Surface charges and ion channel function.

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Annu. Rev. Physiol. 1991.53:341-359. Downloaded from www.annualreviews.org Access provided by University of Manchester - John Rylands Library on 02/27/18. For personal use only.

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1991

/99/. 53:341-59

by Annual Reviews Inc. All rights reserved

SURFACE CHARGES AND ION CHANNEL FUNCTION

William N. Green Department of Cellular and Molecular Physiology, Yale University School of Medicine, 333 Cedar Street, New Haven, Connecticut 06510

Olaf S. Andersen Department of Physiology and Biophysics, Cornell University Medical College, 1300 York Avenue, New York, New York 10021 KEY WORDS:

penneation, selectivity, electrostatics, diffusion limitations, substrate steering

INTRODUCTION Like other proteins, ion channels are charged. The amino acid sequence contains acidic and basic amino acids that are positively or negatively charged at physiologic pH. Other charges are added by posttranslational events such as the addition of sialic acid during complex carbohydrate trimming and phosphorylation. At least some of the charges appear to be essential for ion channel function as part of ligand and permeant ion binding sites and as the voltage sensor of voltage-gated channels (e.g. 35). Surface charges also contribute to channel function in more subtle ways. Any charge on or near the surface of a channel will polarize the surrounding environment, thus establishing an electrostatic potential, i.e. the surface potential, between the channel and aqueous solution (e.g. 54). The surface potential will alter the surrounding ionic atmosphere and change the con centrations of all charged solutes (permeant ions, toxins, or ligands). Op positely charged solutes (counterions) will be attracted to the charge and 341

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GREEN & ANDERSEN

increase their local concentration, while solutes of like charge will be repelled and decrease their concentration (25, 55, 36, 54, 30, 29, 80). In addition, surface potentials will alter the potential difference across the channel. The value of the surface potential will be added to (or subtracted from) the electrostatic potential on one side (or both sides) of the channel. The result is


that the surface potential will offset the potential differences across the channel (25, 10, 36). If surface charges are near important functional sites,

channel function can be modulated solely through the indirect actions of the surface potential on these sites. Changes in the local solute concentrations will alter the single-channel conductance and the channel's sensitivity to agonists, blocking ions and toxins. The offset in the membrane potential created by the surface potential will alter any voltage-dependent processes such as gating or ion channel block. The purpose of this review is to summarize the data indicating that ion channel function is affected by electrostatic potentials emanating from surface charges (see also 35,21). Despite overwhelming evidence for their existence, these charge effects are often overlooked or ignored when interpreting data and constructing models of channel permeation, selectivity and interactions with ligands and toxins. The importance of surface charge for channel gating will not be discussed since it is well established (e.g. 10, 36) and recently reviewed (28). Rather, this review will focus on the evidence that ion permeation is influenced by surface charges.

SOURCE AND DISTRIBUTION OF CHARGE With the purification of many ion channel proteins and the sequencing of even more ion channel cDNAs and genes, substantial information exists about the types and number of charged groups on ion channels. To address the question "what is the chemical identity of ion channel surface charge?" we briefly

examine the amino acid sequences and some posttranslational modifications of three different ion channels: the Torpedo nicotinic acetylcholine receptor (AChR), the bovine y-aminobutyric acid (GABAA) receptor, and the Elec trophorus voltage-sensitive sodium channel (Table 1). The evidence that charges on phospholipids can affect channel function will also be discussed.

Acidic and Basic Amino Acids Although amino acid sequences have been obtained, little is known of ion channel secondary and tertiary structure. Models of channel structure are based primarily on hydropathy profiles of deduced amino acid sequences. In these models charged amino acids and other hydrophilic residues are, for the most part, placed at or near the aqueous surfaces, while stretches of hydrophobic amino acids form lipid bilayer spanning a-helices (37, 49, 23).

