ANNUAL REVIEWS
Further
Quick links to online content
Annu. Rev. Biophys. Biomol. Struct. 1992.21:267-92 Copyright © 1992 by Annual Reviews Inc. All rights reserved
Annu. Rev. Biophys. Biomol. Struct. 1992.21:267-292. Downloaded from www.annualreviews.org Access provided by University of Illinois - Chicago on 10/04/15. For personal use only.
THE PERMEATION PATHWAY OF NEUROTRANSMITTER-GATED ION CHANNELS Henry A. Lester
Division of Biology, California Institute of Technology, Pasadena, California 91125 KEY WORDS:
synapse, nicotinic acetylcholine receptor, neurotransmitter receptor, open-channel blockers, site-directed mutagenesis
CONTENTS PERSPBCTlVES AND OVERVIBW........................................................................................
Transmembrane Topology and Primacy of the M2 Region....... . . . . ............ . . . . . . . . . . . . . . . . . Classes of Molecular Manipulation...... . .................................................................... Practical Relevance ...................................... . ......... .................................................. ........ . ......... . ...... . ......... .............. . ............... . ..... Reversal Potentials .. . ............. ................ ....... . . .. . ..... Current-Voltage Relations ... ......... . . .. ... ....................... .......... Anomalous Mole-Fraction Effects...................... .......... . . . . ......................................... Blockade Within the Channel by Organic Ions and Local Anesthetics ............... ........ Covalent Labeling by Channel Blockers ............. . . ........... . . . . . . ................... . ............... Blockade Within the Channel by Inorganic Ions.. . . . . . . . . . . . . .......................................... Peptide Channel Models .................................... ....... ..... ....... ........... . ........... . ......... Does the Conduction Pathway Move in Galin.q?
CLASSES OF MEASUREMENT ... . . . ... . .. . .......
.
.
.
270 270 273
277
277 284 285 287 288
........................... .. .. . ....... ..... . .. ................... ... ...... ...... .........
288
..................................................... ..... . . .. . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .... . . . . . . . . . .
289
THEORETICAL APPROACHES CONCLUSIONS .
.
267 268 270 270
.
PERSPECTIVES AND OVERVIEW
The group of neurotransmitter-gated ion channels includes (a) cation channels gated by acetylcholine (ACh), glutamate, ATP, and serotonin and (b) anion channels gated by GABA, glycine, ACh (invertebrates), and histamine (invertebrates). I doubt that more than one or two additional neurotransmitters will be found that directly gate ion channels, although 267 1056-8700/92/0610-0267$02.00
Annu. Rev. Biophys. Biomol. Struct. 1992.21:267-292. Downloaded from www.annualreviews.org Access provided by University of Illinois - Chicago on 10/04/15. For personal use only.
268
LESTER
additional receptor subtypes gated by the above known transmitters are often discovered. The same neurotransmitters also activate, with quite different kinetics and pharmacology,a distinct class of receptors that have seven transmembrane helices and in turn activate G proteins; this chapter does not treat the seven-helix receptor family. Ligand-dependent ion chan nels are presently thought to perform several distinct functions: (a) ligand binding; (b) gating,the conformational transitions that open and close the channel; (c) desensitization; and (d) permeation. This chapter focuses on permeation and is motivated by much recent data from site-directed mutagenesis and peptide chemistry showing that permeation occurs in regions of the protein that are at least partially distinct from those sub serving the other three functions. My attempt at a unified treatment is of course based on the structural similarity suggested by recent cDNA sequencing and protein chemical data for neurotransmitter-gated chan nels; these similarities suggest that this group represents a superfamily (see e.g. 22, 145). Many incisive previous reviews have treated channels gated by neurotransmitters and by intracellular ligands (18,22,55,60,97, 125, 145, 154). Most ligand-dependent cation channels are less selective than voltage dependent cation channels. Ligand-gated anion channels are rather non selective as well-indeed,GABA and glycine channels show a measurable permeability for cations (122). As is also true for the voltage-gated channels, no studies report pharmacological or genetic manipulations that grossly affect the selectivity of ligand-gated channels, e.g. that change cation to anion channels. Such manipulations, when successfully per formed and interpreted, will represent a triumph of the site-directed muta genesis approach. Mutations that produce subtle changes in acetylcholine receptor selectivity have,however, recently been studied (29, 30,81,152). Transmembrane Topology and Primacy of the M2 Region
The adult muscle ACh receptor is a pseudosymmetric pentamer with subunit composition (/.2/3yt3. Other members of the neurotransmitter-gated receptor superfamily are probably pen tamers as well ( lOa). Each subunit has at least four putative membrane-spanning regions (Ml through M4). This chapter reviews the evidence that the M2 region (Figure 1) lines at least part of the conduction pathway. Certain contrary facts are also cited below and must be explained. An elegant recent review provides hypothetical diagrams of the M2 region at atomic resolution for the ACh channel and homologous regions for GABA and glycine channels (145). In the view Figure 1 C presents of the ACh receptor in its open confor mation, the five M21X-helices line the conducting pore like sheaves of wheat, slightly askew to create a point of closest appr oach . At the extracellular vestibule, some fixed charges affect ion flux. The open-channel blocker,
Annu. Rev. Biophys. Biomol. Struct. 1992.21:267-292. Downloaded from www.annualreviews.org Access provided by University of Illinois - Chicago on 10/04/15. For personal use only.
