the topography of the AChR (8, 20) and Na channel (19,37, 38). For a voltage-gated channel such as the Na channel, it would be expected that voltage-driven intramolecular structural rearrangements would be associated with channel opening and proposed that intramolecular

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closing (gating). It has been movement of a multiple re-

KRUEGER

peated sequence containing acid in every third position

a positively of the Na

charged amino and Ca channels

may representthe voltage-sensing gating charge movement associatedwith channel opening (38, 39). Another deduction from the primary amino acid sequence concerns which amino acid residues might form the hydrophilic (and possiblycharged) liningof the channel pore. Such a deduction isdifficult to make on the basis of structural models derived from the linear amino acid sequence alone, because regions from differentsubunits or from distant segments of a single large subunit could come together to participatein pore formation due to protein foldingand multiple membrane crossings.Although such predictionsremain largelyspeculative,recentresultswith the AChR offerconsiderable insight into this problem (see below). Information about channel structureand function can be provided by directexperimental manipulation of the channel gene when combined with functionalcharacterization of the resulting modified gene product in an appropriate expression system (40). There are several kinds

of modifications, such as the substitution of a particular amino acid residue or sequence of residueswith another by site-directedmutagenesis, the deletionor insertionof specificsegments of the protein,or the manipulation of the composition, source, or proportions of subunits. So far,these approaches have been confined to studies of the AChR. Using site-directed mutagenesis of the a subunit followed

pressed

by

characterization

in Xenopus

of

oocytes,

nearly all modifications in a functional activity or were tions partially or completely the receptor. Although it is

mutations identify the subunit, mutation-induced could affect Deletions

ACh binding and insertions

the

Mishina subunits without

modified AChR exet al. (41) found that either eliminated all effect. Many muta-

eliminated ACh sensitivity of tempting to think that such AChR binding site on the a alterations in tertiary structure by an allosteric in the c5 subunit

mechanism. of the AChR

have permitted the identification of a portion of the ionconducting, pore-forming region of the channel. Torpedo and bovine AChRs are functionallyand structurally quite similar;however, the bovine channel has a smaller singlechannel conductance. This differenceisdetermined primarily which quence

by the c5 subunit. Using chimeric subunits in one portion of the protein is of the bovine #{244} seand the other is of the Torpedo sequence, Imoto

et al. (42) showed that a small 20-amino-acid segment of b determines whether the single-channel conductance ishigh (Torpedo) or low (bovine). Within thisregion, the Torpedo channel has another negatively charged amino acid, which tance. This (see below).

Sakmann

possibly problem

explains the larger cation has recently been further

et al. (43) produced

hybrid

conducexplored

AChRs

by in-

jecting mRNAs for mixtures of Torpedo and calf subunits. Although the two AChR channel types had similar single-

channel conductances and selectivity among permeant cations,Torpedo channels exhibited unitary opening events of much shorter duration. Hybrid channels in oocytes injectedwith mRNA coding for calf a and Torpedo /3’yb STRUCTURE AND FUNCTION OF ION CHANNELS

had intermediate lifetimes,and hybrids with calfb and Torpedo a(3-y were virtually indistinguishable from normal

calf AChR tributes

with respect to open-state

of .5 and,

to a lesser

extent

duration. of a,

Thus, at-

determine

the

open-state lifetime and thus the closing rate of the channel. The discovery of a fifth (#{128}) type of AChR subunit (44) explained the well-documented observation that, in embryonic and denervated muscle, AChRs are distributed over the entire cell surface, whereas in adult muscle, the

receptors have somewhat different physiological properties and are localized almost exclusively at the motor endplate. Apparently, developing (45) and denervated (44, 45) mammalian muscle produces AChRs with ‘y subunits whereas adult muscle synthesizes receptors with the #{128} subunit replacing -y. Adult (a2/3h#{128}) receptors have a larger single-channel conductance and shorter open lifetimes than receptors from developing (a2f3’y#{244}) muscle. BIOCHEMISTRY