343

ION CHANNEL SURFACE CHARGES

Table 1

Swface charge estimates for the AChR, GABAA receptor, and the Na+ channel

Amino acid charges Total

Ligand-gated channels

Extra-

Intra-

Potential

Sialic acid resi-

phosphorylation sites

dues (references)

PKN

PKCb

TyrosineC

I (38)

1 (38) 1 (38) I (38)


cellular cellular Cationic AChRd a

subunit

{3 subunit 'Y subunit 8 subunit

no (66)

- 7.5 - 8.5 -14 - 6.5

- 5 - 6.5 -16.5 -10

-2.5 -2 +2.5 +3.5

+14 +11 + 9

+ 6 + 3.5 + 4

+8 + 7.5 +5

1 (77)

-37

ND

ND

3 (22)

1 (38) 1 (38)

no (66) yes (66) yes (66)

Anionic GABA receptord a

{3

'Y

subunit subunit

subunit

N De N D

1 (67)

ND

Voltage-gated channels Na+ channelf

yes; 1 1 3

±

1 1 (56)

a Denotes protein kinase A; b denotes protein kinase C; 'denotes tyrosine kinase. d Total, extracellular and intracellular charge estimates based on the folding model with four membrane spanning a-helices (II, 19,64). Glutamate and aspartate amino acids were assigned a charge of -I, arginines and Iysines a charge

of + I, and histadines a charge of

e Not

determined. f Sequence from Noda et al

+ 0.5.

(62).

Table 1 summarizes information on the overall number of charged residues as well as estimates of extra- and intracellular amino acid charges based on current structural models for the AChR (11, 19, 64) and GABAA receptor

(77). The cation-selective AChR and sodium channels are negatively charged and the anion-selective GABAA receptor postively charged, which implies a role for some of the charge in ion selectivity

(77, 85; see below).

Sialic Acid Sialic acid residues are attached as the last step in the trimming of complex asparagine-linked oligosaccharide chains (47). Carbohydrate is added only to the extracellular domain of membrane proteins and may be a source of surface charge asymmetry. Sialic acids can make sizeable contributions to the net surface charge. The Electrophorus sodium channel contains large polysialic acid domains containing -110 negative charges (56, 42). No function has been established for this negative charge, but its removal by the sialidase, neuraminadase, caused a large shift in the average midpoint potential of channel activation to more depolarized potentials and increased the frequency of the subconductance states of the Electrophorus sodium channel (51). The AChR also has a substantial amount of sialic acid on its 'Yand 8 subunits (66),

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GREEN & ANDERSEN

but the amount of charge contributed by sialic acid and its functional signifi cance have not been detennined.


Phosphates All three channels in Table I contain potential phosphorylation sites de termined from sequence analysis and/or in vitro phosphorylation experiments (38, 77, 67, 22). Protein phosphorylation adds two negative charges to a phosphorylation site on the cytoplasmic surface of the channel. Unlike amino acids and sialic acids, which are permanent fixtures on the polypeptide, phosphorylation is a transient event resulting from the combined actions of cellular kinases and phosphatases. The transient nature of phosphorylation is in keeping with its role in ion channel modulation (43). The established view has been that the covalent attachment of a phosphate group affects function through conformation changes. Recent studies indicate that function also can be affected by a direct electrostatic interaction between the negatively charged phosphate and charged substrates or ligands (83, 39), which suggests that a similar mechanism is possible for ion channels.

