Na+, K+, H+ and Cl− permeability properties of rabbit skeletal muscle sarcolemmal vesicles

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 223, No. 1, May, pp. 9-23, 1983

Na+, K+, H+ and Cl- Permeability Properties of Rabbit Skeletal Muscle Sarcolemmal Vesicles JOHN R. GILBERT

AND

GERHARD

MEISSNERl

Departments of Biochemistry and Nutrition, and Physiology, and the Neurobiology Program, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North, Carolin,o 27.516 Received August 2, 1982, and in revised form January

14, 1983

The ion permeability properties of rabbit skeletal muscle sarcolemmal vesicles were investigated by means of radioisotope flux, membrane potential, and light-scattering measurements. An enriched sarcolemmal fraction was obtained from the 22-27% region of sucrose gradients after isopycnic centrifugation. The presence of contaminating sarcoplasmic reticulum was assessed with the use of a purified sarcoplasmic reticulum vesicle fraction. 22Na ’ , “Rb ’ , 36C1 , and [3H]sucrose flux measurements indicated that the sarcolemmal fraction possessed isotope spaces ranging between 1.5 and 4 pl/mg protein. Membrane potential measurements using the voltage-sensitive fluorescent probe 3,3’-dipentyl-2,2’-oxadicarbocyanine iodide (diO-C,-(3)) indicated that sarcolemmal vesicles were impermeable to Ht and Nat but that lo-15% of the vesicles were permeable to K’. Light-scattering measurements indicated a small fraction of sarcolemmal vesicles were permeable to both Kt and Cl-. Whether the low permeability of sarcolemmal vesicles to Na+, K’, and Cl- is the result of a low concentration of ion channels or the inactivation of these channels during isolation is at present uncertain.

The intracellular ion concentrations of muscle cells are regulated through the action of sarcoplasmic reticulum and two sarcolemmal” components, the surface membrane and transverse-tubule. The sarcoplasmic reticulum is a large, specialized intracellular memhranous system which regulates the contraction-relaxation cycle of skeletal muscle by releasing and reabsorbing Ca2+. The release of Ca2+ from sarcoplasmic reticulum is preceded by an action potential of the sarcolemma which is thought to be communicated to the sarcoplasmic reticulum via the trans-

verse-tubular system (l-3), an invagination of the muscle sarcolemma into the muscle cell in the area of the A-I band region in rabbits. A knowledge of ion conductances and their distribution between the sarcolemma1 components should help in understanding the electrical properties of the sarcolemma, including those of the action potential. Electrophysiological studies using frog semitendinosis and sartorious fihers indicate that, in general, the surface membrane and transverse-tubule are impermeable to Na+, except during action potentials; that both the transverse-tubule and surface membrane are relatively permeable to Kt; and that the surface membrane is permeable to Cl- while the transverse-tubule is either Cl impermeable, or possesses very low Cl- permeability (4, 5). Recent experiments, however, indicate that this simple picture is not true in mammals. The transverse-tubule of rat dia-

’ To whom all correspondence should be addressed: Department of Biochemistry 231 H, School of Medicine, University of North Carolina, Chapel Hill, North Carolina 27514. ‘The sarcolemma is defined as consisting of two components: the surface membrane proper and the invaginations of the surface membrane into the muscle cell known as the transverse-tubular system (Tsystem). 9

0003-9861183 $3.00 CopyrIght All nyhts

G 1983 by Academic Press. Inc. of reproduction in any form reserved.

10

GILBERT

AND

phragm (6) and red sternomastoid fibers are Cl- permeable (4). Studies have also shown that amphibian fibers possess active Na+-Na+, K+-K+, Cl-Cl, and Na+Ca2+ ion exchange (5, 7); while mouse soleus muscle possesses H+-Na+ and Cl-HCO; exchange activities (8). Sarcolemmal vesicles have recently been used to study Na+-Ca2+ exchange in cardiac (9-13) and skeletal muscle (14). Barchi and Weigele (15) have used radioactive saxitoxin binding to vesicles derived from rat skeletal muscle to study Naf channel density in sarcolemma. In this paper we use a combination of radioisotope tracer, membrane potential, and light-scattering measurements to study the Na+, K+, H+, and Cl- permeability of a surface membrane vesicle population derived from rabbit skeletal muscle. A preliminary account of part of this work has been presented (16).

MEISSNER

Reagents. The fluorescent dye 3,3’-dipentyl-2,2’-oxadicarbocyanine was the generous gift of Dr. Alan S. Waggoner (Amherst College, Amherst, Mass.). a6Clm, ZtNa’, s’Rb+, 35SO:-, L-[l-3Hlglucose, and Ifructose-l‘HIsucrose were purchased from New England Nuclear, Boston, Massachusetts. D-[U-‘?]Glucose was obtained from ICN Pharmaceuticals, Irvine, California. Other reagents were of reagent or analytical grade. Preparation of vesicles. Rabbit skeletal sarcolemma1 and sarcoplasmic reticulum vesicles were prepared by zonal gradient centrifugation as previously described (14). Briefly, the white muscle of the back and legs of a rabbit were homogenized in aliquots of 40 g in 320 ml of 0.3 M sucrose with two 30-s bursts in a Waring Blendor set at high speed. The homogenate was centrifuged for 12 min at SOOOg.The supernatants (-1500 ml) were centrifuged for 3 h at 29,000 rev/min in a Beckman Ti 15 zonal rotor, and a crude muscle “microsomal fraction” was collected at a discontinuous 20/50% sucrose interface, created by pumping in prior to centrifugation, 100 ml of 20% sucrose followed by 150 ml of 50% sucrose. The crude microsomal fraction was diluted with HzO, sedimented, and resuspended in approximately 25 ml of 0.3 M sucrose, 2 mM Hepes3 (N-2-hydroxyethylpiper-

azine-N’2-ethanesulfonic acid), pH 7.2, and stored at -70°C. In experiments utilizing [aH]ouabain labeling of transverse-tubule, the microsomal fraction was prepared as above from back muscle after backstrips were injected with a [3H]ouabain solution as described by Lau and Caswell (17, 18). Unless otherwise indicated, the crude microsomal fractions from four rabbits were combined after rapid thawing at 32”C, and diluted with KC1 to a final concentration of 0.15 M sucrose, 0.45 M KCI, 5 ITIM Hepes, pH 7.1. The vesicle suspension was kept at 2°C for 4 h, then subjected to sucrose isopycnic zonal centrifugation at 29,000 rev/min in a Beckman Ti 15 for 18 h on a lOOO-ml continuous 20-50% sucrose gradient containing 50 mM KC1 and 10 mM Hepes, pH 7.1. Fractions were collected, diluted with HzO, and centrifuged at 33,000 rev/min (90,OOOg) for 75 min in a Beckman 35 rotor. The pellets were taken up in 0.3 M sucrose, 4 mM Hepes, pH 7.1, and stored at -70°C until use. Control experiments with unfrozen fractions indicated that initial freezing and thawing during vesicle preparation did not appreciably affect radioisotope flux rates. Final gradient fractions were, however, frozen in small aliquots since a decline in the flux rates was seen with repeated thawing and freezing of the purified vesicles. Biochemical assays. Protein was determined by the procedure of Lowry et al. (19) using bovine serum albumin as a standard. Me- or Ca’+-activated (“basic”) ATPase (20) was determined at 32°C in 2 ml of a medium containing 20 mM Hepes, 0.1 M KCI, 2.5 IIIM ATP, 6 mM Mgz+ plus 1 mM EGTA (ethyleneglycol bis-[P-aminoethyl ether]-N,N’-tetraacetic acid), pH 7.2. The reaction was started by the addition of ATP and stopped after 5 and 10 min with 0.7 ml of 1.5 M HCIOl. Inorganic phosphate was determined on 1 ml of the protein-free supernatant (21) using Elon as a reducing agent. The enzyme concentration used resulted in less than 10% hydrolysis of ATP. The concentration of the Ca’+-dependent, 32P-labeled phosphoenzyme intermediate of sareoplasmic reticulum was measured as previously described (22). Isotopejux measurements. Apparent isotope spaces and efflux rates to [3H]sucrose, %a’, %Rb+, %Cl-, and ?jOi- were determined by Millipore filtration as previously described (23). Isotope flux measurements were carried out at 21°C. In ion-exchange studies, vesicles were incubated in the given medium and then centrifuged for 30 min at 20,000 rev/min (33,OOOg) in a Beckman 42.1 rotor. The pellet was taken up in a minimum amount of the original incubation mixture at a concentration of approximately 5 mg protein/ ml and incubated for a minimum of 4 additional hours