OF

Recent

progress

Direct

purification

ION

of ion

CHANNELS

channels,

as assayed

by

the

specificbinding of radiolabeled ligands, enabled the identificationof subunit composition and molecular weight of the nicotinic AChR, the voltage-dependent Na channel, and the voltage-dependent Ca channel (7).Itis generally agreed that the AChR is a pentamer of four different50- to 60-kDa subunits in the ratio a2/3’y.5 or a2/3.5#{128} (see above) (8). Recently, a putative Ca channel (dihydropyridine binding protein) was purified from several tissues (10, 11) and found to consist of two different large (at and a2) subunits ( 170 kDa) and at least two smaller subunits (30-60 kDa). There has also been some controversy about the subunit composition of the Na chanwith reports of only a single large a subunit ( 250 in Electrophorus electricus (46), a plus one small /3 subunit (38 Wa) in mammalian skeletal muscle (47), and a plus two /3 subunits (37 and 39 Wa) in the rat brain (9). nel,

kDa)

As discussed rat

brain

previously,

is sufficient

mRNA to direct

coding

for only a from

expression

of functional

channels, which suggests that only a is required, even in the brain (27, 28). All three channel types are apparently glycoproteins with extensive carbohydrate moieties attached to the extracellular side of the channel (7).

The properties of these purified subunits provide several kinds of valuable information in addition to quaternary structure and subunit mass. 1) Directly determined partial sequences of purified a subunits have led to the identification of complete sequences by cloning techniques (see above). 2) Electron microscopic studies of the AChR have revealed that the subunits are arranged as the staves of a barrel, with the channel protruding into the extracellularand intracellularspaces by - 5.5 and 2.5 nm, respectively (8).Electron microscopic and crosslinking data indicate that the /3 subunit is located between the two a subunits in the intact pentameric con-

figuration channels

(48). have

Ultrastructural not

yet yielded

channel’s structure. 3) Purified as antigens for the production

studies as clear

of purified a picture

a subunits of polyclonal

Na

of that

have served and mono1909

clonal antibodies, which have been used for topographic mapping of the channels in the membrane matrix, immunocytochemical localization of the channel in intact tissues, immunochemical identification as the basis for assaying the channel protein (independently of biological activity), and direct modulation of function. 4) Electrophoretic identification of the a subunits has enabled the determination of covalent regulatory modification of the channel protein by such mechanisms as protein kinase-mediated phosphorylation, which can be correlated with phosphorylation-mediated modulation of function. Much recent work has concentrated on points 3 and 4, which are discussed in more detail below. Channel

immunochemistry

Analysis of the primary amino acid sequences of several channels has led to proposals about the folding of the channel proteins on the basis of the number of hydrophobic, putative membrane-spanning regions (8, 37, 38, 49). Morevoer, certain hydrophilic regions have been proposed as possible elements of the lining of the ion permeation pathway through the channel. These hypotheses can be tested by topographical analyses. For example, if there is an even number of membrane-spanning regions, both amino and carboxyl terminals should be located on the same side of the membrane. Alternatively, for an odd number of crossings, the terminals should be on opposite sides. Monoclonal antibodies were raised to a synthetic peptide corresponding to a sequence near the carboxyl terminus of the AChR. These antibodies bound only to permeabilized, right-side out Torpedo vesicles, indicating that the carboxyl terminus is normally located on the cytoplasmic side of the channel (50). This conclusion was confirmed by in situ immunocytochemical localization of the bound antibody to the cytoplasmic side.Because the amino terminus was known to be extracellular, this result lends support to the idea that models predict five transmembrane crossings in each AChR subunit (8). As discussed by Stevens (20),thisalsoprovides evidence for a hydrophilic membrane-spanning segment that might serve as part of the pore, with each subunit contributing one side, like the staves of a barrel (51). More recently, mapping of immunogenic sites on the Na channel has demonstrated that the carboxyl terminus and (probably) the amino terminus are located on the cytoplasmic side (47). Additional evidence is needed before the topography of the Na channel can be determined with certainty. Channel