Charged Lipids A net charge on the lipid headgroups in the surrounding bilayer can alter channel function. The single-channel conductance of bilayer-incorporated potassium channels from sarcoplasmic reticulum was increased in negatively charged bilayers and decreased in positively charged bilayers (6). The con ductance changes were most prominent at low permeant ion concentrations, which suggests that the lipid charge changes the concentration of permeant ion at the mouth of the channel. The conductance of calcium-activated potassium channels (57) and, to a lesser degree, the L-type calcium channels (12) is also affected by charged lipid. The conductance of voltage-dependent sodium channels was unaffected, which suggests that its channel entrance is farther from the lipid than the other channel types (30). Interestingly, the sodium channel activation curve is affected (14), thus indicating that the voltage sensor lies close to the lipid bilayer. ELECTROSTATIC POTENTIALS AT THE CHANNEL SURFACES

A precise mapping of the surface potential profile for ion channels is not possible without detailed structural information, which is unavailable. De tailed maps of the surface potentials of soluble proteins have been con structed, however, using high-resolution structures available for these pro teins. These surface potential profiles show features that ion channels may have in common with soluble proteins.


345


Lessons from Soluble Enzymes Charged residues are unevenly distributed over the surface of soluble proteins and clusters of charge are common (e.g. 82). The electrostatic potential thus varies with position over the protein surface, and the surface potential is a local, not a global, descriptor of the protein's electrostatic behavior. Detailed electrostatic maps, constructed using the Poisson-Boltzmann equation (e.g. 46), confirm that the electrostatic potential varies widely along the surface of the protein. Functionally charge clusters near the enzyme active site seem to steer substrate flow towards and into the site (61). In this way, local charges affect the binding of substrates to the active site of subtilisin (74), calbindin (53), acetylcholine esterase (65), and Cu, Zn-superoxide dismutase (13). Substrate steering to Cu, Zn-superoxide dismutase is particularly well-characterized (26). At physiologic pH, these enzymes have many more negative than positive charges (75), which should produce an electrostatic barrier for the substrate O2-, Nonetheless, the catalytic (association) rate constant decreases as ionic strength increases (13), which indicates that O2- is attracted to the active site (and that no electrostatic barrier exists). A cluster of positively charged lysines at the entrance of the active site pocket appears to attract O2(82, 46). Removing these charges through an acetylation reaction decreases the catalytic rate (13). After acetylation, the rate constant increases with ionic strength thus indicating that not only is the electrostatic attraction eliminated, but O2- is repelled from the modified enzyme.

Approximate Descriptions In the absence of detailed structural information, the electrostatics of ion channels are usually described by one of two limiting models: the Gouy Chapman theory of the diffuse double layer, or the Debye-Hiickel theory of ionic solutions. For either model, the distribution of the ions (and thus the electrostatic potential profile) is calculated by balancing the electrostatic interactions between surface charge and ions in solution with the tendency of the ions to diffuse from high to low concentrations. At eqUilibrium the local concentrations are given by the Boltzmann equation: 1.

where [I]s and [I]b are the surface and bulk concentrations, respectively, Vs the surface potential, Zi the valence of the ion, e the elementary charge, k Boltzmann's constant, and T the temperature in Kelvin. Ions of the same charge as the surface charge are repelled from the channel, and [1]8 will be less than [I]b' Ions of opposite charge are attracted to the channel, and [I]s will be larger than [l]b'

346

GREEN & ANDERSEN


In the Gouy-Chapman theory, a channel's surface charges are assumed to be unifonnly distributed (smeared) over a planar surface of infinite area. The relation between the surface potential (Vs), the charge density (a), and the aqueous electrolyte composition is given by (e.g. 48) a =

A .

{f:

[n . [exp(

-

zi . e

.

VsfkT) - 1]

o

}o.s

,

2.

where the constant A ( 2'kT' E o'Er) s, Eo is the permitivity of free space, Er the relative dielectric constant, [I]i and Zi the concentration and valence of ion species i. In the Debye-Hiickel theory, the surface charge is assumed to be distributed over a conducting, impenetrable sphere of radius a and valence ZS' The potential at the surface of the sphere is given by (e.g. 48). =

.