3 Abbreviations used: Hepes, N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid; EGTA, ethylene glycol bis(&aminoethyl ether) N,N’-tetraacetic acid; Pipes, 1,4-piperazinediethanesulfonicacid; diO-C,-(3),

3,3’-dipentyl-2,2-oxadicarbocyanine iodide; DIDS, 4,4’-diisothiocyanostilbene-2,2-disulfonic acid; MEA, monoethanolamine; FCCP, carbonylcyanide-ptrifluoromethoxyphenyl hydrazone.

MATERIALS

AND

METHODS

SARCOLEMMAL

ION

at 21°C. After vesicle dilution, ion flux was followed by placing aliquots at the given time intervals on a 0.45-Frn HAWP Millipore filter followed by rapid rinsing (3X) with unlabeled medium. The radioactivity retained on the filters was counted in 4.5 ml of a scintillation liquid which completely dissolved the Iiters (14). Membrane potential meusurements. Membrane potentials were generated by gradients of permeant ions between the intravesicular cavity and the medium into which the vesicles were diluted. Membrane potentials (negative inside) were detected by the use of the fluorescent dye 3,3’-dipentyl-2,2’-oxadicarbocyanine iodide [diO-C,-(3)] as described by McKinley and Meissner (23). The sign of the membrane potential is reported according to standard convention, i.e., reference (ground) is extravesicular. Fluorescence assays were carried out at 20°C under stirring in a Farrand Model 801 fluorometer. Excitation was at 470 nm and emission was recorded at 495 nm. Vesicle concentrations were used which produced negligible perturbation of the fluorescence emission during dilution with incubation medium. Light-scattering measurements. Osmotically induced volume changes in sarcoplasmic reticulum and sarcolemmal vesicles were detected by monitoring the changes in light-scattering intensity at 400 nm at right angle to the incoming beam using a Farrand Model 801 fluorometer. Membrane vesicles (30 rg/ml protein) were equilibrated at 21°C in 2.7 ml of 10 mM K or Na Pipes (1,4-piperazinediethanesulfonic acid), pH 7.2. Osmotic volume changes were induced by adding to the membrane vesicle suspension under rapid stirring one-tenth of a volume (0.3 ml) containing 10 mM K or Na Pipes, pH 7.2 and 1000 mosm of the test compound. RESULTS

Isolaticm and analysis of sarcolemmal fractions. In preliminary studies the distribution of vesicles possessing sarcolemma1 marker activity on sucrose gradients was found to be dependent on salt concentration and pH. The effects of KC1 on sarcolemmal vesicle isolation were studied by treating the microsomal crude fraction with KC1 and following the distribution of the two sarcolemmal markers, “basic” ATPase activity and [3H]ouabain labeling, on continuous isopycnic sucrose gradients. “Basic” ATPase has been shown to be a membrane bound “marker” enzyme for skeletal muscle sarcolemma (20), while [3H]ouabain labeling of transverse-tubule has been used to follow the movement of

11

PERMEABILITY

A lh

25 20

0 mM KCI , [‘H]owbain

0 25

%

20 ;!

20

a

20

lo

3S”CROSE 450

mM KCI

15

0 40

30

%

SUCROSE

FIG. 1. Effect of KC1 on distribution of protein and sarcolemmal markers in sucrose gradients. Crude rabbit skeletal microsomal fractions in 0.3 M sucrose buffer were incubated with (A) 0 mM KC1 or (B) 450 IIIM KC1 at pH 7.0 for 4 h at 2°C. Vesicles were then layered over a 200-ml 20-50X (w/w) continuous sucrose gradient containing 10 InM K Hepes, pH 7.1, and (A) 0 or (B) 50 mM KC1 and centrifuged at 100,OOOy for 16 h in a Ti14 Beckman rotor. lo-ml aliquots were collected from the gradient and assayed for protein (0), “basic” ATPase activity (m), and [aH]ouabain labeling (0). Results are presented as percentage of total activity recovered from the gradient within a given aliquot.

transverse-tubule derived membrane vesicles (17, 18). In Fig. 1, we show the effect of treating the microsomal crude fraction with 0 (A) and 450 mM (B) KC1 for 4 h before isopycnit sucrose centrifugation. In the absence of KC1 the greater portion of the microsomal protein and sarcolemmal vesicles were found at sucrose concentrations ranging from roughly 30 to 42% (Fig. 1A). On treatment with 450 mM KC1 (Fig. 1B) two distinct protein peaks corresponding to approximately 33 and 20% sucrose emerged, while the sarcolemmal-associated “basic” ATPase and [3H]ouabain peaks found at approximately 41% in the absence of KC1 became substantially smaller and migrated toward the 22-30% sucrose region of the gradient. The protein

12

GILBERT

AND TABLE

MEISSNER I

YIELD AND ENZYMATIC MARKER ACTIVITIES OF SUCROSEGRADIENT FRACTIONS DERIVED FROM RABBIT SKELETAL MUSCLE

Fraction CF 1 2 3 4 5 6

Sucrose” (76, W/W) 19-22 22-27 27-29 29-31 31-36 36-45

Yield (mg protein/1000 g muscle) 2392 9 27 46 286 620 628

“Basic” ATPase (pmol/mg proteinlmin) 0.28 3.06 4.48 2.70 0.66 0.23 0.14

2 f * * + f f

0.06 0.52 0.99 0.70 0.20 0.04 0.03

32P-Labeled phosphoenzyme (nmol/mg protein) 3.06 0.42 0.57 1.41 2.96 4.42 3.12

k 0.75 f 0.14 f 0.30 f 0.72 f 0.82 3~ 0.34 + 0.75

Note. Crude microsomal fractions (CF) and sucrose gradient fractions (fractions l-6) were obtained and enzymatic assays were carried out as described under Materials and Methods. The data are the average of 6 preparations. Standard deviations are as shown. a Region of gradient from which fractions l-6 were obtained.

