LI-YEN MAE HUANG, WILLIAM A. CATTERALL, and GERALD ... that can be measured electrophysiologically (Fischbach, 1972) or by isotopic flux methods ...
Selectivity of Cations and Nonelectrolytes for Acetylcholine-Activated Channels in Cultured Muscle Cells L I - Y E N MAE H U A N G , W I L L I A M A. C A T T E R A L L , and G E R A L D EHRENSTEIN From the Laboratory of Biophysics, National Institute of Neurological and Communicative Disorders and Stroke, and the Laboratory of Biochemical Genetics, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20014. Dr. Catterall's present address is Department of Pharmacology, School of Medicine, University of Washington, Seattle, Washington 98195.
A B S T R ACT The selectivity of acetylcholine (ACh)-activated channels for alkali cations, organic cations, and nonelectrolytes in cultured muscle cells has been studied. To test the effect of size, charge, and hydrogen-binding capacity of permeant molecules on their permeability, we have obtained the selectivity sequences of alkali cations, compared the permeability of pairs of permeant molecules with similar size and shape but differing in charge, and studied the permeability of amines of different hydrogen bonding capacity. ACh-activated channels transport alkali cations of small hydration radii and high mobility. The molecules with positive charge and (or) a hydrogen-bond donating moiety are more permeable than the ones without. On the other hand, several nonelectrolytes, i.e., ethylene glycol, formamide, and urea, do have a small, but measurable, permeability through the channels. These results are consistent with a model that ACh-activated channel is a water-filled pore containing dipoles or hydrogen bond accepting groups and a negative charged site with a pK of 4.8. INTRODUCTION
T h e binding o f acetylcholine (ACh) to its r e c e p t o r in the muscle endplate induces an increase in the permeability o f the m e m b r a n e to small ions (Fatt a n d Katz, 1951). From tracer-flux studies in d e n e r v a t e d rat d i a p h r a g m muscle, J e n k i n s o n and Nicholls (1961) observed that A C h increases both sodium and potassium permeability. Studying the reversal potential at frog n e u r o m u s c u l a r j u n c t i o n by voltage clamp technique, T a k e u c h i and T a k e u c h i (1960) and T a k e u c h i (1963) f o u n d that Erev varies significantly with changes o f external concentration o f sodium a n d potassium ions, whereas chloride ions have no effect on Erev. Recent evidence obtained f r o m the studies o f equilibrium potential (Kordas, 1969), the observation o f monotonic decay o f endplate c u r r e n t (Magleby and Stevens, 1972), and the m e a s u r e m e n t o f e n d plate c u r r e n t variance from noise analysis (Dionne and Ruff, 1977) strongly suggests that both T H E J O U R N A L OF G E N E R A L P H Y S I O L O G Y " V O L U M E
71, 1978
- pages
397-410
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THE JOURNAL OF GENERAL P H Y S I O L O G Y " VOLUME 7 1 '
1978
sodium and potassium ions use one channel for their permeation. Besides sodium and potassium ions, various organic cations are also permeable to the ACh-activated channel (Furukawa and Furukawa, 1959; Koketsu and Nishi, 1959; Maeno et al., 1977; Guy et al., 1977). ACh-activated channels are much less selective than the Na + channels responsible for the action potential in nerve and muscle. Large organic cations such as biguanide and bis (2-hydroxye t h y l ) d i m e t h y l a m m o n i u m can pass t h r o u g h the ACh-activated channels. T h e single ACh-activated channel conductance derived f r o m fluctuation analysis is approximately 25 pS (Anderson and Stevens, 1973; C o l q u h o u