3. where the Debye length, LID [(2'e/A)/�[Ikzi2]O.5, is a measure of how far into the solution the potential change extends. The magnitude of the surface potential (and the Debye length) decreases with increasing ionic strength. The reduction in surface potential is caused by shielding or screening of surface charges by the increased concentration of counterions nearby. Since ionic strength (I[Ikzi2) increases as a second order function of an ion's valence (zJ. divalent or multivalent ions will be much more effective than monovalent ions at screening surface charges. At very high ionic strength both models predict that the surface potential approaches o mV and [I]s approaches [I]b' The essential difference between the models occurs when the ionic strength approaches zero. According to the GouyChapman theory (Equation 2), Vs will approach ±oo in such a way that the interfacial counterion concentrations approach finite limiting values (e.g. 54). In contrast, the Debye-Hiickel theory (Equation 3) predicts that Vs will approach zs'e/(4''lT'Eo'Er'a), and the interfacial counterion concentrations will approach zero. Either model represents a simplification, but they nevertheless provide semi-quantitative estimates on the importance of surface potentials on channel function (see below). The most glaring deficiency is the neglect of structural features. Recently, models incorporating more realistic structural detail have been proposed (15, 7). For example, low resolution structural data for the AChR show that the extracellular entrance to the channel has an unusual topology. The entrance consists of a large funnel that extends out -65 A perpendicular to the plane of the membrane (45, 84). Electrostatic potentials at the channel entrances will be enhanced by this funnel shape (15). The =

J


347

electric field, believed to arise from rings of negative charge at the bottom of the funnel (40), is concentrated by the funnel walls and steers permeant ions to the channel entrance and thus increases the local cation concentration.


PERMEATION

Overcoming Diffusion Limitations The primary function of ion channels is to provide selective yet rapid ion movement. The rates at which ions pass through a channel are limited by physical constraints. Ions cannot enter a channel faster than allowed by diffusion, which rate limits the approach to the channel entrance to 108_109(m sec)-l. Still, channels achieve ion translocation rates on the order of 107_108 (sec)-l (35), values close to the diffusion-limited constraint, given the physiologic concentrations of permeant ions such as K+ ,Na+, and Cl-. Such high translocation rates suggest that ions pass through the channels as fast as they reach the entrance. Interactions between ions and channel thus appear to be limited, and the channel has little time to discriminate among different ions. How then can a channel interact with and select among different ions if ion translocation is so fast? Like other proteins, ion channels are subject to evolutionary, selective pressures to maximize function. Nature has molded the design of soluble enzymes to maximize the rate of catalysis (1). In a similar way, ion channels have evolved to deal with the problem of diffusion limitations and allow both maximal ion translocation rates and ion specificity. A major structural adapta tion may be the large funnel-shaped entrances that are found in the AChR (45, 84) and in voltage-dependent sodium channels (18). It has been proposed that funnel-shaped entrances serve to increase the capture radius for incoming ions as compared to channels with long narrow pores (50). An increased capture radius will increase the overall translocation rate across the channel, but it will not increase the rate at which ions can access the region where the channel protein narrows and interacts with ions. In fact, the sterlc constraints imposed by a funnel will decrease the rate at which an ion diffuses to this part of the channel (76). If, however, the funnel is endowed with means to concentrate incoming ions, such as negative charge for a cation-selective channel, the funnel will increase ion access to the pore (2). In short, for a funnel-shaped entrance to increase ion access to a channel it must contain charges. The question of how charges in the funnel affect the conductance has been analyzed (15). The low-dielectric protein walls of the funnel serve to con strain the field lines, and guide the ions down towards fixed charges at the entrance to the narrow region of the channel. These charges serve a similar function as the charges at the entrance to the active site channel in soluble enzymes. A charged channel entrance is thus similar to a charged catalytic site

348

GREEN & ANDERSEN

on a soluble enzyme, by increasing ion entry through an increase in the local ion concentration.


Evidence that Ion Channel Conductance is Increased by Surface Charge Here we review experimental evidence that surface charges on ion channels enhance ion permeation and discuss problems involved in interpreting these data. The information is categorized by experimental technique. We start with the most general evidence that surface charges affect permeation and move through experiments designed to specify the chemical identity of the charged groups and their location on the polypeptide.