peak at 20% sucrose was largely attributable to soluble proteins (data not shown). The 4-h incubation was found to be optimal for sarcolemmal vesicle isolation. Longer incubations in 450 mM or higher KC1 concentrations tended to lower enzymatic activity without resulting in greatly improved purity or yield, whereas lower KC1 concentrations resulted in less complete separation of sarcolemmal and sarcoplasmic reticulum membranes (data not shown). The 22-2’7% area of the sucrose gradient registered highest in sarcolemmal associated activities and contained relatively little sarcoplasmic reticulum contamination, as indicated by the low levels of [32P]phosphoenzyme intermediate (Table I). Above 27% sucrose, sarcoplasmic reticulum content of the gradient fractions rose sharply; while below 22% sucrose only small amounts of membranes with decreased “basic” ATPase activity were recovered. Since the possibility existed that some sarcoplasmic reticulum vesicles might be in an inside-out configuration (24) resulting in substrate inaccessibility, phosphoenzyme assays were measured in the presence of the detergent dodecyloctaoxyethylene glycol monoether (&Es) (25). In the presence of &Es [32P]phosphoenzyme levels increased approximately 25% in fraction 2 and were

unaffected in fraction 5; indicating that estimates of sarcoplasmic reticulum contamination of fraction 2 might be slightly underestimated using the standard [““PIphosphoenzyme assay in the absence of detergent. The 22-27% region was enriched in sarcolemmal-associated leucyl P-naphthylamidase activity, capable of enhanced Dversus L-glucose uptake, and essentially free of inner mitochondrial membranes (14). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (26) showed also that the 22-27% sucrose region of the gradient had a low Ca2+-ATPase content (-20% of that of the 31-36% sucrose region), supporting our marker enzyme data (not shown). The ion permeability of rabbit skeletal muscle sarcolemmal vesicles with a buoyant density corresponding to 22-27% sucrose (fraction 2 of Table I) to K+, Na+, H+, and Cl- was investigated using three techniques: radioisotope flux, membrane potential, and light-scattering measurements. The presence of contaminating sarcoplasmic reticulum in ion-permeability measurements was assessed with the use of a vesicle fraction enriched in sarcoplasmic reticulum (fraction 5 of Table I). Fraction 5 displayed a low level of “basic” ATPase activity, indicating that the sar-

SARCOLEMMAL

ION

13

PERMEABILITY

coplasmic reticulum vesicles were slightly contaminated with sarcolemmal membranes. Radioisotopeflux. Previous radioisotope measurements have shown that sarcolemma1 vesicles can trap *‘Rb+ (a substitute for K+), 22Nat, 45Ca2+, and [3H]sucrose. These studies further demonstrated that a small fraction of the sarcolemmal vesicles were capable of Nat-Na+, Na+-Ca2+, and K+-K+ exchange (14). Figure 2 shows a similar experiment for 36C1l. Vesicles were incubated for 6 h at 22°C in a medium containing 20 mM Na 36C1and then diluted 150-fold into unlabeled isoosmolal dilution media containing 20 mM NaCl or KC1 or an equivalent amount of the sodium or potassium salts of Pipes-. Radioactivity remaining with the vesicles at various time intervals was determined by collecting the vesicles on Millipore filters. On dilution of the vesicles into media containing Cl-, an

O5

II

/

2

5 TIME

10

(mln)

FIG. 2. a%- efl’mx from sarcolemmal vesicles. Vesicles of fraction 2 were incubated for 2 h at 22°C in media containing 20 mM Na %I, 270 mM sucrose, and 10 mM Hepes, pH 7.2. Vesicles were then centrifuged for 30 min at 20,000 rev/min (33,000g) in a Beckman 42.1 rotor. The pellet was taken up at a concentration of 5 mg protein/ml in the original incubation medium and incubated for an additional 4 h at 21°C. Aliquots of vesicles were diluted 150-fold into isoosmolal solutions of 270 mM sucrose, 10 mM Hepes, pH 7.2, and either 20 mM KC1 (O), K Pipes (O), NaCl (O), or Na Pipes (m). At the indicated time points vesicles were caught on Millipore filters, rinsed (3X), and radioactivity remaining with the vesicles on the filters was determined.

1

I 0

I I6

1 24 TIME

I 32

I 40

J 40

(h)

FIG. 3. Measurement of [aH]sucrose, 86Rbt, %a+, ““SOAR, and 36Clm isotope spaces. Fraction 2 vesicles (5 mg/ml) were incubated at 21°C in media containing 5 mM Hepes, 10 mM Tris maleate, 0.3 M [aH]sucrose (0), and either 0.1 M =NaCl (A), or 0.1 M %RbCl (m), or 0.1 M Naa %O, (+), or 0.1 M Na3%1 (O), all at pH 7.2. Vesicles were diluted 200-fold into unlabeled media of identical composition at 21°C. Aliquots of 0.4 ml were applied to 0.45-Frn Millipore filters, rinsed (3X), and the amount of radioactivity remaining with the vesicles on the filters was determined. Apparent isotope spaces were determined by taking 30-, ‘75., and 120-s time points after vesicle dilution and extrapolating back to zero time (cf. Fig. 2). Spaces are the average of 4 separate experimental determinations. Standard errors are indicated by error bars.

increased initial 36C1l efflux rate was observed suggesting the possibility that a small population of the vesicles were capable of Cll-Cl- exchange. Increased 36C1l efflux was not due to sarcoplasmic reticulum. In sarcoplasmic reticulum 36C11 passes very rapidly across the membrane so that no radioactivity remains within the vesicles 20 s after dilution into the exchange medium (23). Identical [3H]sucrose efflux rates from vesicles double-labeled with [3H]sucrose and 36C11suggested that the difference in 36C1l efflux rates, in the presence of extravesicular Cl- or Pipes-, was not due to nonspecific osmotic effects (not shown). Apparent isotope spaces of sarcolemmal vesicles for “Na+ “Rb+ 36Cll 35S02p and 4 [3H]sucrose are shown id Fig. 4. Sarcolemma1 vesicles were incubated in buffered 100 mM salt media containing 0.3 M sucrose and trace amounts of the radioactive isotopes of the substance studied. Apparent ion

14

GILBERT

AND

spaces were determined by removing sample aliquots, at the indicated time points, and diluting them into an identical medium without the radioactive tracer. Radioactivity associated with the vesicles was determined by collecting the vesicles on Millipore filters (cf. Fig. 2). Radioactivity retained by the vesicles on the filters rose steadily during the first 16 h, reached a peak during the 16- to 30-h incubation period, varying with preparation, then declined slowly as the vesicular membrane barrier deteriorated. Apparent isotope spaces for 36C1- and 35SOzp were smaller than for [3H]sucrose and =Na’. “Rb+ spaces increased more rapidly than the values for either 22Na+, 36C1-, or 35SO&-, reaching a peak of approximately 3.75 pl/mg protein at about 24 h. Maximum spaces found for *‘Rb+ were about 1.5 times those of “Na+ and [3H]sucrose. After 24 h *‘Rb+ spaces declined quickly, decaying more rapidly than the other isotope spaces. Differences between ionic spaces for “Rb’ and 22Na+ were not significant for sarcoplasmic reticulum (data not shown). Since, as demonstrated below, our sarcolemma1 fractions did not seem to be very permeable to Na+ and lower Na+ spaces cannot be accounted for by very rapid Na’ efflux due to, for example, the presence of a Na+ channel, the reason for the higher Rb+ space was unclear. One possibility would be specific Rb+ binding to the sarcolemmal vesicles. To test for this possibility sarcolemmal spaces were taken at early incubation time points (at 30 s and 3 min as opposed to 3-40 h in Fig. 3). Rb+ binding to sarcolemmal vesicles would presumably be a fast process relative to influx into vesicles and a rapid initial increase in the Rb’ space might be indicative of a binding process. It was found that *‘Rb+ ionic spaces have a slightly faster initial rise than those for [“HIsucrose (0.35 vs 0.27 pl/mg at 30 s) and “Na+ (0.30 pl/ mg at 30 s), but there was no immediate extravesicular binding of Rb+ sufficient to explain the differences observed. Rb+ binding to internal sites was not excluded, however, and could explain the abnormally large Rb+ spaces. Another possibility was that at least some of the sarcolem-