PERMEATION AT LIGAND-GATED CHANNELS
269
QX-222, penetrates from the external solution partially into the tapering channel, so that its nonpolar aromatic moiety and its polar charged moiety interact most strongly with residues at position 10' and 6', respectively. Permeant ions also interact with residues at position 6'. The taper of the channel prevents QX-222 from reaching the next turn,at p�sition 2'. Here and at the next turn-position -l'-thechannel is narrowest, and amino acid side chains interact most strongly with permeant ions. As ions con tinue to flow inward, they experience a broadening channel, but con ductance can still be affected by residues at position -4'. Classes of Molecular Manipulation
The rapid progress of recent years derives directly from four new classes of molecular manipulation: (a) DNA sequencing and site-directed mutag enesis, (b) protein sequencing, (c) patch-clamp measurements of channel conductance, and (d) patch-clamp measurements of single channels during local anesthetic molecule block. This chapter shows how these experiments are used to test the postulated structures and mechanisms, particularly for the ACh receptor. It also provides a comparative view of the comp lementary information from other neurotransmitter-gated ion channels. Practical Relevance
Biophysical and structural analysis of the permeation pathway may lead to knowledge that is useful in neurobiological or clinical contexts. Rec tification in the current-voltage relation of neurotransmitter receptors might influence encoding properties of neurons (92). The flux of Ca2+ through neuronal ACh channels might influence many processes ranging from transmitter release to gene activation (reviewed in 38). At N-methyl D-aspartate (NMDA) receptors, Ca2+ influx might underlie some forms of memory and learning (79) as well as ischemic damage (28). CLASSES OF MEASUREMENT
This section describes the conclusions that can be drawn from several classes of experiments on neurotransmitter-gated channels. The organ ization follows a recent chapter describing permeation through voltage gated channels (90). The reader should consult that chapter and the book by Hille (62) for the theoretical basis of the electrophysiological experiments. Reversal Potentials The measurement of reversal potentials is a null technique that gives a single value; it has wide application because it is sensitive to neither block nor saturation in the channel. For instance, macroscopic reversal potential measurements are possible under conditions in which single-channel cur-
270
A ACh ex1
LESTER
-4 '
-1 '
2'
D:: �E �KAGGQK eE K «7 DD D S G RV SAT3 GAAB s VP A T V o A A PAR V G GLY D Q
Annu. Rev. Biophys. Biomol. Struct. 1992.21:267-292. Downloaded from www.annualreviews.org Access provided by University of Illinois - Chicago on 10/04/15. For personal use only.
K
GluRl
COND
SL E QX Ncr
N
G
X X
r c
6'
L
g' 10'
�L::::: L AQ T V F L AQ V F LTVFMLLVA T LG Y VF V D V T T V T M TTL AR N T V M TT GA Q 'i
N
W
F
G
0
L
R
C
F
X
20'
10'
6'
2'
-1'
-4'
A D
Annu. Rev. Biophys. Biomol. Struct. 1992.21:267-292. Downloaded from www.annualreviews.org Access provided by University of Illinois - Chicago on 10/04/15. For personal use only.
