Ryanodine does not affect the potassium channel from the sarcoplasmic reticulum of skeletal muscle

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 148, No. 3, 1987

Pages 1137-1143

November 13, 1987

RYANODINE

DOES

NOT

AFFECT

FROM THE SARCOPLASMIC

A.C.

THE

POTASSIUM

CHANNEL

RETICULUM OF SKELETAL MUSCLE

CAMPOS de CARVALHO and SAMUEL CUKIERMAN

(*)

INSTITUTO DE BIOFISICA, UNIVERSIDADE FEDERAL DO RIO DE JANEIRO, ILHA DO FUNDAO, 21941, RJ, BRASIL * University of Maryland, Department of Physiology, 660 W Redwood Street, Baltimore, 21201, MD

Received September 21, 1987

Single

K

channels

were incorporated either

side

conductance that

the

( I ),

The regulation is

a

in

the

of calcium transport

complex

physiological

known t h a t

experimental

Ryanodine

applied to

Ca

These results

is limited only membrane

of

nor

the

suggest to

the

sarcoplasmic

by the sarcoplasmic

reticulum

process

different

ion

proteins play important

influence

reticulum

© 1987 Academic Press, Inc.

movement accross

( * )

present

such as ionic gradients,

regulatory

is

membranes.

site of action of ryanodine

reticulum

It

into artificial

muscle sarcoplasmic

properties of those channels.

channels

factors

skeletal

of the membrane did not affect the gating

calcium

(SR)

from

accumulation conditions,

Ca-ATPase

and interdependent

of

the

SR

decamethonium

(2). and

and

and

roles.

interfering

l i k e decamethonium

by

which

channels,

substances capable the SR,

in

with

K

valinomycin,

Under

defined

valinomycin

have

ADDRESS FOR CORRESPONDENCE.

0006-291X/87 $1.50 1137

Copyright © 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.

Vol. 148, No. 3, 1987

opposite

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

effects,

-vesicles

the

first

t o accumulate Ca.

one decreasing t h e a b i l i t y Decamethonium i s a l s o

b l o c k e r o f K - s e l e c t i v e SR channels (3). present i n t h e SR at a f a i r l y

of

an

SR

effective

Since these channels are

high d e n s i t y (4),

the

involvement

o f K channels i n the modulation o f Ca t r a n s p o r t accross t h e SR i s a

plausible

hypothesis.

In

this

sense,

by

changing

the

d i s t r i b u t i o n o f charges

across the SR membrane the t r a n s p o r t

Ca

Recently,

could

be a f f e c t e d .

important e x p e r i m e n t a l support. Ca

movements

t h a t h y p o t h e s i s has

i n muscle c l e a r l y depend on

is

transport general,

a

neutral

alkaloid

whether

closed

which

vesicles.

increases

the

Ca

sarcoplasmic

state

(5).

interferes

both i n s k e l e t a l and c a r d i a c muscle SR ryanodine

received

Fink and Stephenson showed t h a t

r e t i c u l u m K channels are i n t h e open o r

Ryanodine

of

content

with

Ca

membranes.

In

inside

Regardless o f t h e p r e p a r a t i o n t h e a c t i o n o f

the

SR

ryanodine

has not been u n r a v e l l e d but t h e Ca pump i s not a l i k e l y c a n d i d a t e since

the

effects

conditions prompted

ryanodine

are

still

where the pump i s i n h i b i t e d (6). us

channels.

of

observed Those

under

observations

t o analyse t h e i n f l u e n c e o f ryanodine on t h e

SR

K

We s t u d i e d t h e e f f e c t s o f t h i s drug on t h e g a t i n g and

conductance

properties

o f s i n g l e K channels

from

SR

vesicles

inserted i n t o planar l i p i d b i l a y e r s .