Changing Ionic Strength: Screening Surface Charge CONDUCTANCE-CONCENTRATION RELATION: DEVIATIONS FROM SIMPLE

Surface potentials vary as a function of the ionic strength of the surrounding solution (see above). Increasing the ionic strength screens surface charge, which reduces the magnitude of the surface potential and the distance over which it affects permeating (or blocking) ions. Measurements of conductance as a function of the concentration of permeant and impermeant ions is, in principle, a simple test for surface charge effects on conductance. In the absence of electrostatic effects, the conductance-concentration relation for a singly occupied channel is described by a Michaelis-Menten curve (33, 34) SATURATION

g=gmaxl( l +Kg/[permeant ion)),

4.

where g is the channel conductance, gmax is the maximal conductance value, and Kg is the permeant ion concentration at half-maximal conductance. The conductance of AChRs (16) and batrachotoxin-modified sodium chan nels (30) deviate considerably from the simple rectangular hyperbola pre dicted by Equation 4. When ionic strength is varied by changing permeant ion concentration, the single-channel conductance at low permeant ion con centrations (20-100 mM) is larger than expected and is almost independent of concentration (see Figure 1). This is the behavior expected if negative charges are increasing permeant ion concentrations at the channel entrances. The conductance-concentration relation is well-described by several electrostatic models relating variations in bath concentrations with changes in the electrostatic potential at the channel entrances (15, 30, 7). Similar ex periments on inward rectifying potassium channels (44), L-type calcium channels (69), and calcium-activated potassium channels (52) have led to similar conclusions about the existence of fixed charges near the entrances of these channels.


349

40 30


20

(f) a. Q) u c: 0

ti "

"0 c: 0 u

10 0

0

40

2 [Na+j (Ml

3

4

30 20 10 0

Figure 1

1000

0

Single-channel conductance of batrachotoxin-modified sodium channels as a function

of Na+ concentration. (top) Conductance-[Na+] relation for channels in phosphatidylethanol amine: phosphatidylcholine (4: 1) membranes (no net charge). The points denote means ±S.D.

a fit of Equations 1,2, and 4 to the results assuming gm"x 45 pS; Kg 1.5 M, and 0.38 e·mn-2 • (bottom) An Eadie-Hofstee plot of the same data. The solid line is a transform of the curve above. The dotted line is a nonlinear least-squares fit of Equation 4 to the results for 20 mM :$ [Na+] :$ 100 mM, assuming a = 0; gm., 21 pS; and Kg 3 mM. Reproduction from Green et al (30), J. Gen. Physiol. 1987. 89: 841-872, by copyright permission from The

The curve is

=

=

(j =

=

=

Rockefeller University Press.

For most of the channels, the negative charges must reside on the channel itself, not on the lipid. This is known for sodium channels and calcium activated potassium channels since the channels were incorporated into planar bilayers composed of lipids carrying no net charge. For the AChR, the extracellular channel entrance is 6--7 nm from the membrane surface (84), too far for lipid charges to significantly affect permeant ions entering the AChR. Additional evidence that surface charges increase channel conductance is obtained by measuring the conductance-concentration relation at constant ionic strength. Changing permeant ion concentrations at a constant ionic strength will keep the electrostatic potential profile constant, and the con ductance-concentration relation should conform to the predictions of Equation 4. When the ionic strength was held constant, using the impermeant ion

350

GREEN & ANDERSEN

12 1.0

"

OB

,


0.6

,

0.4 «

Q.

0.2 01--+--+--A'---l----l--1

C

OJ

� -02

(.)

-0.4 -0.6 -0.8 -1.0 -

, 1 2 �'"=' ----=�---=-----='=---:'-:::-----=': -=' :-80 -60 -40 -20 0 20 40 60 80 .