PERMEATION AT LIGAND-GATED CHANNELS
27 1
rents are too small to be measured (29, 30). For ligand-gated channels in particular, permeabilities measured by reversal potentials decrease mono tonically with hydrated radius or other measures of molecular size (6, 42); therefore the narrowest region of the channel is usually identified with the highest energy barrier that an ion must traverse in crossing the membrane. Because several previous studies of ACh receptors suggested that single channel conductance was most strongly affected by mutations at the 2' or the -I' position (26, 7 1), these positions seemed likely candidates for the narrowest region of the channel. It also seemed most promising to examine effects of mutations on the relative permeability PTris/PNa, as Tris is a slightly permeant ion. Several mutations at the 2' position, but not at the 6', 10', nor 14' positions, did in fact render Tris relatively less permeant than in the wild-type receptor, without appreciable changes in the per meability to Na+ (29, 30). The total range of manipUlations in these experiments was roughly threefold, from PTris/PNa 0.36 to 0.1 1. Thus, the 2' position is apparently near the narrowest region of the channel. A surprising result was the ratio of 0.36 for wild-type mouse ACh channels, compared with the ratio of 0. 18 obtained for the frog muscle channels (6, 42). Yet Cohen et al (29) measured PTris/PNa for the wild-type Torpedo cali/arnica ACh channel in the oocyte system and obtained 0.22, close to the value for frog; thus among wild-type ACh channels, this parameter displays differences that cannot be explained by the M2 sequence alone. The amino acids at the -I' position were not systematically mutated by Cohen et al (29); however, the single mutation reported, bE-l'Q, decreased PTris/PNa nearly threefold. Interestingly, the mouse rxf3y receptor (obtained by omitting (j-subunit mRNA from the injection mixture), but not the rxf3(j =
Figure 1
The M2 region of neurotransmitter-gated channels.
M2 region of the mouse muscle a,
(A) Aligned sequences in the
/3,]1, and (j subunits, the chick neuronal a7 subunit [which
forms homooligomeric channels (33)], a SHT 3 receptor (10Sa), a subunits from the GABA
and glycine receptors, and the GluRI kainatelAMPA receptor (the latter sequence is the least similar in the group and the alignment is therefore tentative). Underlining shows a periodicity of 3.5, which would align the residues in a stripe along one face of the helix. Underlining agrees well with the location of boxed residues, or their homologs in the T.
californica
receptor, which have been localized to the conduction pathway by types of D. (D) Abbreviations are: COND, conductance; SEL, selectivity; QX, QX-222 blockade; NCI, noncompetitive inhibitor binding. (C) Schematic diagram of the experiments shown in
region surrounding the permeation pathway. The five rods each represent an a-helix that extends from position - 4' to 20'. The distance from the center of each helix to the center of the channel is arbitrarily set to 5 A at position l' (dUlled circle). Each helix is tilted 20" in a plane tangent to the circumference at this point, yielding a taper in both directions away from position I'. Assuming 3.6 residues/turn and 5.4 Altum, the diagram shows that position
20' is 26.8 A away axially from the narrowest region and has a diameter of 10.9 A ; position -4' is 7.5 A away and has a radius of 5.5 A . The vertical scale at right shows positions
discussed above (A) and in the text.
Annu. Rev. Biophys. Biomol. Struct. 1992.21:267-292. Downloaded from www.annualreviews.org Access provided by University of Illinois - Chicago on 10/04/15. For personal use only.
272
LESTER
receptor, was characterized by a very low PTris/PNa ratio (29, 30). Other work suggests that the omitted y or b subunit is replaced by the b or y subunit, respectively (P. Charnet, C. Labarca & H. A. Lester, submitted). Because the y subunit has a Gin residue at the - I' position, the apy receptor is like the bE-I'Q m� tation at the - I' position; the result is thus consistent with the large effect the -I' position has on selectivity. The mutations generated by Imoto et al (69) (described in detail below) were later subjected to reversal potential analyses that clearly showed the primacy of the - l ' position versus the -4' and 20' positions (81). The bE-l'Q mutation changed the PCs/PK ratio to 0.77 from the wild-type value of l . 1O; smaller changes also occurred in the PNa/PK ratio. On the other hand, at the -4' and the 20' positions, four charge changes were required to affect the PNa/PK ratio. CALCIUM PERMEABILITY Table 1 summarizes some studies that determine the Ca2 + permeability of neurotransmitter-gated channels, mostly on the basis of reversal potential measurements. A useful generalization is that there appear to be three classes of Ca2 + permeabilities: (a) undetectably low, (b) comparable to that for Na +, and (c) much higher than Na+ (the last class includes NMDA receptors only). Friel & Bean (50) have presented a lucid discussion of the possible artifacts in measuring Ca2 + permeability of channels gated by extracellular ATP, which would be expected to chelate Ca2 +. Although non-NMDA glutamate receptors are often considered impermeable to Ca2 +, the recent study by Hollmann et al (63) should be consulted for literature references to previous studies of non-NMDA receptors that reported Ca2 + -permeable non-NMDA responses in neurons.