MEISSNER

ma1 vesicles were simply more permeable to Rb+ than Na+ or [3H]sucrose. Thus, Rbf equilibrated across the sarcolemmal vesicles before the membrane barrier began to deteriorate, while Na+ and [3H]sucrose, entering the vesicles more slowly, did not fully equilibrate. Figure 3 shows that 36C1l and 35SOip spaces were smaller than those observed for “Rb+, “Na+ or [3H]sucrose. One explanation for the smaller Cll and SOi- spaces would be that a portion of the vesicles contained a specific permeation system for anions. Therefore, these vesicles were able to release 36C1l and 35SOip before the first time point was taken. The question of whether a portion of fraction 2 was readily permeable to Cll and SOi- was tested with the use of the anion channel blocker 4,4’diisothiocyanostilbene-2,2’-disulfonic acid (DIDS). DIDS has been used extensively by Rothstein and co-workers to study the anion channel of red blood cells (27) and more recently by Kometani and Kasai to study the anion permeability of sarcoplasmic reticulum (28). Prior treatment of vesicles with DIDS at concentrations ranging from 0 to 1 mM increased the apparent 35SOip spaces of fraction 2 vesicles by about 20-40% (Fig. 4A). At approximately 1 mM DIDS, the 3”SOim spaces approached the one observed for 22Na+, then decreased again slightly at higher DIDS concentrations. 36C1- spaces were also increased at 1 mM DIDS, but not to the extent observed for the slower penetrating 35SOip (Fig. 4A). In control double-labeling experiments utilizing [3H]s~crose in conjunction with 35S042por “Na+ the effects of DIDS on 22Na+ and [“HIsucrose spaces or elllux rates were insignificant. In sarcoplasmic reticulum, prior treatment with 1 mM DIDS increased 35SOim spaces approximately fourfold, a greater increase than was seen with sarcolemmal vesicles. Sarcoplasmic reticulum “YJl spaces were small, consituting 5 to 10% of the whole, and in agreement with a previous study (28), were little affected by 1 mM DIDS. Prior treatment with 1 mM DIDS had no significant effect on sarcoplasmic reticulum [3H]sucrose and 22Na+ spaces.

15

SARCOLEMMAL ION PERMEABILITY

2 TIME

3 hni

4

3

nhc

4

hn)

FIG. 4. Effect of DIDS on sarcolemmal and sarcoplasmic reticulum isotope spaces. In Fig. 4A the effect of DIDS on apparent vesicle spaces and efflux rates of %Oz-, pNa+, and %- from fraction 2 vesicles are shown. Fraction 2 vesicles (7 mg/ml) were incubated for 4 h at 22°C then 20 h in ice at 2°C in a medium containing 200 IXIM [aH]sucrose, 50 mM KCl, and either 10 mM %aCl, or 20 mM Na 36C1or 5 mM Naz a5S04, 5 mM Hepes and 10 mM Tris-Pipes, pH 7.2. After this incubation the %Of- vesicles were reacted with either 0 mM (0). 0.2 IIIM (A), 0.6 tIIM (m), 1.2 mM (V), or 2.0 mM DIDS (+) for 30 min at 37°C. (a, 0) %a+- and (0, 0) 36C1m-containing vesicles were incubated for 30 min at 37°C at either 0 (A, Cl) or 1 ttIM DIDS (0, 0) in the presence of radioisotopes in incubation media. In Fig. 4B, fraction 5, sarcoplasmic reticulum vesicles were incubated for 4 h at 22°C in 200 mM [3H]sucrose (0, n ), 50 mM KCI, 5 InM Hepes, 10 mM Tris-Pipes, pH 7.2, and either 20 mIM Na “%I (0, l ), or 50 mM Nas “SO:- (A, A). After the incubation sarcoplasmic reticulum vesicles were reacted with either 0 !TIM (m, 0, A) or 1 mM (17, 0, a) DIDS, respectively for an additional 30 min at 37°C. Aliquots of vesicles were diluted 200-fold at 22°C into dilution media identical to incubation media except they lacked radioactive tracers. Vesicles were caught at the given time points on Millipore filters, rinsed (3x), and radioactivity remaining with the vesicles on the filters was determined. All data are expressed as the calculated ionic space of the vesicles on the filter

In conclusion, the investigation of anion permeability of our sarcolemmal preparation showed that a fraction of the sarcolemmal vesicles could be rendered impermeable to 3”SO%mby DIDS. 36C11spaces were only slightly increased by DIDS, presumably due to incomplete blockage of the fast penetrating 36C1- ion. Whether the increase in 3”SOim space shown by DIDS was due to sarcolemmal vesicles with a 3”SO&mpermeability mechanism, to sarcoplasmic reticulum contamination of the sarcolemmal fraction, or to a combination of both was further investigated using the light-scattering techique. Light-scattering measurements. Osmotically induced changes in vesicle volume, as detected by light-scattering intensity measurements have been used to determine the permeability of sarcoplasmic reticulum vesicles (28, 29). The advantage of this technique vis a vis radioisotope tracer techniques is its general applicability and time resolution (2-3 s).

Figure 5 shows that the addition of various test solutes to sarcoplasmic reticulum and sarcolemmal vesicles resulted in transient changes in the light-scattering intensity of the vesicles. Vesicles were initially present at a concentration of 30 pg protein/ml in 2.7 ml of 10 mM K Hepes, pH 7.1. The extravesicular osmolality was increased by the addition of 0.3 ml of a buffered 1000 mosm solution of the indicated test compounds under rapid mixing. In control experiments the decrease in signal intensity due to vesicle dilution from 30 to 27 pg protein/ml was determined by adding 0.3 ml of 10 mM K Hepes, pH 7.1. Maximal light-scattering signals were observed for both sarcoplasmic reticulum (Fig. 5A) and sarcolemmal vesicles (Fig. 5B) on addition of 0.3 ml of 1000 mosm choline Cl or K gluconate. Initially, the vesicle suspensions shrank as water left the vesicles increasing the light-scattering signal. The signals then slowly returned to the one observed for control vesicles as

16

GILBERT

AND

8.