n et al., 1975; N e h e r and Sakmann, 1976). Muscle cells cultured in vitro form multinucleate myotubes which have a high density o f acetylcholine receptors distributed over the entire cell surface (Fischbach, 1972; Vogel et al., 1972). T h e s e cells r e s p o n d to acetylcholine or o t h e r cholinergic agonists with a large increase in permeability to small cations that can be m e a s u r e d electrophysiologically (Fischbach, 1972) or by isotopic flux methods (Catterall, 1975). Like Ach-activated channels at the endplate, Achactivated channels in cultured muscle cells are permeable to both Na + and K + (Ritchie and F a m b r o u g h , 1975). T h e i r single-channel conductance is 31-39 pS at 25~ (Sachs and Lecar, 1977; Lass and Fischbach, 1976). T o u n d e r s t a n d the factors i m p o r t a n t for the postsynaptic m e m b r a n e selectivity, we have studied the effect o f charge o f the t r a n s p o r t e d molecules on the rate o f their permeation. T h e specific questions considered here are whether the charge on the molecules plays an essential role in their permeability and w h e t h e r the h y d r o g e n binding ability o f test molecules affects their permeation. We have a p p r o a c h e d these questions by studying different series o f c o m p o u n d s which differ in size, charge, or chemical properties. T h e selectivity ratios for both cations and nonelectrolytes were d e t e r m i n e d by isotopic flux methods in cultured muscle cells, using a modification o f the methods described previously (Catterall, 1975). MATERIALS
AND
METHODS
Materials The chemicals and media used were purchased from the following sources: DulbeccoVogt modification of Eagle's minimal essential medium (DMEM) and Dulbecco's phosphate-buffered saline from the Media Unit of National Institutes of Health, horse serum from Microbiological Associates, Walkersville, Md.; fetal calf serum from Grand Island Biological Co., Grand Island, N. Y.; recrystallized trypsin from Worthington Biochemical Corp., Freehold, N. J.; carbamylcholine, D-arabinofuranosylcytosine, ouabain, and dtubocurarine from Sigma Chemical Co., Saint Louis, Mo.; calf tail collagen from Calbiochem, San Diego, Calf.; 2ZNaC1, [1,2-14C]ethanolamine, [1,2-14C]ethylene glycol, [1,3-14C]glycerol, [1-14C]-mannitol, [14C]urea, [14C]methylamine, and [1-14C]ethylamine from New England Nuclear, Boston, Mass.; labelled [14C]Tris and [U-~4C]ethylenediamine from Amersham/Searle Corp., Arlington Heights, Ill.; and [~4C]guandine, [14C]formamide, and [l-14C]acetamide from ICN Corp., Irvine, CA. Chick embryo extract was prepared as described by Cahn et al. (1967). ~-Bungarotoxin was purified from the venom ofBungarus multicinctus (Miami Serpentarium, Miami, Fla.) according to the method of Mebs et al. (1971).
HtrANG E'r AL. Cation and Nonelectrolyte Selectivityfor ACh-Activated Channel~
399
Muscle Cultures Suspension of single muscle cells from thigh muscle of 10 or ll-day-old chick embryos was prepared as described by Fischbach (1972). Cells were then seeded at a density of 75,000 cells/cme in collagen coated multi-well plates (16-mm diameter, Costar Co. Mass.). The growth medium contained 91% DMEM, 2% fetal calf serum, 5% horse serum, 2% chick embryo extract, 50 U/ml penicillin, and 10 /zg/ml streptomycin. The cells were grown in a humidified atmosphere of 10% CO2-90% air, and the growth medium was changed every 2-3 days. 10/zM D-arabinofuranosylcytosine was added in the medium on day 3 or day 4 to reduce the n u m b e r of fibroblasts in the culture (Fischbach, 1972).[3H]leucine (0.2 /zCi/ml) was added with growth medium 24 h before the experiment to allow the protein recovery to be measured from the 3H counts per minute.