Table 1
Measured Ca2+ permeabilities for some neurotransmitter-gated channels
Agonist
Tissue/source
PCa/PNa
GCa/GN, 0.4--0.6
ACh
Muscle/electroplaque
0.6- 1 .0
ACh
Muscle
0.2
ACh
Neuronal
0.93
ACh
A p/ysia neurons
Kainate/AMPA
GluR2+GluRI or
Kainate/AMPA
GluRI +GluR3 or GluR l
GluR2+GluR3
Reference 38, 85, 95 6
47
�0.5 0.\3
14
N.D.'
N.D."
63
� Ib
0.3-0.7
63
or GluR3 NMDA
Mouse neurons
7.S 10.6
12, 1 1 2
ATP
Smooth muscle
�3
20
5-HT
NG108-15 cells
40 j1M. First, the blocked state displayed a double exponential distribution of lifetimes, and second, the time integral of the openings decreased to less than the control value. Apparently, the channel can close on the bound QX-222 molecule, perhaps by the trapping effect described below. QX-222 BINDING SITE In the mid-1980s, site-directed mutagenesis and oocyte expression became the appropriate techniques for investigating the interaction between open-channel block ers, exemplified by QX-222, and the receptor channel. Researchers hoped that the specificity of site-directed mutagenesis would complement the molecular precision of the single-channel measurements on open-channel blockade. To identify promising residues for mutagenesis, the Caltech group noted the body of literature on noncompetitive inhibitors at ACh channels. When we began the site-directed mutagenesis experiments in 1987, position 6' was the appropriate first choice, as outlined in the next major section. Mutant subunits of the mouse muscle ACh receptor (a, /3, y,
Further
Quick links to online content
Annu. Rev. Biophys. Biomol. Struct. 1992.21:267-92 Copyright © 1992 by Annual Reviews Inc. All rights reserved
Annu. Rev. Biophys. Biomol. Struct. 1992.21:267-292. Downloaded from www.annualreviews.org Access provided by University of Illinois - Chicago on 10/04/15. For personal use only.
THE PERMEATION PATHWAY OF NEUROTRANSMITTER-GATED ION CHANNELS Henry A. Lester
Division of Biology, California Institute of Technology, Pasadena, California 91125 KEY WORDS:
synapse, nicotinic acetylcholine receptor, neurotransmitter receptor, open-channel blockers, site-directed mutagenesis
CONTENTS PERSPBCTlVES AND OVERVIBW........................................................................................
Transmembrane Topology and Primacy of the M2 Region....... . . . . ............ . . . . . . . . . . . . . . . . . Classes of Molecular Manipulation...... . .................................................................... Practical Relevance ...................................... . ......... .................................................. ........ . ......... . ...... . ......... .............. . ............... . ..... Reversal Potentials .. . ............. ................ ....... . . .. . ..... Current-Voltage Relations ... ......... . . .. ... ....................... .......... Anomalous Mole-Fraction Effects...................... .......... . . . . ......................................... Blockade Within the Channel by Organic Ions and Local Anesthetics ............... ........ Covalent Labeling by Channel Blockers ............. . . ........... . . . . . . ................... . ............... Blockade Within the Channel by Inorganic Ions.. . . . . . . . . . . . . .......................................... Peptide Channel Models .................................... ....... ..... ....... ........... . ........... . ......... Does the Conduction Pathway Move in Galin.q?
CLASSES OF MEASUREMENT ... . . . ... . .. . .......
.
.
.
270 270 273
277
277 284 285 287 288
........................... .. .. . ....... ..... . .. ................... ... ...... ...... .........
288
..................................................... ..... . . .. . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .... . . . . . . . . . .
289
THEORETICAL APPROACHES CONCLUSIONS .
.
267 268 270 270
.