Potential

(mV)

Figure 2 Effects of Ba2+ on the single-channel current of batrachotoxin-modified sodium 2+ channels. (a) i-V relation in symmetrical 20 mM Na+ with 5 mM Ba added to the extracellular solution. (0) i-V relation in symmetrical 20 mM Na+ with 5 mM Ba2+ added to both solutions. 2+ The dashed line represents the average i-V relation in 20 mM Na+ in the absence of Ba . Reproduction from Green et al (30) 1. Gen. Physiol., 1987. 89: 841-872, by copyright permis sion from The Rockefeller University Press.

TEA+, the deviations from the ideal conductance-concentration relation for sodium (30) and L-type calcium channels (69) disappeared. CHANGING DIVALENT CATION CONCENTRATION: CHANNEL BLOCK OR SCREENING OF SURFACE CHARGE. Changes in divalent cation concentra tion alter negative surface potentials to a much larger extent than the same change in monovalent cation concentration (see above). Another test for surface charge effects on conductance, therefore, has been to vary divalent cation concentrations. But divalent cations also can bind within the pore and block the channel (e.g. 86). Under certain experimental conditions, channel current reductions caused by divalent cation screening of surface charge and channel block are indistinguishable. This difficulty is illustrated in Figure 2 where the addition of 5 ruM Ba2+ reduces the sodium channel conductance. 2 When Ba + is added only to the extracellular solution (closed triangles), the current-voltage (i-V) relation rectifies as if the BaH causes a voltage-



351

dependent block. When Ba2+ is applied symmetrically, however, the i-V relation is linear (closed circles). and the conductance is reduced by 70%. In contrast, symmetric application of the extracellular channel blockers Zn2+ and Ca2+ causes much larger reductions in the inward going current than in the outward current (58, 30). The symmetry of the Ba2+-induced current reduction suggests that Ba2+ does not block the sodium channel, but simply screens surface charges at the channel entrances. According to this interpretation, the Ba2+-induced i-V rectification is caused by a combination of reduced local permeant ion con centration at the extracellular entrance and an offset of the transmembrane potential (25). Even this interpretation is too simple. At the concentrations Zn2+ and Ca2+ block the channel, they also must be screening surface charge. Further, divalent cations can directly bind to surface charges and reduce the charge density in addition to screening (e.g. 36). Conversely, it is likely that Ba2+ blocks the channel to some extent. Screening (along with binding to) charges and channel block are thus two extremes in a continuum. At the single-channel level, block can be distinguished from screening by resolving discrete blocking events (59), or channel flicker caused by the blocker (88). When discrete blocking events are not evident, screening and block, in principle, can be distinguished by the shape and position of the dose-response curve for a suspected blocker on the channel conductance. With increasing concentration an impermeant blocker will reduce the con ductance to zero over a concentration range highly dependent on the chemical identity of the blocker. If only screening is involved, the conductance de crease will approach a nonzero value (Figure 3) and the dose-response curve will be independent of the ion used. (The dose-response curve will vary with the ion species if the ion is binding to the surface charges.) This test was used to demonstrate the surface charge effects on AChR conductance. Increasing Ca2+, Ba2+, S�+, or Mg2+ concentrations over the same range equally reduced the AChR single-channel conductance by only one half (41). Increasing divalent cation concentrations at inward rectifying (44) and cal cium-activated potassium channels (52; Figure 3) yielded similar results and conclusions.

Changing pH: Neutralizing Surface Charge Another approach is to study conductance as a function of pH. This relation can provide some information about the chemical identity of the surface charge. Lowering or raising pH will titrate acidic and basic groups that contribute to ion channel surface charge. One or more proton dissociation curves are fit in relation to those obtained from the changes in conductance as a function of pH (32, 27, 60, 36). The curve fit yields characteristic pKa values that provide clues as to the chemical nature of the charge groups. The

352

GREEN & ANDERSEN


A

-=

24

12

o +-------+---� o 100 200

Figure 3

Reduction of the outward single-channel K+ current through calcium-activated potas

sium channels by Mg2+. The intracellular solution contained 150 mM KCl and the extracellular solution 4 mM MOPS-NaOH, pH 7.4, plus the indicated [Mg2+J. Reproduction from MacKin non et al (52), Biochemistry 1 989, 28: 8092-8099, by copyright permission from the Am. Chem.