A. SOLUTE 4

MEISSNER

ADDED

-

Speesanged 3s

3 min

-

3s

1 3rfiIl

FIG. 5. Effect of an increase in osmolality on the light-scattering intensity of sarcoplasmic reticulum (A) and sarcolemmal (B) vesicles. Vesicles (30 pg protein/ml) were equilibrated in 2.7 ml of 10 mM K Hepes (pH 7.2) for 30 min at 21°C. Where used valinomycin was added to the equilibrated vesicle suspension at a concentration of 5 pg/ml prior to solute addition. Vesicle volume changes were initiated at 21°C under rapid stirring by adding 0.3 ml of medium containing 10 mM K Hepes, pH 7.1, and 1000 mosm of choline-Cl (0), K gluconate (O), K gluconate (into valinomycin) (B), (0). Light-scattering monoethanolamine Cl (V), NaCl (A), KCI (0), or KC1 (into valinomycin) intensity changes were followed at a right angle to the incoming beam and are expressed as the percentage change of the signal AI/I,. The final light-scattering intensity caused by the addition of 0.3 ml of the test compound to the vesicle suspension was determined by adding 0.3 ml of 10 mM K Hepes, pH 7.2.

solute followed by water entered and the vesicles reexpanded. The rate of swelling was a function of the permeability of the vesicles to the solutes, the slower penetrating ion being rate limiting (28). As indicated by the slower return rates of their light-scattering signals, sarcolemmal vesicles were less permeable to choline Cl and K gluconate than sarcoplasmic reticulum vesicles. The addition of the K+-ionophore valinomycin did not affect the return rate, indicating that gluconate- was the slower ion of the K gluconate ion pair in sarcoplasmic reticulum and sarcolemmal vesicles. All sarcoplasmic reticulum vesicles are permeable to Cl- (23). K+, Nat, and monoethanolamine+ can pass rapidly through the Kf, Na+ channel which is present in about two-thirds of sarcoplasmic reticu-

lum vesicles (23). Light-scattering signals of reduced size were therefore recorded when the osmolality of sarcoplasmic reticulum vesicle was increased by the addition of media containing KCl, NaCl, or monoethanolamine Cl (Fig. 5A). The reduction in the light-scattering signals indicated vesicle populations that were permeable to KCl, NaCl, or monoethanolamine Cl. The K+, Nat ion-permeable vesicle population contracted and reexpanded too fast to be observed on the time scale of the light-scattering experiments (2-3 s). The further decline in the light-scattering signal, seen on KC1 addition in the presence of valinomycin, indicated the presence of K+, Na+-impermeable sarcoplasmic reticulum vesicles that in the absence of valinomycin were permeable to Cl- but not to K+ (Fig. 5A). The small remaining sig-

SARCOLEMMAL

ION

nal in the presence of KC1 plus valinomycin was presumably due to a residual population of Cll-impermeable vesicles derived from contaminating sarcolemma or mitochondria. Addition of KCl, NaCl or monoethanolamine Cl to sarcolemmal and sarcoplasmic reticulum vesicle suspensions produced light-scattering signals that differed in several ways. First, light-scattering signals for sarcolemmal vesicles decayed more slowly, indicating the greater overall “tightness” of the KCl-, NaCl-, and monoethanolamine Cl-impermeable sarcolemmal vesicle populations. Second, a greater proportion of the light-scattering signal for sarcolemmal vesicles was contributed by K+- and/or Cll-impermeable vesicles than in sarcoplasmic reticulum. Third, the large remaining signal seen with

17

PERMEABILITY

KC1 in the presence of valinomycin indicated a significant population of Cll-impermeable vesicles in the sarcolemmal fraction, in agreement with the radioisotope measurements (Fig. 3). Fourth, addition of NaCl and monethanolamine Cl to sarcolemmal vesicle suspensions elicited signals that were greater than those observed with KCl. The difference in lightscattering signals suggested the presence of a population of sarcolemmal vesicles intrinsically permeable to K+ and Cl- but not to monoethanolamine+ or Na+ and Cl-. NaCl, and monoSimilar KCl, ethanolamine Cl signals in Fig. 5A showed that the K+- and Cl--permeant vesicle fraction was not of sarcoplasmic reticulum origin. Membrane potential measurements. In experiments described below, isolated VESICLES

VESICLES 1 K GLU-K

GLU

A

K&U-KGLU KGUJ+KGLUtVAL

A

c.

/

J/==$

Speed Changed 3s 3s

2mm

FIG. 6. The effect of K+ diffusion potentials in sarcolemmal vesicles on the fluorescence emission of diOC,-(3). Fraction 2 vesicles were incubated for 16 h at 20” in 400 mosm K gluconate plus 10 mM K Pipes (pH ‘7.2), then diluted to a final concentration of 45 pg protein/ml into isoosmolal gluconate media at 20°C containing the indicated cation, 1.5 FM diO-Cs-(3), and 0 or 0.5 fiM valinomycin (val). An initial loo-fold (inside-out) K+ gradient was maintained in all experimerits. X537A was used at a concentration of 3 pg/ ml to render all vesicles permeable to Na+ and Tris+, causing a rapid collapse of membrane potentials formed by dilution.

3 ml”

FIG. 7. Effect of K+ gradients in K+, Na+-permeable and -impermeable sarcoplasmic reticulum vesicles on the fluorescence emission of diO-C,-(3). The graphs show the time course of fluorescence emission changes when skeletal muscle sarcoplasmic reticulum vesicles (fraction 5 of Table l), after incubation for 4 h at 20°C in 400 mosm K gluconate plus 10 mM K Pipes (pH ‘7.2), were diluted loo-fold into isoosmolal gluconate media at 20°C. Dilution media contained the indicated cation, 1.5 pM diO-C&-(3) and 0 or 0.5 pM valinomycin (val). After dilution the final protein concentration was 15 pg/ml. An initial loo-fold (inside-out) K gradient was maintained in all experiments.

18

GILBERT

AND

membrane vesicles were first incubated in a solution of known ionic composition. Diffusion potentials, negative inside, were then generated by the dilution of the vesicles containing a permeant cation and impermeant anion into an isoosmolal medium containing an impermeant cation and anion. Changes in potential caused by the efflux of the permeant cation were recorded as a decrease in the fluorescence emission of the dye 3,3’-dipentyl-2,2’-oxadicarbocyanine [diO-C,-(3)]. The decrease in fluorescence emission has been shown to be roughly proportional to the magnitude of the developed membrane potential and the amount of polarized vesicles present (23). The permeability of sarcolemmal vesicles to K+, Na+, and H+ was investigated by these procedures. Since our sarcolemmal vesicle fraction was contaminated with sarcoplasmic reticulum, sarcoplasmic reticulum vesicles were used in control experiments. In Fig. 6A, sarcolemmal vesicles in K gluconate were diluted into K gluconate and K gluconate plus valinomycin. As expected, in the absence of a K+ gradient little or no fluorescence change was observed. On dilution of K gluconate-containing vesicles into Na gluconate (Fig. 6B) no polarization was seen, indicating that either no K’ channels or no channels specific for K’ over Na+ were present. Another possibility was that the majority of sarcolemmal vesicles contained both functional K’ and Na+ channels. On dilution of K gluconate vesicles into Na gluconate plus valinomycin a substantial change in fluorescence was observed. In the presence of Naf plus valinomycin only those sarcolemmal vesicles without a functioning Na+ permeability mechanism and contaminating sarcoplasmic reticulum vesicles without a Kf, Na+ channel (Fig. 7B) should polarize, since any Na+ permeable channel would presumably allow NaC counterflow into the vesicles as the intravesicular K+ flowed out. Fluorescence signals returned to the initial baseline as Na+ moved into the vesicles (23). Sarcoplasmic reticulum vesicles were fully depolarized by 3-min postdilution (Fig. 7B) whereas the sarcolemma1 vesicle fraction could maintain a