MATERIAL AND METHODS P l a n a r b i l a y e r s were formed by t h e p a i n t i n g technique. (30-50

mg/ml

polystirene

in

decane

partition

Asolectin

) was spread o n t o a 0.2mm h o l e

separating

two

different

on

a

compartments

c o n t a i n i n g t h e same s o l u t i o n (potassium s u l f a t e - lOOmM, HEPES lOmM,

pH

- 7.4).

were

added

SR v e s i c l e s prepared by c o n v e n t i o n a l

t o o n l y one s i d e ( c i s - s i d e ) which i s a l s o

1138

methods

the

side

Vol. 148, No. 3, 1987

where

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

the

(trans

membrane p o t e n t i a l was c o n t r o l l e d .

side)

The

was maintained at v i r t u a l ground

by

other a

side

home-made

c u r r e n t - v o l t a g e converter. The experiments were performed at room temperature (22 -24 'C). untill a

A f t e r a d d i t i o n o f SR v e s i c l e s , we waited

a v e s i c l e i n c o r p o r a t i o n was observed. This was detected by

s u d d e n j u m p in transmembrane conductance followed by

openings

and

c l o s i n g events o f a s i n g l e channel.

(>40

minutes)

Prof.

Sutko)

c o n t r o l recording, at

After a

ryanodine (a kind

gift

d i f f e r e n t concentrations (0.5nM t o

obtained.

histograms rate

With

help of a

deduced.

microcomputer,

dwell

for opening and closing of the channel

Also,

conductance

voltage relationships

measurements

were

from

recording

for both open and closed events were constructed.

constants

current

the

long

50uM) was

added t o e i t h e r the c i s o r t r a n s side and another long

was

defined

time The

were

thus

obtained

from

for the channel.

RESULTS AND DISCUSSION Figure

shows

1

side.

recording affected ryanodine

This

shows by

the

was

characteristics. added

to

in

(A) and i n the presence o f 4.2uM ryanodine

conditions the c i s

a t y p i c a l s i n g l e channel recording

representative

that

the conductance

drug.

of a long

of this

channel

Even at higher concentrations

ineffective The

segment

in

altering

single

control added t o

duration is

not

(45

uM)

channel

same conclusion holds when ryanodine

the t r a n s side and when present in both sides

of

was the

membrane. Figure IC shows a complete c u r r e n t - v o l t a g e r e l a t i o n s h i p in lack

the presence and absence o f ryanodine.

We conclude t h a t

the

o f e f f e c t o f ryanodine does not depend on the transmembrane

voltage.

The same r e s u l t s were a l s o obtained when ryanodine at a

lower concentration (0.5nM) was used.

1139

Vol. 148, No. 3, 1987

A

B IO C H E M IC AAND L BIOPHYSICAL RESEARCH COMMUNICATIONS

-u LP JLF L JS JL

I

,.J

L

j

12-

C i/pA

I -60

I

I

I

I

I

6o V/mV

-12 FIGURE

1 .

Single channel recordings in control

(A) and in

the

presence o f ryanodine (4.2uM) on the c i s side (B).C is a c u r r e n t voltage

relationship

from

a different

experiment

conditions ( c i r c l e s ) and in the presence o f 50

in

control

uM ryanodine

added t o both sides o f the membrane ( t r i a n g l e s ) . A l l the points can

of

be well f i t t e d by the same s t r a i g h t l i n e with a

200

pS.

Calibration

bars

:

9.5pA

conductance

(vertical)

and

19s

(horizontal).

Figure events

2 shows cumulative histograms of open in

conditions

presence the

SR

and

absence

channel can be

between two different states

kl\

closed \

open

k_l

1140

of

(A) and closed

ryanodine.

represented

as

In

(B)

control

oscillating

Vol. 1 4 8 , No. 3, 1 9 8 7

BIOCHEMICAL A N D BIOPHYSICAL RESEARCH C O M M U N I C A T I O N S

CLOSED

OPEN

1.0

1.0 =~x

o 0.9 0

¸

0.9

&x 0.6'

0.7

6

0.8

°ix Do&

6

EIx& oXX x

xoa

0.7-

O

X 0 X~

x

0.6"

84 aoA

GO

0.5

o

0.6

A X

o 0

..=

~ 0.5"

A OOX

Ill

oO x

0.4-

O

0.4

x

0.3,

x

0.3

o~ 0.2"

0.2

0.1

0.1

0.0

0.0

2

4

6

8

10 12 14 16 18 20 2 2 2 4 2 6

~m~m 0

A

~

rrn

O

g

I 0

2

4

6

8

10 12 14 16 18 2 0 2 2 2 4 26 2 8 3 0

Time (sec) FIGURE

2 .