PERSPECTIVES AND OVERVIEW
The group of neurotransmitter-gated ion channels includes (a) cation channels gated by acetylcholine (ACh), glutamate, ATP, and serotonin and (b) anion channels gated by GABA, glycine, ACh (invertebrates), and histamine (invertebrates). I doubt that more than one or two additional neurotransmitters will be found that directly gate ion channels, although 267 1056-8700/92/0610-0267$02.00
Annu. Rev. Biophys. Biomol. Struct. 1992.21:267-292. Downloaded from www.annualreviews.org Access provided by University of Illinois - Chicago on 10/04/15. For personal use only.
268
LESTER
additional receptor subtypes gated by the above known transmitters are often discovered. The same neurotransmitters also activate, with quite different kinetics and pharmacology,a distinct class of receptors that have seven transmembrane helices and in turn activate G proteins; this chapter does not treat the seven-helix receptor family. Ligand-dependent ion chan nels are presently thought to perform several distinct functions: (a) ligand binding; (b) gating,the conformational transitions that open and close the channel; (c) desensitization; and (d) permeation. This chapter focuses on permeation and is motivated by much recent data from site-directed mutagenesis and peptide chemistry showing that permeation occurs in regions of the protein that are at least partially distinct from those sub serving the other three functions. My attempt at a unified treatment is of course based on the structural similarity suggested by recent cDNA sequencing and protein chemical data for neurotransmitter-gated chan nels; these similarities suggest that this group represents a superfamily (see e.g. 22, 145). Many incisive previous reviews have treated channels gated by neurotransmitters and by intracellular ligands (18,22,55,60,97, 125, 145, 154). Most ligand-dependent cation channels are less selective than voltage dependent cation channels. Ligand-gated anion channels are rather non selective as well-indeed,GABA and glycine channels show a measurable permeability for cations (122). As is also true for the voltage-gated channels, no studies report pharmacological or genetic manipulations that grossly affect the selectivity of ligand-gated channels, e.g. that change cation to anion channels. Such manipulations, when successfully per formed and interpreted, will represent a triumph of the site-directed muta genesis approach. Mutations that produce subtle changes in acetylcholine receptor selectivity have,however, recently been studied (29, 30,81,152). Transmembrane Topology and Primacy of the M2 Region
The adult muscle ACh receptor is a pseudosymmetric pentamer with subunit composition (/.2/3yt3. Other members of the neurotransmitter-gated receptor superfamily are probably pen tamers as well ( lOa). Each subunit has at least four putative membrane-spanning regions (Ml through M4). This chapter reviews the evidence that the M2 region (Figure 1) lines at least part of the conduction pathway. Certain contrary facts are also cited below and must be explained. An elegant recent review provides hypothetical diagrams of the M2 region at atomic resolution for the ACh channel and homologous regions for GABA and glycine channels (145). In the view Figure 1 C presents of the ACh receptor in its open confor mation, the five M21X-helices line the conducting pore like sheaves of wheat, slightly askew to create a point of closest appr oach . At the extracellular vestibule, some fixed charges affect ion flux. The open-channel blocker,
Annu. Rev. Biophys. Biomol. Struct. 1992.21:267-292. Downloaded from www.annualreviews.org Access provided by University of Illinois - Chicago on 10/04/15. For personal use only.