modulation:

phosphorylation

Considerable evidence has accumulated to demonstrate that AChR and Na and Ca channel proteins are phosphorylated by cyclic nucleotide-dependent protein kinase and/or Ca and phospholipid-dependent protein kinase (PKC) (9, 52). Evidence that agents such as cyclic AMP, forskolin, and phorbol esters, which stimulate phosphorylation, modulate the channels in situ suggests that the AChR and several kinds of K channels may be phosphorylated,although the effectsof these agents may not always be mediated by phosphorylation (53, 54). Although 1910

Vol. 3

June 1989

the effect of phosphorylation on AChR function had not been directly demonstrated until recently, reports now indicate that the phosphorylated AChR desensitizes much more rapidly than controls (55). Huganir et al. (56) demonstrated this by using purified AChR reconstituted in phospholipid vesicles and concluded that the change in desensitization rate was determined by the level of phosphorylation of the -y and.5 subunits. The voltage-dependent Na channel a subunit is phosphorylated at several sites by cyclic AMP-dependent protein kinase and PKC (9). The functional role of phosphorylation is not clear, although a small decrease in channel-mediated Na influx was noted after phosphorylation. Regulation of Ca channels by phosphorylation in situ has been observed in many cases, notably in cardiac muscle where the process probably mediates the positive inotropic effect of /3-adrenergic stimulation. Recent evidence has directly demonstrated phosphorylation of the putative Ca channel protein (dihydropyridine binding site) (10, 57, 58) and shown that phosphorylation of the channel protein increases the channel open probability (58). Channel

modulation: G proteins

Over the lastseveralyears,ithas become apparent that guanine nucleotide-binding proteins (G proteins) in biologicalmembranes act as transducers that linkan extracellular signal at a membrane receptor with a cellular response. In many cases, G proteins may couple the receptor to the generation of a second messenger such as diacylglycerol or cyclic AMP (see above). The same or similar G proteins can also directly link an activated receptor to an ion channel, leading to stimulation or inhibition of channel function (59, 60). For example, G proteins (probably the a subunit) appear to mediate the activation of cardiac muscarinic, cholinergic receptorlinked K channels by ACh and that of cardiac Ca channels by /3-adrenergic and angiotensin activation (60). In neurons, a number of transmitters and neurohormones (e.g., catecholamines, ACh, opioids, -y-aminobutyric acid) inhibit Ca2 influx through voltage-gated Ca channels (59). This effect is thought to be mediated by another G protein, and may function via presynaptic autoreceptors in a negative feedback system to terminate transmitter release.The potentialimplicationsof dual regulation(i.e., by both G protein-mediatedsecond messenger production and direct channel modulation by G proteins) are not presentlyunderstood, nor isthe molecular nature of the interactionsbetween G protein subunits and the channel proteins. The possible difficulties in maintaining selectivity with similar G proteins throughout the transduction process are discussed by Neer and Clapham (61).

INTERDISCIPLINARY ON

THE

APPROACH:

A WINDOW

FUTURE

The progressof the past 10 years has largelyresultedfrom innovative work in the fieldsof channel biophysics, biochemistry, or molecular biology. We can expect that in the next decade the merging of two or all three of these

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KRUEGER

Closed

disciplines will result in a more detailed and profound understanding of channel structure and its relationship to function. Several recent reports in the literature illustrate what may be in store for the future. Pore-forming

segments

within

the channel

component

related

to Na channel

During a sustained depolarizing pulse, Na channels spontaneously enter a nonconducting inactivated state, facilitating the repolarization of nerve and muscle membranes during the action potential. The minimal voltage dependence and susceptibility to internal proteases of the inactivation process suggested that a component of the channel attached to the internal side plugs the inside mouth of the pore (64). Vassilev et al. (65) screened a series of monoclonal antibodies directed toward several putative, internally exposed sequences of the Na channel (49) for functional effects on channels in muscle cells. Antibody directed to a short, probably intracellular, segment (SP19) slowed Na channel inactivation, which suggests that it is bound to a part of the channel accessible to large internal macromolecules that must move to inactivate the channel. Moreover, maintaining the channels in the inactivated state retarded the action of the