MEISSNER

membrane potential for 15 min and longer (Fig. 6B). The addition of the ionophore X537A, which rendered the vesicles permeable to both K+ and Na+, immediately collapsed the membrane potential remaining by 3-min postdilution. Together the results of Figs. 6B and 7B suggest the presence of a population of sarcolemmal vesicles intrinsically impermeable to both K+ and Na+. In Fig. 6C, K gluconate-loaded sarcolemma1 vesicles were diluted into Tris gluconate and Tris gluconate plus valinomytin. The signal seen on dilution into Tris gluconate in the absence of valinomycin was expected to be derived from contaminating sarcoplasmic reticulum vesicles with K+, Na+ channels (23; Fig. 7C) and sarcolemmal vesicles which were permeable to K+ but not Tris+. The fluorescence signal of the sarcolemmal fraction, demonstrated at least two major recovery phases: a small rapid recovery phase and a slow recovery phase (Fig. 6C). Rapid breakdown of the membrane potential in sarcoplasmic reticulum vesicles (Fig. 7C) as well as mixing experiments involving sarcoplasmic reticulum and sarcolemmal vesicles indicated that the initial rapid transient signal was due in large part to sarcoplasmic reticulum contamination of the sarcolemmal fraction (data not shown). The slow recovery phase of the signal seen on dilution of sarcolemmal vesicles into Tris gluconate suggested that there was a small population of otherwise highly impermeable vesicles that possessed a K+ permeability mechanism. Control experiments with the ionophore X537A, which rendered the vesicles permeable to Tris+, supported this conclusion. The addition of X537A, at 3-min postdilution, immediately collapsed the remaining potential. The signal returned to baseline (Fig. 6C), minus the small quenching effect seen in Fig. 6A, suggesting that a small population of sarcolemmal vesicles had indeed developed a potential. On dilution of K gluconate-containing vesicles into Tris gluconate plus valinomycin all of the vesicles were expected to be polarized. Accordingly, maximal fluorescence signals were observed (Figs. 6C

SARCOLEMMAL

and ‘7C). The signal produced by dilution of the sarcolemmal vesicle fraction into Tris+ plus valinomycin, like that by dilution into Tris+ alone, demonstrated a rapid transient phase, which correlated with the extent of sarcoplasmic reticulum contamination, and a slow phase due to sarcolemma1 vesicles. As previously observed for the dilution of K gluconate-loaded vesicles into Tris gluconate, the addition of X537A caused a rapid return of the signal to baseline. In Table II we have summarized the fluorescence decreases seen for sarcoplasmic reticulum and sarcolemmal vesicles when diluted from K+ medium into Na+, Tris’ or monoethanolamine+ (MEA) media, in the presence or absence of valinomytin. Monoethanolamine+ passes rapidly through the K+, Na+ channel of sarcoplasmic reticulum vesicles (23), whereas it is impermeant to the sarcolemmal K+ and Na+ channels (30). When K gluconate-filled sarcoplasmic reticulum vesicles were diluted into MEA gluconate no membrane potential was formed, since K+ efflux from vesicles was balanced by MEA+ influx (Table II). On dilution of K gluconate-loaded sarcolemmal vesicles into monoethanolamine gluconate, however, a small longlived decrease in fluorescence, amounting to lo-15% of the total signal, was observed. This finding was in agreement with the data of Figs. 5 and 6C which also indicated a small population of sarcolemmal vesicles, constituting lo-20% of the total vesicle fraction, to possess a K+ permeability mechanism. The majority of the sarcolemmal vesicles did not appear to possess an intrinsic K+ permeability mechanism, as indicated by the large increase in long-lived fluorescence signals seen when K gluconate-filled sarcolemmal vesicles were diluted into Na, Tris, or monoethanolamine gluconate media containing valinomycin. Sarcolemmal Na+ permeability was studied similarly to K+ permeability (Table III, Exp. l-3). On dilution of Na gluconate-containing vesicles into Na gluconate and K gluconate no significant decrease in fluorescence was observed. Dilution of Na gluconate-filled sarcolem-

ION

19

PERMEABILITY

TABLE

II

EFFECT OF K+ GRADIENTS IN SARCOPLASMIC RETICULUM AND SARCOLEMMAL VESICLE FRACTIONS ON THE FLUORESCENCE EMISSION OF DiO-CS-(3) ‘70 Decrease in fluorescence Incubation medium

Dilution medium

-VAL

+VAL

Sarcoplasmic reticulum (at 0 min) K Glu

K Glu NA Glu TRIS Glu MEA Glu

0 0 29 0

0 16 42 18

Sarcolemma (at 3 min) K Glu

K Glu Na Glu Tris Glu MEA Glu

0 0 2 2

0 11 13 13

Note. K+ diffusion potentials were created in sarcoplasmic reticulum (fraction 5 of Table I) and sarcolemmal vesicles (fraction 2 of Table I) by diluting vesicles loo-fold from a 400 mosm K gluconate medium at pH 7.2 into the indicated isoosmolal dilution media, in the presence and absence of 0.5 PM valinomycin. Dilution media contained 1.5 PM diO-C,-(Y). Fluorescence decreases for sarcoplasmic reticulum vesicles were obtained by back-extrapolation to the time of vesicle addition (cf. Fig. 7) and by substracting the signal seen for nonpolarized vesicles on dilution into K gluconate medium. Sarcoplasmic reticulum signals returned within 3 min to those seen for nonpolarized vesicles (Fig. ‘7). Sarcolemmal vesicle fluorescence decreases indicate those seen at 3 min after vesicle dilution in the absence of X537A (cf. Fig. 6).

ma1 vesicles into Tris gluconate caused a decrease in fluorescence, but the signal collapsed quickly and was therefore attributed to sarcoplasmic reticulum contamination of the sarcolemmal fraction. The ionophore gramicidin D was found to demonstrate a preferential permeability to Na+ over Tris+ on the time scale used for these experiments. On the dilution of Na gluconate-loaded vesicles into Tris glu-

20

GILBERT

AND TABLE

MEISSNER III

EFFECTOFN~+ AND H+ GRADIENTSINSARCOPLASMICRETICULUMANDSARCOLEMMAL VESICLESONTHE FLUORESCENCE EMISSIONOFDiO-C,-(3) Sample

Experiment

Vesicle medium

SL

1 2 3

Na glu

SL

4 5 6 7

Tris-Pipes,

pH 6.5

Tris-Pipes,

pH 6.5

SR

Dilution

medium

-Gram 0 0 0

Na glu K glu Tris glu Tris-Pipes, Tris-Pipes, Tris-Pipes, Tris-Pipes,

% Fluorescence

pH pH pH pH

6.5 8.0 6.5 8.0

-FCCP 0 0 0 8.0

D

decrease +Gram D 0 0 7 +FCCP 0 9 0 10.0

Note. Sarcolemmal (SL) (fraction 2 of Table I) and sarcoplasmic reticulum (SR) (fraction 5 of Table I) vesicles present in 400 mosm Na gluconate, 10 mM Na Hepes medium at pH 7.2 or 400 mosm Tris-Pipes, pH 6.5, were diluted loo-fold into the indicated isoosmolal dilution media containing 1.5 PM diO-CB-(3) and the indicated ionophores. Gramicidin D and carbonylcyanide p-trifluoromethoxyphenyl hydrazone (FCCP) were used at a concentration of 5 and 1 PM, respectively. Fluorescence decreases were calculated as described in Table II.