Measurement of Influx Rates Cell cultures were removed from the incubator and the growth medium was replaced with a medium consisting of 135 mM NaC1, 5.4 mM KCI, 1.8 mM CaC12, 0.8 mM MgSO4, 5.5 mM glucose, and 50 mM HEPES (adjusted to pH 7.4 with Tris base). Cells were equilibrated with this buffer medium at 36~ (15-30 min), and assays for influx were then carried out. In some experiments the acetylcholine receptors of the muscle cells were inhibited by incubation with 10 nM ot-bungarotoxin in the same medium for 60 min at 37~ before assay. This treatment is sufficient to inhibit all ACh receptors (Catterall, 1975). Influx rates were measured at 2~ because desensitization of the receptor is slower at low temperature (Catterall, 1975). Cell cultures were transferred to an ice bath. Isotopic influx measurements were initiated by addition of assay medium consisting of 142.5 mM KCI, 1.8 mM CaC12, 0.8 mM MgSO4, 5.5 mM glucose, and a buffer mixture of 10 mM Nhydroxyethylpiperazine-N'-ethane sulfonic acid (HEPES, pKa = 7.5), 10 mM cyclohexyla m i n o p r o p a n e sulfonic acid (CAPS, pKa = 10.4), 10 mM 2(N-Morpholino) ethane sulfonic acid (MES, pKa = 6.2), 10 mM Tris (hydroxymethyl)methylaminopropane sulfonic acid (TAPS, pKa = 8.4), and 10 mM glycylglycine (pKa~ = 3.1, pKa, = 8.1). 5 mM ouabain was added to inhibit active extrusion of Na + (Catterall, 1975). The assay media contained test permeant molecules at concentrations from 2 to 1 mM (5 t~Ci/ml) as indicated in the figure legends. Carbamylcholine, at a final concentration of 10 mM, was included in the medium to activate ACh receptors in the muscle cells. T h e mixture of buffers used was chosen to provide adequate buffer capacity over the range from pH 4 to pH 11. T h e desired pH was obtained by titration with KOH. Uptake measurements were terminated after 15-s incubation at 2~ by aspirating the assay medium and washing the cells four times at 2~ with 3 ml of wash medium consisting of 142.5 mM KC1, 10 mM choline CI, 1.8 mM CaCle, 0.8 mM MgSO4, 5 mM HEPES (adjusted to pH 7.4 with Tris base), and 1 mM d-tubocurarine to inhibit the activation of ACh-activated channels. Cells were suspended in 0.6 ml of0.4N NaOH or 1% sodium lauryl sulphate and transferred to scintillation vials containing 1 ml 1 M Tris HC1, pH 7.4, and 10 ml scintillation fluid consisting of 5.5% (vol/vol) RPI scintillation cocktail, 61.4% toluene, and 33.1% Triton X100. Protein concentration was determined by the method of Lowry et al. (1951) on representative cultures and the protein content of the remaining samples was calculated from the recovered [3H]leucine counts per minute. The results are expressed as nanomoles of permeant c o m p o u n d influx per minute per milligram cell protein. In each experiment, the rate of influx of each permeant c o m p o u n d in cells not treated with carbamylcholine or in cells treated with c~-bungarotoxin plus carbamylcholine was determined and subtracted from the rate of influx in cells treated with carbamylcholine alone. This difference represents the increase in permeability caused by activation of the acetycholine receptors (Catterall, 1975).
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RESULTS
Selectivity for Alkali Metal Cations Previous results showed (Catterall, 1975) that carbamylcholine and o t h e r cholinergic agonists cause a 20-fold increase in initial rates o f 22Na+ uptake into cultured muscle cells which is blocked specifically by nicotinic acetylcholine r e c e p t o r antagonists. T h o s e results d e m o n s t r a t e d the feasibility o f using ion flux p r o c e d u r e s to measure changes in permeability o f cultured muscle cells due to activation o f nicotinic acetylcholine receptors. T h e flux d e p e n d s on external ion concentration, m e m b r a n e potential, and permeability. In these e x p e r i m e n t s , we have modified the conditions by using a m e d i u m with potassium concentration (142.5 mM K § identical to the intracellular K + concentration in these cells (Catterall, 1975; Ritchie and F a m b r o u g h , 1976). U n d e r these conditions, the m e m b r a n e potential is zero and the large increase in Na + and K + permeability caused by cholinergic agonists has no effect on m e m b r a n e potential. T h u s , according to the G o l d m a n - H o d g k i n - K a t z equation (Goldman, 1943; H o d g k i n and Katz, 1949), the m e a s u r e d flux (J~) is simply p r o p o r t i o n a l to the concentration o f the p e r m e a n t cation and the permeability (P~). Permeabilities were d e t e r m i n e d by measuring the initial rate o f influx o f p e r m e a n t ions or nonelectrolytes at six d i f f e r e n t concentrations and calculating P~ f r o m the slope o f the plot o f influx vs. concentration. Z2Na+ influx was m e a s u r e d in each e x p e r i m e n t and the data are presented as the ratio (R) o f the permeability o f the test cation or nonelectrolyte to that of Na +. In all cases the rate o f influx o f the test cations and nonelectrolytes in the absence o f carbamylcholine has been subtracted from the data. T h e rate o f densensitization o f ACh receptors depends on carbamylcholine concentration as well as cations in the assay m e d i u m (Nastuk and Parsons, 1970; Manthey, 1972). T o test w h e t h e r the estimated permeability is affected by the densensitization o f the ACh receptor, we preincubated the cells in m e d i u m containing various test molecules and 10 mM or 1 mM carbamylcholine for various times, and then m e a s u r e d the rate o f 2ZNa+ uptake. T h e 22Na+ uptake r e m a i n e d constant for at least 25 s o f preincubation time. Desensitization does not obscure the permeability m e a s u r e m e n t with the e x p e r i m e n t a l p r o c e d u r e s employed. An e x p e r i m e n t illustrating results for the alkali metal cations Na +, K +, Rb +, and Cs + is p r e s e n t e d in Fig. 1. In each case the d e p e n d e n c e o f flux on concentration is linear verifying that the m e m b r a n e potential remains constant and that flux coupling can be ignored. T o study the permeability o f K + at low ionic concentration, and relatively constant m e m b r a n e potential, the uptake o f alkali cation in some e x p e r i m e n t s is m e a s u r e d in m e d i u m containing 285 mM sucrose instead o f KCI (Fig. 1). Replacement o f KC1 by sucrose changes the m e m b r a n e potential o f cells d u r i n g the assay. Inasmuch as the m e m b r a n e potential o f cells is no longer maintained at 0 mV in this case, uptake o f test molecule vs. its concentration d e p e n d s not only on permeability but also on the m e m b r a n e potential. W h e n the concentration o f the test ions is below 12 raM, the change in m e m b r a n e potential caused by replacing KCI by sucrose is about the same for all test ions. T h e r e f o r e , even t h o u g h the individual permeabilities
401
HUANG ET AL. Cation and Nonelectrolyte Selectivityfor ACh-Activated Channels
m i g h t be affected by the c h a n g e in m e m b r a n e potential, the permeability ratio R = PJPsa should not be affected. We have tested this a s s u m p t i o n by r e p e a t i n g the same kind o f e x p e r i m e n t s in m e d i a containing either high K § or high Na § T h e results for R o b t a i n e d u n d e r these various e x p e r i m e n t a l conditions agree within e x p e r i m e n t a l e r r o r . T h e m e a n e x p e r i m e n t a l R --- SEM o f these ions are given in T a b l e I. T h e selectivity sequence we observe in 285 m M sucrose is Cs > Rb > K > Na (1.91: 1.52: 1.47:1.00). T h e s a m e selectivity sequence for the alkali ions is o b t a i n e d in m e d i u m containing 142.5 m M KC1. T h e experim e n t s illustrated below, unless specified, were all d o n e in high K § m e d i u m .
p
CESIUM
o 300--
"~ E
9
9
Cs +
RUBIDIUM POTASSIUM SODIUM
J / / ~
z24O
/
/ i
/
/
S.
U-
Rb+
.dlK+
/-"
/ -
/
-'+
91
- ~
i oL~W 'v
I
2
i
4
I
I
I
I
6 8 10 12 CONCENTRATION (raM)
L
I
FIGURE 1. Effect of concentration on alkali ion uptake. Two identical sets of muscle cells were incubated for 60 rain at 36~ in preincubation medium containing either 0 or 10 nM a-bungarotoxin. The cells were transferred to an ice bath and rinsed to remove excess Na +. The uptake of Na +, K +, Rb +, and Cs + in this specific experiment was measured for 15 s at 2~ in assay medium containing 285 M sucrose, instead of KC1, and various indicated concentrations of alkali cations. The uptake of alkali ion in the presence of a-bungarotoxin was then subtracted from uptake data obtained from cultures without toxin treatment.