PERMEATION AT LIGAND-GATED CHANNELS
269
QX-222, penetrates from the external solution partially into the tapering channel, so that its nonpolar aromatic moiety and its polar charged moiety interact most strongly with residues at position 10' and 6', respectively. Permeant ions also interact with residues at position 6'. The taper of the channel prevents QX-222 from reaching the next turn,at p�sition 2'. Here and at the next turn-position -l'-thechannel is narrowest, and amino acid side chains interact most strongly with permeant ions. As ions con tinue to flow inward, they experience a broadening channel, but con ductance can still be affected by residues at position -4'. Classes of Molecular Manipulation
The rapid progress of recent years derives directly from four new classes of molecular manipulation: (a) DNA sequencing and site-directed mutag enesis, (b) protein sequencing, (c) patch-clamp measurements of channel conductance, and (d) patch-clamp measurements of single channels during local anesthetic molecule block. This chapter shows how these experiments are used to test the postulated structures and mechanisms, particularly for the ACh receptor. It also provides a comparative view of the comp lementary information from other neurotransmitter-gated ion channels. Practical Relevance
Biophysical and structural analysis of the permeation pathway may lead to knowledge that is useful in neurobiological or clinical contexts. Rec tification in the current-voltage relation of neurotransmitter receptors might influence encoding properties of neurons (92). The flux of Ca2+ through neuronal ACh channels might influence many processes ranging from transmitter release to gene activation (reviewed in 38). At N-methyl D-aspartate (NMDA) receptors, Ca2+ influx might underlie some forms of memory and learning (79) as well as ischemic damage (28). CLASSES OF MEASUREMENT
This section describes the conclusions that can be drawn from several classes of experiments on neurotransmitter-gated channels. The organ ization follows a recent chapter describing permeation through voltage gated channels (90). The reader should consult that chapter and the book by Hille (62) for the theoretical basis of the electrophysiological experiments. Reversal Potentials The measurement of reversal potentials is a null technique that gives a single value; it has wide application because it is sensitive to neither block nor saturation in the channel. For instance, macroscopic reversal potential measurements are possible under conditions in which single-channel cur-
270
A ACh ex1
LESTER
-4 '
-1 '
2'
D:: �E �KAGGQK eE K «7 DD D S G RV SAT3 GAAB s VP A T V o A A PAR V G GLY D Q
Annu. Rev. Biophys. Biomol. Struct. 1992.21:267-292. Downloaded from www.annualreviews.org Access provided by University of Illinois - Chicago on 10/04/15. For personal use only.
K
GluRl
COND
SL E QX Ncr
N
G
X X
r c
6'
L
g' 10'
�L::::: L AQ T V F L AQ V F LTVFMLLVA T LG Y VF V D V T T V T M TTL AR N T V M TT GA Q 'i
N
W
F
G
0
L
R
C
F
X
20'
10'
6'
2'
-1'
-4'
A D
Annu. Rev. Biophys. Biomol. Struct. 1992.21:267-292. Downloaded from www.annualreviews.org Access provided by University of Illinois - Chicago on 10/04/15. For personal use only.
PERMEATION AT LIGAND-GATED CHANNELS
27 1
rents are too small to be measured (29, 30). For ligand-gated channels in particular, permeabilities measured by reversal potentials decrease mono tonically with hydrated radius or other measures of molecular size (6, 42); therefore the narrowest region of the channel is usually identified with the highest energy barrier that an ion must traverse in crossing the membrane. Because several previous studies of ACh receptors suggested that single channel conductance was most strongly affected by mutations at the 2' or the -I' position (26, 7 1), these positions seemed likely candidates for the narrowest region of the channel. It also seemed most promising to examine effects of mutations on the relative permeability PTris/PNa, as Tris is a slightly permeant ion. Several mutations at the 2' position, but not at the 6', 10', nor 14' positions, did in fact render Tris relatively less permeant than in the wild-type receptor, without appreciable changes in the per meability to Na+ (29, 30). The total range of manipUlations in these experiments was roughly threefold, from PTris/PNa 0.36 to 0.1 1. Thus, the 2' position is apparently near the narrowest region of the channel. A surprising result was the ratio of 0.36 for wild-type mouse ACh channels, compared with the ratio of 0. 18 obtained for the frog muscle channels (6, 42). Yet Cohen et al (29) measured PTris/PNa for the wild-type Torpedo cali/arnica ACh channel in the oocyte system and obtained 0.22, close to the value for frog; thus among wild-type ACh channels, this parameter displays differences that cannot be explained by the M2 sequence alone. The amino acids at the -I' position were not systematically mutated by Cohen et al (29); however, the single mutation reported, bE-l'Q, decreased PTris/PNa nearly threefold. Interestingly, the mouse rxf3y receptor (obtained by omitting (j-subunit mRNA from the injection mixture), but not the rxf3(j =
Figure 1
The M2 region of neurotransmitter-gated channels.