Soc.

sodium channel conductance, for example, is reduced by lowering the ex tracellular pH (32). The data were well fit by a single H+ dissociation curve with a pKa of 5.2, which suggests that an acidic group is important for sodium channel conductance. In subsequent studies it was debated whether protons titrate surface charge at the channel entrance (32,20, 8), or charge within the channel thus resulting in a voltage-dependent block (86, 8), It is ex perimentally difficult to distinguish between these two possibilities, and they are not mutually exclusive (17; see above).

Chemical Modifications: Eliminating SUlface Charge Using Group Specific Agents The chemical identity of surface charge groups can be probed using group specific chemical reagents. Extracellular application of the carboxyl-specific reagents, carbodiimides, to sodium channels abolishes the channels' sensitiv ity to tetrodotoxin (78,5, 71). Another carboxyl-specific reagent, trimethylo xonium (TMO), abolishes tetrodotoxin sensitivity and also reduces the single channel conductance without affecting selectivity (81, 79, 31, 87). The latter results suggest that one or more carboxyl groups lie at the extracellular channel entrance where they contribute to the negative surface charge. Carbo diimides can reduce the single-channel conductance without altering tetrodo toxin sensitivity (9), further indicating that some of the carboxyl groups



353

involved in the conductance change differ from those affecting tetrodotoxin sensitivity. TMO modification of calcium-activated potassium channel conductance indicates that carboxyl groups are contributing to the electrostatic potential at that channel entrance (52). It was concluded that more than a single carboxyl group is involved because the single-channel conductance decrease occurred in discrete steps. The TMO effect on conductance depended on ionic strength. At low ionic strength, TMO caused a marked decrease in conductance, and a progressively smaller effect occurred with increasing ionic strength.

Site-Directed Mutations: Adding or Removing Specific Surface Charges Specific charges that affect channel conductance have been identified using methods that alter the ion channel amino acid sequence. This approach was first applied to gramicidin A channels, which long have served as prototypical transmembrane ion channels (3). At the channel entrance, pyromellitic acid was attached to the ethanolamine, which is part of the carboxyl terminus of the peptide (4). This modification added three negative charges at the channel entrance. The single-channel conductance was increased at low permeant ion concentrations, but not at high concentrations, consistent with an increase in permeant ion concentration resulting from the negative charge. Essentially similar results were obtained with desethanolamine gramicidin A, which has a free carboxyl group at the entrance (72), and [taurine16] gramicidin A, where the neutral ethanolamine has been replaced with the sulfur amino acid taurine (73). Specific charge groups serving a similar function on AChRs have been located using recombinant DNA methods. A series of point mutations were performed that removed negatively charged amino acids thought to be situated at the extracellular and intracellular mouths of the AChR channel (40). Three sets of negatively charged amino acids, located on homologous positions on each of the four AChR subunits, were mutated by substituting neutral or positively charged amino acids. Because of the pentameric symmetry of the AChR, these acidic amino acids are thought to form rings of negative charge around the channel entrances. The results of the single-channel current measurements of the mutated AChRs support this structural hypothesis and are consistent with the charge rings increasing permeant ion concentrations through electrostatic interactions. Mutations in the extracellular ring of charge reduced the cell-inward current,and mutations in an intracellular ring reduced the cell-outward current. Each decrease in negative charge caused a decrease in current. The decrease depended primarily on the charge of the substituted amino acid, and to a lesser extent on its size and the subunit on which it was located. Additionally, the mutated channels were much less sensitive to the effects of divalent cations.