antibody. lustrated

A possible explanation for these results is ilin Fig. 1. SP19 may be a part of the channel

protein (exposed to the internal side) that is involved in inactivation, perhaps by serving as a tethered blocking particle (hexagon), as suggested by Armstrong and Bezanilla (64). Antibody would bind to an antigenic determinant (notch in hexagon) that is less accessible in the inactivated state. Thus, with the antibody bound, inactivation would proceed more slowly, and the antibody effect would develop more slowly when the chanSTRUCTURE

AND

FUNCTION

OF ION CHANNELS

Inactivated

structure

Oiki et al. (62) synthesized a 22-amino-acid peptide that had the sequence of part of the voltage-gated Na channel thought to form part of the channel pore. When incorporated into planar bilayers, this peptide spontaneously formed channels with a single-channel conductance ( 20 pS) similar to that of intact channels (63). Although more selectivefor cationsthan for anions, these channels were not significantly selective forNa over K, nor was it demonstrated that this 22-amino-acid peptide was unique in itsabilityto form cation channels. The gating of the peptide-induced channels was not voltage dependent; however, this could be explained by the lack of the remaining channel protein (some 2000 amino acids). Computer simulation of 3-dimensional folding and aggregation showed that four of these peptides might aggregate like the staves of a barrel to form an aqueous pore down the middle, as has been proposed for the 3-dimensional arrangement of the four homologous repeating domains of the whole channel (19, 20). Although it is too early to tell whether this potentially productive strategy will provide useful information about channel structure, this approach may be an alternate way to study the functional consequences of altering the molecular structure of channels. A structural inactivation

Open

Figure

1. Illustration

of a hypothetical

mechanism

for Na channel

inactivation and how it may be modulated by antibodies to a segment (SP19) of the channel thought to be on the cytoplasmic side (65). Left: a closed (nonconducting) channel at rest. Center: an open (conducting) noninactivated channel. A negatively charged site has appeared on the cytoplasmic side. Right: an inactivated (nonconducting) Na channel. The positively charged tethered inactivation particle (64) (hexagon), which may be associated with SP19, plugs the open pore in the inactivated state. Antibody to SP19, shown bound

to the inactivating and

binds

poorly

particle (center), impedes the plugging process to already

inactivated

channels.

nels were inactivated. This is strong, presumptive evidence that a specific part of the channel (SP19) is associated with inactivation and is positioned in such a manner that its action can be affected by internally applied antibody. Voltage

sensor-lipid

charge

relationships

Incorporation of channels into planar lipid bilayers provides the opportunity to examine the effect of lipid composition, especially head group charge, on channel function. The effect of lipid charge on ion permeation through channels has been studied for sarcoplasmic reticulum K channels, Ca-activated K channels, Ca channels, and Na channels (66). For all but the Na channels, the mouth of the channel pore appears to be 1-2 nm from the negatively

charged

lipid head groups;

the Na channel

pore appears

to be at least 3-4 nm from the lipids. Probably the large mass of the Na channel isolates the pore from the lipids. Recently, the effects of surface charges on Na channel gating have been studied in planar bilayers of defined lipid charge (67). External or internal Ca2 altered channel gating as if the membrane potential were being hyperpolarized or depolarized, respectively. This was observed in bilayers containing only neutral lipids, which indicated that Ca2’ could affect gating by interacting directly with the channel protein on both sides of the channel, possibly by asymmetrically altering the surface potential. The effect of internal and external Ca2 was consistently larger in negative lipids, which demonstrated that the gating machinery is dose enough to the edge of the channel to be affected by the electric field across the lipid. These results are illustrated in Fig. 2. Dimension a is the distance between the mouth of the channel pore and the charged lipid head groups, which is probably at least 3 nm. Dimension b is the effective distance from the lipid surfaces to the gating machinery (potrayed as positively charged helices) (see refs 38, 39). These results place ap1911