The Effect of pH As p a r t o f o u r study o f the effect o f c h a r g e on permeability, we p l a n n e d to study c h a r g e d a n d u n c h a r g e d species o f the same p e r m e a n t molecules by altering p H . In p r e p a r a t i o n for those e x p e r i m e n t s , we studied the effect o f p H on the acetylcholine r e c e p t o r a n d the A C h c h a n n e l using Na + as the p e r m e a n t ion. We find that the Na § permeability increase d u e to c a r b a m y l c h o l i n e is m a r k e d l y r e d u c e d as the p H is r e d u c e d below 5.5, b u t is little affected by increasing p H above 7 (Fig. 2). At p H 3.0, the permeability increase is completely blocked. T h e data are fit well by a theoretical dissociation c u r v e with a pK o f 4.8 (Fig. 2). T h e inhibition is reversible at all p H ' s studied. T h e rate o f Na + influx in the absence o f agonist or in the presence o f b o t h agonist a n d otb u n g a r o t o x i n is u n a f f e c t e d by p H . T h e s e results indicate that e x t e r n a l p r o t o n s
402
THE JOURNAL OF GENERAL PHYSIOLOGY ' VOLUME 71 " 1978 TABLE
PERMEABILITY
RATIO
OF
ALKALI
I
IONS
AND
ORGANIC
MOLECULES
R = permeability o f test molecules permeability o f Na + T r a n s p o r t i n g molecules
Molecular f o r m u l a
Mkali ion Sodium Potassium Rubidium Cecum Calcium
Molecular d i m e n sion*
Na +
r ffi 0.90
K+
r = 1.33
Rb*
r ffi 1.48 r ffi 1,69 r = 0.99
Cs + Ca *+
) n e - or two-carbon c o m p o u n d Ethanolamine Ethylenediamine
Ethylamine M ethyl a mi ne Ethylene glycol
Uncharged form
C h a r g e d form
(n)
(n) t.0 1.47 1.52 1.91 0.22
N H,--(CH=)f--OH N H2-"(CH,),--NH ,
3 , 8 4 x 4.11 x 7.93
NH2----(CH2)2--H OH----(CH2---OH
3 5 4 x 4.11 x 5.98 3.79 x 3.87 x 4,32 3.84 x 4.11 x 6.50
N Hs +
3.00 x 5.12 x 5.49
N HI----C H s
pK,
3.84 x 4.11 x 6.82
• -+ • •
0.26 0.41 0.65 0.08
(5)$ (6) (7) (7)
9.5 9.98, 7.52
~0 s0
0.72 - 0.17 (9) 0.63 • 0.20 (15) 0.57 • 0.14{ (5)
10.65 10.62 14.77
~0
~0
0.45 • 0.16 (7) 0.82 • 0.30 (61
0.19 •
0.05
(9)*
3arbonyl and related c o m p o u n d 0.92 • 0,21 (S)
/ NHfC
Guanidinium
\ NH2 N~
3.00 x 4.38
5.35
0,17 • 0.05 (4)
3.00 x 4.97 x 5.34
0.12 -+ 0.05 (4)
3.76 x 5.12 x 5.30
~0
3.77 x 5.77 x 5.90
0.04 • 0 0 1 6 (3)
x
/ Formamide
0= \ H NHI / 0=C
Urea
\ N H= NH~ / 0=C
Acetamide
\ GHs NH~ / Thiourea
S=C \ NH2
)ther compounds T r i s (hydroxy-methyl) methane Glycerol
amino
(C H f - - O H ) ~ - - - C - - N H3 + 6.03
x
6.89
C H ~ - - G H ---CH 2
x
5.99 • 6.90
I
I
I
OH
OH
OH
4.69
Maon, o,
,,.xSI,
/ [ I] OH \ o n / ,
•
7.71
8,1
~0
0.II • 0.04(4)
0.05 +- 0.02 (3)
xl,.
! o.
* r - radius of the crystal size o f alkali ion. Molecular d i m e n s i o n s o f organic molecules were estimated f r o m CPK model.
N u m b e r in p a r e n t h e s e s r e p r e s e n t s the n u m b e r of e x p e r i m e n t s d o n e for the specific molecules. w Doubly-charged form.
HUANG IrT AL. Cation and Nonelectrolyte Selectivityfor ACh-Activated Channels
403
specifically inhibit the t r a n s p o r t o f Na + t h r o u g h ACh-activated channels in tissue c u l t u r e d muscle cells. T h e r e are at least two i n t e r p r e t a t i o n s o f this observation. (a) T h e r e may be c h a r g e d group(s) n e a r or in the channels. T h e ACh-activated channels are blocked by the p r o t o n a t i o n o f the c h a r g e d group(s). (b) C h a r g e d group(s) m a y exist on the acetylcholine receptors. T h e p r o t o n a t i o n o f these g r o u p s p r e v e n t s o p e n i n g the ACh-activated channels, p e r h a p s by inactivation o f the r e c e p t o r . T o test the possibility that p r o t o n a t i o n prevents carbamylcholine binding, we have studied the titration curves o f the activation by c a r b a m y l c h o l i n e at p H 4.75 a n d 7.4 (Fig. 3). T h e a p p a r e n t Ko changes very little with p H , w h e r e a s the
2 i
1.0
~
T
--
z
< ~0.8 e~ 0.6
i
0.4
D 0.2
FIGURE 2.