conate plus gramicidin D (Table III, Exp. 3) a signal was formed by Na+-impermeable “silent” sarcolemmal vesicles. This total Na+ signal, however, while demonstrating a population of Na+ impermeable vesicles, was never as large as that observed when sarcolemmal vesicles are transferred from K gluconate medium into Tris gluconate medium plus valinomycin. The reduced signal seen in sarcolemmal and sarcoplasmic reticulum (not shown) vesicles was likely due to the rapid influx of Tris+ as indicated by the rapid collapse of the membrane potential in the presence of gramicidin D. Proton permeability. The free H+ permeability of sarcolemmal vesicles was investigated as previously described for sarcoplasmic reticulum vesicles (31). A 30-fold H’ gradient (inside-out) was established across the vesicle membranes by diluting vesicles in Tris-Pipes, at pH 6.5, into an identical solution at pH 8. The resulting H+ diffusion potentials (negative inside) were detected with the use of the fluorescent probe diO-C,-(3). Absence of a significant signal on dilution of vesicles from pH 6.5 to 8 indicated that sarcolemmal vesicles (Exp. 4 and 5 of Table III), unlike sarcoplasmic reticulum vesicles (Exp. 6 and

7 of Table III; Ref. (31)), were essentially impermeable to H+. H+-impermeable vesicles were revealed with the use of the H+ionophore carbonylcyanide p-triIluoromethoxyphenyl hydrazone (FCCP). DISCUSSION

In this study we have used isotope flux, membrane potential, and light-scattering measurements to show that a sarcolemmal-enriched membrane fraction was impermeable to Na+ and protons, and that it exhibited passive ion fluxes far slower than those observed for sarcolasmic reticulum. About lo-20% of sarcolemma derived vesicles were found to be permeable to Cland Ki. As previously outlined (14), enzymatic marker studies showed rabbit skeletal muscle membranes found between 22 and 27% sucrose on isopycnic gradients to be enriched in sarcolemma. The chief contaminant of the sarcolemmal fraction was sarcoplasmic reticulum. The origin of the sarcolemmal fraction, that is whether it was predominantly derived from the surface membrane or transverse-tubule portion of the surface membrane, was not established in the present study. However, the ability of vesicles to trap [3H]ouabain

SARCOLEMMAL

ION PERMEABILITY

(17, 18), as well as recent immunological (32) and freeze-fracture (33) studies, suggest that the sarcolemmal vesicles prepared by the method used in the present study are largely derived from the transverse-tubule portion of the surface membrane. Three techniques were used to measure the membrane permeability of sarcolemma1 vesicles derived from rabbit skeletal muscle. The radioisotope tracer-Millipore filtration technique was useful for determining vesicle spaces, ionic exchange, and efflux rates for slowly permeating solutes. Light-scattering experiments gave similar information to radioactive tracer studies, but possessed the additional advantage of allowing the study of ion pair movements and solute fluxes on the time scale of 2-3 s, as compared to 30 s for our tracer studies. Membrane potential measurements were valuable in determining the free cation permeability of isolated membrane vesicles. It should be pointed out, however, that the different techniques measured different vesicle parameters. For example, radioisotope tracer and membrane polarization measurements reflected internal vesicle spaces and the surface area of the polarized vesicles, respectively. Using these three techniques, it was possible to differentiate sarcolemma from contaminating sarcoplasmic reticulum. This was accomplished by using a sarcoplasmic reticulum vesicle preparation as a control. Radioisotope measurements showed similar [“HIsucrose spaces for sarcoplasmic reticulum (23) and sarcolemmal vesicles (Fig. 2). “Rb+, =Naf and 36C11 spaces were, however, all appreciably smaller for sarcoplasmic reticulum than for sarcolemmal vesicles, in agreement with the presence of specific permeability mechanisms for K+, Na’, and Cl- in the sarcoplasmic reticulum membrane. The presence of these channels resulted in the efflux “Rb+, “Na+, or 36C1l within 20-30 s, i.e., the first time point in the Millipore filtration experiments. A limitation of the ion space technique is that radioisotopes have to equilibrate across the vesicle membrane before the vesicular membrane barrier deteriorates. In sarcoplasmic re-

21

ticulum vesicles radioisotopes including [3H]sucrose equilibrate rapidly, but in the case of sarcolemmal vesicles this could not be clearly shown. Therefore, for sarcolemma1 vesicles the technique only allowed the determination of the minimum ion impermeable spaces. In the case of SO&-, it was possible to use the anion channel blocker DIDS to estimate the size of the SO:--permeable space. One of the advantages of the radioisotope technique is that it can measure ion exchange. Previously we found that rabbit skeletal sarcolemmal vesicles are capable of Na+-Na+, Na+-Ca’+, and K+-K+ exchange (14). This study showed that external Cll stimulated 36C1l efflux from a small population of sarcolemmal vesicles. The increased rate may indicate that sarcolemmal membranes also possessed Cll - Cl- exchange activity. An alternative explanation would be that slower 36C11efflux in Pipes- medium was due to formation of a membrane potential, positive inside. A Cll exchange activity was not observed in sarcoplasmic reticulum vesicles. Differences in the permeability of sarcoplasmic reticulum and sarcolemmal vesicles toward individual ions such as monoethanolamine+ allowed us to dissect light-scattering and membrane potential signals into their respective components. In vesicles lacking channels, sarcoplasmic reticulum and sarcolemmal signals could be separated by utilizing differences in the “tightness’ of the two membranes. In K+ membrane potential measurements, for example, sarcolemmal signals were differentiated by taking advantage of the fact that Tris+ equilibrated across the sarcoplasmic reticulum membrane within 3 min, eliminating the sarcoplasmic reticulum signal, while sarcolemmal K+ signals, in the presence of external Tris+, lasted up to 1 h. One perhaps surprising result was the paucity of observable sarcolemmal “channels.” Membrane potential and light-scattering data yielded no evidence of Na+ “channels” in our sarcolemmal vesicle preparations. Assuming that there are 30 Na+ channels/ym’ [as determined by saxitoxin binding to sarcolemmal vesicles iso-