354

GREEN & ANDERSEN


A single point mutation, changing (a glutamate to glutamine, dramatically reduces the sodium channel's sensitivity to tetrodotoxin and decreases the cell inward current substantially more than the outward going current (63). The latter result indicates that the mutated glutamate could be near the ex tracellular channel entrance where it contributes to a negative electrostatic potential. Is

Channel Conformation Altered?

The experimental manipulations described above (changes in ionic strength, composition, or pH, chemical modifications or site-directed mutations) may impose strains on the channel structure. Conductance changes resulting from these manipulations may, therefore, be caused by changes in channel con formation instead of changes in the surface potential. Among the evidence that conformational changes can occur is the observation of single-channel subconductance states. At high ionic strength (1.0--3.5 M), batrachotoxin modified sodium channels from dog brain exhibit a subconductance state about 20% of the size of the fully open state (30). A similar subconductance

state is observed when Zn2+ is added to batrachotoxin-modified sodium

channels from dog heart (70). Increasing the Zn2+ concentration causes the channel to spend progressively more time in a subconductance state, 15-25% as large as its fully open state. While these examples are most likely caused by conformational changes, it is possible that they result from changes in surface charge at the channel entrance. This is not the case for a subconductance state of the L-type calcium channel. A subconductance state, 25% as large as the fully open state, is observed when extracellular pH is lowered (68). A similar ion concentration dependence for both the fully open and subconductance state indicates that the subconductance state does not occur when surface charge at the channel entrance is titrated (69). Subconductance states appear in many channel types. To the extent that their frequency is affected by changes in ionic composition (or the other experimental changes listed above), it is important to distinguish between conductance changes that are the direct result of changes in surface potential from those that result from channel conformational changes.

Relevance to Channel Selectivity Charged groups at the entrance of an ion channel are a structural feature that helps determine whether a channel is cationic or anionic. A negatively charged entrance will increase the cation to anion concentration ratio at the entrance; a positively charged entrance will have the opposite effect. (As an example,a potential of -80 mV at the entrance will increase cation concentra tion over anion concentration by 400-fold, or vice-versa if the potential is + 80 mV.) Early evidence for the importance of this mechanism came from experiments on polyene-doped planar bilayers (24). Additional evidence in



355

support of such a selection mechanism comes from a comparison of the charged regions at the entrance of the cationic AChR (40) with their structural counterparts from the anionic GABAA receptor (see Table 1). At both its extracellular and intracellular entrances, the Torpedo AChR is thought to have rings containing three negative charges. In the corresponding positions on the GABAA receptor (77, 67) there are at least seven positive charges at the extracellular entrance (0': +2, {3: +2, y: +3) and two positive charges at the intracellular entrance (0': + 1, {3: 0, y: +1). Thus, the cation selective AChR has negatively charged entrances, and the anion selective GABAA receptor has positively charged entrances.

CONCLUSIONS There is substantial evidence that ion permeation (and possibly ion selectivity) of many channels is enhanced by charges at the channel entrances. A striking parallel exists between the effect of these channel surface charges and the charges on soluble enzymes that steer substrate into the enzyme's active site. In both cases the effects of strategically located charge groups suggest that they result from evolutionary adaptations to overcome diffusion limited ac cess to either the channel entrance or the enzyme active site. Given the very high ion translocation rates exhibited by ion channels, we believe that most, if not all, ion channels use surface charges to increase the rate in which ions enter the channel. Charge clusters at the entrances of ion channels, therefore, should be a common structural motif and could provide a landmark for locating a channel's entrance within its amino acid sequence. ACKNOWLEDGMENTS

We thank Dr. E. Moczydlowski for providing data prior to publication. Preparation of this review was supported by a OssermaniMcClure Fellowship from the Myasthenia Gravis Foundation (WNG) and National Institutes of Health grant GM40062 (OSA).

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