proximate limitson the distancesbetween the negatively charged head groups and functionallyimportant locations within the channel protein associated with voltagedependent gating. Ion binding sitesin the ACh

receptor

channel

Previous work (42) had identified the region around the M2 segment of the a subunit as important in determining the rate of ion permeation (see above). Recently Imoto et al.(68) systematically altered the charge on aspartate and glutamate carboxyl residues in the vicinity of M2 by using site-directed mutagenesis. By monitoring the single-channel current-voltage relations of ACh receptors expressed in oocytes injected with the altered gene transcripts, they found that removing the negative charge reduced the single-channelconductance in proportion to the amount of charge removed. Moreover, asymmetric block by Mg2 ions revealed that some of those charged residuesare near the outsideor insideregionsof the channel pore, whereas some are located within the channel pore. Imoto et al. (68) suggested that in the intact a2f3y.5 pentamer, carboxyl residues in the vicinity of the M2 region of each of the subunits align to form three negatively charged rings through which entering ions must pass. This structural information is illustrated in Fig. 3, which shows a cross section of the AChR pore through the two a subunits and the relative locations of the carboxyl residuesidentifiedby Imoto et al.(68).The innerand outermost rings may serve as divalentblocking sites and prefiltersto facilitate entry of permeant cations, whereas the centralring may function as a selectivity filter

Figure

3. A hypothetical

model

of the AChR

showing

a cross

sec-

tion through the two a subunits. This diagram illustrates the conclusions of Imoto et al. (68), which were based on site-directed mutagenesis of certain aspartate or glutamate residues to asparagine, glutamine, or lysine, that a permeant ion would encounter a series of three rings of negatively charged groups as it traversed the channel pore. The external and internal rings may serve as prefilters and divalent blocking sites; the central site may function as a selectivity filter to select among permeant and impermeant cations. An Na ion is shown in the middle site; a divalent cation (e.g., Mg2) is shown in the intracellular site. Dimensions are not to scale.

only permeant cations. Although this idea has long hypothesized (1, 13), this is the firstdirect demonstration of a role for negatively charged amino acid residues in channel permeation and ion selectivity. to admit

been

CONCLUSIONS

Figure 2. A possible transparent view of a voltage-gated Na channel portrayed as a cylinder just spanning the membrane. The central pore is so far away from the negatively charged lipid head groups (dimension a) that entering cations are unaffected by lipid charge (66). As suggested by the results of Cukierman et al. (67), the voltage sensing machinery, which is shown as parts of transmembrane helices (possibly S4) containing repeating charged groups, can be affected by the electric field established by negatively charged groups (divalent cation binding sites) on both sides of the channel protein and by the charges on the lipid head groups. Thus, the voltage sensing apparatus is close enough to the edge of the channel to be influenced by the electric field between charged lipid head groups (dimension

1912

b).

Vol. 3

It is apparent from the surge of information that has appeared in the last few years that a satisfactory understanding of ion channel structure and function will require concerted and coordinated interdisciplinary research efforts from many fields. Investigators may have to learn new techniques and will certainly need to be conversant in the complex principles and terminologies of disciplines other than their own. Nevertheless, many laboratories are diversifying either independently or in collaboration with others. If the progress of 1988 alone is an indication, a clear understanding of the molecular correlates of ion channel function may be achieved much sooner than has been anticipated. Work from the author’s NS16285 and NS20106 from

laboratory the National

was

supported

by

grants

Institutes of Health and by

contract DAMD17-85-C-5283 from the U.S. Army Medical Research and Development Command. Special thanks to Dr. RobertJ. Bloch for helpful discussions and to Dr. Robert Guy for providing a preprint of ref 38.

June 1989

The FASEB Journal

KRUEGER

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