22

GILBERT

AND

lated from rat skeletal muscle (Barchi and Weigle (15)) and that approximately 30 vesicles of an average diameter of 1000 A are derived from 1 pm2 of surface membrane, it would be expected with random distribution that about two-thirds of the sarcolemmal vesicles should contain a Na+ channel. Since we see no evidence of a functioning Na+ channel, it is concluded that we have either isolated a subfraction of sarcolemmal vesicles that do not possess a Na+ channel, or that the Na+ channels are “inactive” under our experimental conditions, not surprising given the fact that sarcolemmal Na+ channels are known to be voltage gated (34). Experiments with batrachotoxin and veratridine, two Na+ channel “activators” (35), also showed no evidence of a functioning Na+ channel (unpublished results). In the case of K+, membrane potential and light-scattering measurements revealed a small fraction (approximately 15%) of the sarcolemmal vesicles to be permeable to K+. Assuming that the K+permeable vesicles contain a K+ channel, it can be calculated, again assuming 1000 A vesicle diameter, that maximally 5 K+ channels are present per pm2 of membrane surface. In this regard, it is of interest that in squid axons the number of K’ channels constitute lo-20% of the number of Na+ channels (36). Vesicle space studies with the anion channel blocker DIDS and 35SOiP presented evidence that a portion of the sarcolemmal fraction was Cl- permeable (lo40% of the total ion space depending upon the individual preparation). Light-scattering experiments using KC1 and monoethanolamine Cl demonstrated that part of these Cl--permeable vesicles could be ascribed to sarcoplasmic reticulum contamination of the sarcolemmal fraction. The other portion, however, since it was monoethanolamine+-impermeable and possessed a signal of long duration, was most likely due to Cl--permeable vesicles derived from sarcolemma. Light-scattering experiments suggested that the Cl-permeable sarcolemmal fraction was also permeable to K+. Since the K+-permeable and Cl--permeable sarcolemmal vesicles

MEISSNER

each constituted only a fraction of the total vesicle population, this finding was surprising if it is assumed that K+ and Clpermeability mechanisms distribute randomly. Two explanations for this phenomenon may be given. It is possible that the K+ and Cl- permeability mechanisms are physically linked in the sarcolemma and therefore found in the same vesicles. An alternate possibility was that K+ and Clpermeability mechanisms are randomly distributed, but are derived from a portion of the sarcolemma with high K+ and Cll channel density. Experiments with amphibian twitch fibers have shown that Clpermeability in white muscle was confined to the surface membrane and was not found in the transverse-tubule (4, 5, 37). K+ channel density has also been shown to be higher in the surface membrane than transverse-tubule of frog semitendinosis fibers (38). With the higher concentrations of Cl- and K+ channels in the surface membrane, there would be a corresponding higher probability that each would be found in the same sarcolemmal vesicle. This hypothesis would be in agreement with the suggestion that our sarcolemmal fraction contains K+- and Cll-impermeable vesicles derived from transverse-tubule and a small fraction of K’- and Cl--permeable vesicles derived from the surface membrane portion of the sarcolemma. ACKNOWLEDGMENT This research was supported by a grant U. S. Public Health Service (AM18687).

of the

REFERENCES 1. BASTIAN, J., AND NAKAJIMA, S. (1974) J. Gen. Physiol 63, 257-278. 2. CONSTANTIN, L. L. (1975) Fed. PKJC. 34,1390-1394. 3. HODGKIN, A. L., ANDHOROWICZ, P. (1960) J. PhysioL (Lvndon) 153,370-385. 4. DULHUNTY, A. F. (1979) J. Memtn-. BioL 45, 293310. 5. VENOSA, R. A. (1979) in Membrane Transport in Biology (Giebisch, G., Tosteson, D. C., and Ussing, H. H., eds.), pp. 211-263, Springer-Verlag, Berlin/Heidelberg/New York. 6. PALADE, P. T., AND BARCHI, R. L. (1977) J. Gen. PhysioL 69, 325-342.

SARCOLEMMAL

ION

7. CAPUTO, C., AND BOLANOS, P. (1978) J. Membr. Biol. 41, 1-14. 8. AICKIN, C. C., AND THOMAS, R. C. (19’77) J. Physiol. 273, 295-316. 9. PHILIPSON, K. D., AND NISHIMOTO, A. Y. (1981) J. BioL Chem 255, 6880-6882. 10. PHILIPSON, K. D., AND NISHIMOTO, A. Y. (1981) J. BioL Chem. 256,3698-3702. 11. PITTS, B. J. R. (1979) J. BioL Chem. 254,6232-6235. 12. REEVES, J. P., AND SUTKO, J. L. (1979) Proc. Nat. Acud Sci. USA 76, 590-594. 13. REEVES, J. P., AND SUTKO, J. L. (1980) Science 208, 1461-1463. 14. GILBERT, J. R., AND MEISSNER, G. (1982) J. Membr. BioL 69, 77-84. 15. BARCHI, R. L., AND WEIGELE, J. B. (1979) J. Physiol. 295, 383-396. 16. GILBERT, J. R. (1980) Fed. Proc. 39, 2176a. 17. CASWELL, A. H., LAU, Y. H., AND BRUNSCHWIG, J-P. (1976) Arch. Biochem. Biophys. 176, 417430. 18. LAU, Y. H., CASWELL, A. H., AND BRUNSCHWIG, J-P. (1977) J. BioL Chem. 252, 5565-5574. 19. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. BioL Chem. 193, 265-275. 20. MALOUF, N. N., AND MEISSNER, G. (1979) Exp. Cell Res. 122, 233-250. 21. FISKE, C. H., AND SUBBAROW, Y. (1925) J. BioL Chem. 66, 375-400. 22. MOORE, B. M., LENTZ, B. R., AND MEISSNER, G. 17, 5248-5255. (1978) Biochemistry

PERMEABILITY

23

23. MCKINLEY, D., AND MEISSNER, G. (1978) .I Membr. BioL 44, 159-186. 24. SARZALA, M. G., AND MICHALAK, M. (1978) B&him. Biophys. Acta 513, 221-235. 25. MOORE, B. M., LENTZ, B. R., HOECHLI, M., AND MEISSNER, G. (1981) Biochemistry20,6810-6817. 26. MEISSNER, G. (1975) Biochim. Biophys. A&. 389, 51-68. 27. ROTHSTEIN, A., AND RANJEESINGH, M. (1980) Ann. N. Y. Acad. Sci. 358, 1-12. 28. KOMETANI, T., AND KASAI, M. (1978) J. Membr. BioL 41, 295-308. 29. TAGIJCHI, T., AND KASAI, M. (1980) B&hem. Bier phys. Res. Commun. 96, 1088-1094. 30. HILLE, B. (1972) J. Gen. PhysioL 58, 599-619. 31. MEISSNER, G., AND YOUNG, R. C. (1980) J. BioL Chem. 255, 6814-6819. 32. ROSEMBLATT, M., HIDALGO, C., VERGARA, C., AND IKEMATO, N. (1981) J. BioL Chem. 256, 81408148. 33. SCALES, D. J., AND SABBADINI, R. A. (1979) J. Cell BioL 83, 33-46. 34. HILLE, B. (1977) in Handbook of Physiology, Vol. 1, pp. 108-136, Williams & Wilkins, Baltimore. 35. CATTERALL, W. A. (1977) J. BioL Chem. 252,86698676. 36. CONTI, F., DE FELICE, L. J., AND WANKE, E. (1975) J. Physiol. (LondmL) 248, 45-82. 37. EISENBERG, R. S., AND GAGE, P. W. (1969) J. Gen. PhysioL 53, 279-297. 38. KIRSCH, G. E., NICHOLS, R. A., AND NAKAJIMA, S. (1977) J. Gen. PhysioL 70, 1-21.