0'
'I' 3
4
5
6
7
1
8
I
9
I
10
I
11
J
pH
Effect o f p H on 22Na+ uptake. Muscle cells were first i n c u b a t e d in
preincubation medium at 36~ for 15 min and then transferred to an ice bath. The preincubation medium subsequently was removed and replaced by a medium containing 142.5 mM KCI, 1.8 mM CaCI2, 0.8 mM MgSO4, 5.5 mM glucose and good buffers (described in Materials and Methods). Cells were equilibrated in this medium which was buffered at the indicated pH, for 2-3 min. The initial rate of Na + uptake is measured at the same pH in the assay medium for 15 s. Each point represents the average of 7-12 separate measurements. The vertical bars indicate the experimental variations. Solid line is the least-squares fit to the theoretical dissociation equation with a pK at 4.8. m a x i m u m influx rate is lower at p H 4.75. T h u s , the inhibition by h y d r o g e n ions is n o n c o m p e t i t i v e with respect to carbamylcholine binding, indicating h y d r o g e n ions bind at a site separate f r o m the carbamylcholine binding site. T h e p r o t o n a t i o n site could be located elsewhere on the r e c e p t o r or within the channel itself.
Selectivity for Organic Cations and Nonelectrolytes Organic molecules, particularly a m i n e s , replacing the s o d i u m in assay m e d i u m may have a p h a r m a c o l o g i c a l effect on A C h channels. T o test this, =ZNa+ u p t a k e was m e a s u r e d in assay m e d i u m containing different concentrations o f the organic molecules studied. We f o u n d that a low concentration o f organic molecules ( e t h y l e n e d i a m i n e > ethylamine. At p H 11.5, w h e r e m o r e than 90% o f each o f the above c o m p o u n d s is 60 _
o 9
pH = 4.75 pH = 7.50
50
9
pH = 10.00
9 9 /
./"
J vm
9
30-
9
0
,
NH2 > H. I n a s m u c h as this difference in the h y d r o g e n b o n d i n g ability o f these three molecules may alter the interaction with ACh-activated channels d u r i n g their transport, it is likely that the permeability to ethanolamine and ethylenediamine is e n h a n c e d because they are better h y d r o g e n bond d o n o r s than ethylamine. F u r t h e r evidence for this is provided by some o f the u n c h a r g e d molecules tested. Ethylene glycol, f o r m a m i d e , and urea are measurably permeant and have some h y d r o g e n b o n d i n g capacity. Ethylene glycol, in having two - - O H groups, is much more p e r m e a n t than u n c h a r g e d ethanolamine, ethylenediamine, and ethylamine (Table I). In all these cases, there is a positive correlation between permeability and the h y d r o g e n - b o n d i n g ability o f the molecules. F o r m a m i d e and urea have m o d e r a t e dipole m o m e n t due to electronegative character o f the oxygen atom in carbonyl groups. T h u s , in addition to h y d r o g e n - b o n d i n g , the dipole m o m e n t o f these two c o m p o u n d s can result in favorable interactions with the channel structure, increasing their permeability. Steric restriction and mobility also play i m p o r t a n t roles in d e t e r m i n i n g the permeability o f molecules t h r o u g h ACh-activated channels. We observed a selectivity sequence for alkali cations to be Cs + > Rb + > K + > Na + (Fig. I). In o u r system, K + is more p e r m e a n t than Na +, which is opposite to Takeuchi's (1963) results, but is in a g r e e m e n t with the findings o f Lassignal and Martin (1977). 1 Na + has the smallest crystal radius and the lowest mobility o f o u r series; Cs +, the largest ion, has the highest mobility. T h u s , ACh-activated channels are selective for the cations o f highest mobility in free solution (Fig. 6). F r o m the selectivity studies o f ACh-activated channels for large organic cations in postsynaptic m e m b r a n e s , Maeno et al. (1977) estimated the m i n i m u m internal d i a m e t e r o f ACh-activated channels to be 6.4A. This channel d i a m e t e r is large e n o u g h to allow cations to p e r m e a t e in their h y d r a t e d forms. O u r observation that the selectivity sequences are in the o r d e r o f the mobilities o f the h y d r a t e d alkali cations is consistent with this estimate o f channel diameter. O u r analysis suggests that the ACh-activated channel is a relatively large water-filled pore containing hydrogen-accepting groups and at least one negative-charged site. This charged site may be a carboxyl g r o u p with a pK o f 4.8 inasmuch as protonation o f a g r o u p with pK = 4.8 inhibits Na + t r a n s p o r t without changing the affinity o f carbamylcholine for its receptor. H y d r o g e n bond-accepting groups are likely present in the channel because h y d r o g e n b o n d - d o n a t i n g moieties e n h a n c e the permeability o f t r a n s p o r t e d molecules. T h e h y d r o g e n b o n d acceptors may interact with the water o f hydration o f cations or f o r m h y d r o g e n bonds with the cations, themselves, or with nonelectrolytes d u r i n g their transport. A m o n g the ion channels studied in biological m e m b r a n e s , the most detailed i n f o r m a t i o n on selectivity is available for Na + channels o f nerve axons (Hille, t Lewis, C., and C. F. Stevens. 1977. Personal communication.