M2 region of the mouse muscle a,
(A) Aligned sequences in the
/3,]1, and (j subunits, the chick neuronal a7 subunit [which
forms homooligomeric channels (33)], a SHT 3 receptor (10Sa), a subunits from the GABA
and glycine receptors, and the GluRI kainatelAMPA receptor (the latter sequence is the least similar in the group and the alignment is therefore tentative). Underlining shows a periodicity of 3.5, which would align the residues in a stripe along one face of the helix. Underlining agrees well with the location of boxed residues, or their homologs in the T.
californica
receptor, which have been localized to the conduction pathway by types of D. (D) Abbreviations are: COND, conductance; SEL, selectivity; QX, QX-222 blockade; NCI, noncompetitive inhibitor binding. (C) Schematic diagram of the experiments shown in
region surrounding the permeation pathway. The five rods each represent an a-helix that extends from position - 4' to 20'. The distance from the center of each helix to the center of the channel is arbitrarily set to 5 A at position l' (dUlled circle). Each helix is tilted 20" in a plane tangent to the circumference at this point, yielding a taper in both directions away from position I'. Assuming 3.6 residues/turn and 5.4 Altum, the diagram shows that position
20' is 26.8 A away axially from the narrowest region and has a diameter of 10.9 A ; position -4' is 7.5 A away and has a radius of 5.5 A . The vertical scale at right shows positions
discussed above (A) and in the text.
Annu. Rev. Biophys. Biomol. Struct. 1992.21:267-292. Downloaded from www.annualreviews.org Access provided by University of Illinois - Chicago on 10/04/15. For personal use only.
272
LESTER
receptor, was characterized by a very low PTris/PNa ratio (29, 30). Other work suggests that the omitted y or b subunit is replaced by the b or y subunit, respectively (P. Charnet, C. Labarca & H. A. Lester, submitted). Because the y subunit has a Gin residue at the - I' position, the apy receptor is like the bE-I'Q m� tation at the - I' position; the result is thus consistent with the large effect the -I' position has on selectivity. The mutations generated by Imoto et al (69) (described in detail below) were later subjected to reversal potential analyses that clearly showed the primacy of the - l ' position versus the -4' and 20' positions (81). The bE-l'Q mutation changed the PCs/PK ratio to 0.77 from the wild-type value of l . 1O; smaller changes also occurred in the PNa/PK ratio. On the other hand, at the -4' and the 20' positions, four charge changes were required to affect the PNa/PK ratio. CALCIUM PERMEABILITY Table 1 summarizes some studies that determine the Ca2 + permeability of neurotransmitter-gated channels, mostly on the basis of reversal potential measurements. A useful generalization is that there appear to be three classes of Ca2 + permeabilities: (a) undetectably low, (b) comparable to that for Na +, and (c) much higher than Na+ (the last class includes NMDA receptors only). Friel & Bean (50) have presented a lucid discussion of the possible artifacts in measuring Ca2 + permeability of channels gated by extracellular ATP, which would be expected to chelate Ca2 +. Although non-NMDA glutamate receptors are often considered impermeable to Ca2 +, the recent study by Hollmann et al (63) should be consulted for literature references to previous studies of non-NMDA receptors that reported Ca2 + -permeable non-NMDA responses in neurons.
Table 1
Measured Ca2+ permeabilities for some neurotransmitter-gated channels
Agonist
Tissue/source
PCa/PNa
GCa/GN, 0.4--0.6
ACh
Muscle/electroplaque
0.6- 1 .0
ACh
Muscle
0.2
ACh
Neuronal
0.93
ACh
A p/ysia neurons
Kainate/AMPA
GluR2+GluRI or
Kainate/AMPA
GluRI +GluR3 or GluR l
GluR2+GluR3
Reference 38, 85, 95 6
47
�0.5 0.\3
14
N.D.'
N.D."
63
� Ib
0.3-0.7
63
or GluR3 NMDA
Mouse neurons
7.S 10.6
12, 1 1 2
ATP
Smooth muscle
�3
20
5-HT
NG108-15 cells
40 j1M. First, the blocked state displayed a double exponential distribution of lifetimes, and second, the time integral of the openings decreased to less than the control value. Apparently, the channel can close on the bound QX-222 molecule, perhaps by the trapping effect described below. QX-222 BINDING SITE In the mid-1980s, site-directed mutagenesis and oocyte expression became the appropriate techniques for investigating the interaction between open-channel block ers, exemplified by QX-222, and the receptor channel. Researchers hoped that the specificity of site-directed mutagenesis would complement the molecular precision of the single-channel measurements on open-channel blockade. To identify promising residues for mutagenesis, the Caltech group noted the body of literature on noncompetitive inhibitors at ACh channels. When we began the site-directed mutagenesis experiments in 1987, position 6' was the appropriate first choice, as outlined in the next major section. Mutant subunits of the mouse muscle ACh receptor (a, /3, y,