Normalized cumulative event histograms f o r s i n g l e

SR-channel

at

conditions

(

diamonds

the

30mV.

A- Open time

squares ) ; ,

triangles

Time (sec)

) ;

0.174

by

4.2uM ryanodine i n both sides o f the

time

points

side

4.2 uM ryanodine i n t r a n s and 50 uM ryanodine The experimental p o i n t s can

corresponding t o a mean open time distribution.

are also well f i t t e d

constant

control

( kI

)

(

membrane

a s i n g l e exponential w i t h a r a t e constant

sec - 1 ,

Closed

in

4.2 uM ryanodine i n the t r a n s

c i s side ( crosses ).

fitted

distribution

K

of

be

( in

well

( k_l )

of

5.8s.

B-

Symbols are the same as i n

A.

by a s i n g l e exponential with a

The rate

o f 0.114 sec-lcorresponding t o a mean closed

time o f 8.7s. On average 70 events were computed f o r each channel s t a t e i n a defined experimental c o n d i t i o n .

The rate constants measured from the plots in figure 2 were found to be :

kI

=0.114

were

not

trans

s i d e s and a r e

sec-land

k_1

=0.174

sec -I . These values

changed when r y a n o d i n e was p r e s e n t i n good agreement w i t h

1141

in the

previous

cis

and/or

measurements

Vol. 14.8, No. 3, 1987

(7).

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

The

proposed

presence

of

simple k i n e t i c scheme i s s t i l l

ryanodine

and the

v a l i d in

the

finding

the

p r o b a b i l i t i e s of

channel in e i t h e r the open or shut c o n f i g u r a t i o n are not a l t e r e d , i e , 40 and 60%, r e s p e c t i v e l y at 30mV. We conclude

t h a t ryanodine does not a f f e c t the conductance

nor

the k i n e t i c p r o p e r t i e s of the SR K channel thus l i m i t i n g the s i t e of

its

action

in

the

SR t o

calcium

channels

or

to

some

intermediary molecule involved in the c o n t r o l of Ca accumulation by the SR.

ACKNOWLEDGMENTS: This work was supported by grants from CNPq, FINEP and CEPG. We are g r a t e f u l t o Mr. Ricardo Pires Coelho f o r h i s generous assistance.

REFERENCES 1.Suarez-Isla BF, calcium

C Orozco,

PF H e l l e r and JP Froehlich.

Single

channels in n a t i v e sarcoplasmic reticulum membranes from

s k e l e t a l muscles. Proceedings of the National Academy o f Sciences (USA) 83, 7741-7745, 1986. 2_.

Hall,PJ,

1984.

Besch HR J r . ,

Monovalent

Henry BG, Anderson T and Maddock SW.

c a t i o n channel r e g u l a t i o n of calcium t r a n s p o r t

by sarcoplasmic reticulum v e s i c l e s . Pharmacologist 26: 128. 3. Coronado R and C M i l l e r . 1982. Conduction and block by organic cations

in

a K s e l e c t i v e channel

incorporated

intoo

planar

from

sarcoplasmic

phospholipid

bilayers.

reticulum

Journal

of

General Physiology 79: 529-547. 4.

Garcia

cation

AM and C M i l l e r .

fluxes

in

isolated

1 9 8 4 . Channel -mediated monovalent sarcoplasmic

Journal o f General Physiology 83:819-839.

1142

reticulum

vesicles.

Vol. 148, No. 3, 1987

5_. by

B I O C H E M I C AAND L BIOPHYSICAL RESEARCH COMMUNICATIONS

Fink RHA and DG Stephenson. Ca-movements in muscle modulated the

state

of

K-channels

in

the

sarcoplasmic reticulum

membranes. Pflugers Archives 409: 374-380, 1987. 6. Besch HR Jr.

1985. Effects of ryanodine on cardiac s u b c e l l u l a r

membrane f r a c t i o n s . Federation Proceedings 44: 2960-2963. 7~. Labarca P, kinetic from

the

R Coronado and C M i l l e r .

1980. Thermodynamicand

studies of the gating behavior of a K-selective sarcoplasmic reticulum membrane. Journal

Physiology 76:397-424.

1143

of

channel General