408
THE
JOURNAL
or
GENERAL
PHYSIOLOGY
9 VOLUME
71
9 1978
1972). B o t h A C h - a c t i v a t e d a n d N a c h a n n e l s a r e c a t i o n - s e l e c t i v e . B o t h c h a n n e l s a r e also s o m e w h a t p e r m e a b l e to n o n e l e c t r o l y t e s , b u t m u c h less so t h a n to c o m p a r a b l e m o l e c u l e s w i t h p o s i t i v e c h a r g e . S e v e r a l e x a m p l e s o f this f o r A C h a c t i v a t e d c h a n n e l s a r e s h o w n in T a b l e I. W e h a v e also e x a m i n e d t h e selectivity o f n o n e l e c t r o l y t e s in b a t r a c h o t o x i n - a c t i v a t e d N a + c h a n n e l s o f n e u r o b l a s t o m a cells, a n d f o u n d t h a t f o r m a m i d e a n d u r e a h a v e s m a l l b u t m e a s u r a b l e p e r m e a bility. H o w e v e r , t h e s e u n c h a r g e d m o l e c u l e s a r e m u c h less p e r m e a n t t h a n c h a r g e d g u a n i d i n i u m ( H u a n g et al., u n p u b l i s h e d r e s u l t ) . T h e m o s t s t r i k i n g differences between ACh-activated channels and Na + channels are that the l a t t e r c h a n n e l s a r e m u c h m o r e selective, a n d h a v e a selectivity s e q u e n c e f o r a l k a l i c a t i o n s o f N a + > K + > R b + > Cs + ( C h a n d l e r a n d M e v e s , 1965; H i l l e , 1972; 2.5
8
m
/
2.0-
,5/i
F
9 0.5
1.0
1.5
2.0
(mobility of alkali ion) (mobility of Na +)
FIGURE 6. Permeability ratio (R) for alkali ions as a function of their relative mobility (relative to Na +) in aqueous solution. T h e R values are taken from Table I; the relative mobility values are from Robinson and Stokes (1959). T h e vertical bars represent the experimental variations. The solid line has a slope o f 1 representing the perfect correlation between R and relative mobility. C a h a l a n a n d B e g e n i s i c h , 1976; E b e r t a n d G o l d m a n , 1976). T h e s e d i f f e r e n c e s a r e c o n s i s t e n t with t h e r e l a t i v e l y s m a l l e r (3 x 5,~) cross s e c t i o n e s t i m a t e d f o r N a + c h a n n e l s ( H i l l e , 1972). We would like to thank Dr. Harold Lecar for his interest, stimulating discussions, and comments throughout this investigation, and Drs. Charles Stevens, Robert Taylor, and Thomas Smith for their comments on the manuscript. Dr. Huang is a recipient of National Research Service Award F32 HL 05049-1 from National Heart, Lung, and Blood Institute, National Institutes of Health. Received for publication 17 November 1977. REFERENCES ANDERSON, C. R., and C. F. STEVENS. 1973. Voltage clamp analysis of acetyicholine p r o d u c e d end-plate current fluctuation at frog neuromuscular junction. J. Physiol. (Lond. ). 235:655-691.
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