C o , o q u e S.B.C.F. , R o u e n 1991

ATYPICAL CLEAVAGE OF THE GAP JUNCTIONS IMPLICATED IN TRIADS IN A BIOLUHINESCENT

S¥STEM.Gis~le NICOLAS, L a b o r a t o i r e de Technologie Appliqu~e ~ La Microscopie E l e c t r o n i q u e , CNRS, 105 Bd

Raspa i l , 75000 Pa r i s (F rance ) .

Gap j u n c t i o n s were found i n c l u d e d in t r i a d s b a s i c a l l y s i m i l a r to t hose o f the s t r i a t e d muscle

f i b r e s , in the b io luminescen t e p i t h e l i a l c e l l s o f a scale-worm, Harmothoe l u n u l a t a . Under e L e c t r i c

s t i m u l a t i o n muscle f i b r e s r espond by c o n t r a c t i o n , t h e s e c e l l s by emiss ion of f l a s h e s of l i g h t . The

i n t r a c e L L u l a r sources o f l i g h t a r e p a r a c r y s t a l s o f endoplasmic r e t i cu lum named photosomes. To be abble

to f l a s h , they have to be coup led wi th the plasma membrane by t r i a d i c t r a n s i e n t j u n c t i o n s which

d i f f e r e n t i a t e and d e d i f f e r e n t i a t e w i t h i n m i l l i s e c o n d s f rom the su r round ing ER. UnLike what e x i s t s in

muscLe, , o f o o t p rocesses a r e found in these t r i a d s between the plasma membrane and the membrane of the

ER. But gap j u n c t i o n s l i n k the i n f o l d i n g s o f the plasma membrane a t the l e v e l of the t r i a d s . When

s t u d i e d by the f r e e z e - f r a c t u r e t e c h n i q u e , t he se gap j u n c t i o n s show a c h a r a c t e r i s t i c c leavage d i f f e r e n t

from the c leavage u s u a l l y observed in most gap j u n c t i o n s as wel l as in the gap j u n c t i o n s s i t u a t e d

o u t s i d e the pho togen ic complexes o f t he se c e l l s . I n s t e a d o~ s p l i t t i n g one of the two plasma membranes a t

the l e v e l o f the gap j u n c t i o n , the f r a c t u r e p lane passes through the u n d e r l y i n g ER membrane.

S ince the luminous r e a c t i o n p ropaga tes to the ne ighbour ing photosomes under repea ted s t i m u l a t i o n

wi thou t t a k i n g the c e l t | i m l t s i n t o account , the h y p o t h e s i s t ha t these unique gap j u n c t i o n s can be

Involved in the propagation of the exci tat ion can be considered.

IMMUNOLOGICAL CHARACTERIZATION OF RECONSTITUTED LIVER GAP JUNCTION PROTEINS INTO LIPOSOMES AND INCORPORATION INTO PLANAR BILAVER. Frangolse MAZET (a), Jean-Luc MAZET (a), Daniel GROS (b) and Th~r~se JARRY (b). (a) Laboratoire de Physiologie Compar~e, (CNRS URA1121), Bat. 443, Universit~ Paris-Sud, 91405-ORSA¥ (France) and (b} Laboratoire de Biologic de la Diff6rentiation Cellulaire (CNRS URA179), Facult6 des Sciences de Lumigny, 13288-MARSEILLE (France).

Isolated rat liver gap junctions were solubilized by a mixture of digitonin and octylglucoslde (1) and reincorporated after dialysis into liposomes. Observation of these liposomes by freeze-fracture have demonstrated the restoration of gap junctions like-structures (2).

The presence of the major protein of the gap junctions into the reconstituted liposome~ was shown by immunoblotting. Samples of liposomes were fractionated by SDS-PAGE and transferred onto nitrocellulose membrane. The membrane was incubated with an affinity purified antibody directed to the 20 peptides located at the C-terminus of the rat liver connexin 32. The primary 8ntibody was revealed with a peroxidase conjugated secondary antibody.

The proteoliposomes were incorpnrated into a planar bilayer preformed with an asolectin/cholesterol mixture. Liposomes fusion occurs spontaneously and the transmembrane currents appear as single channels wlth potential- Indeppndent conductsace, ranglD 9 from 800 to 2000 pS in the presence of 1H Natl. The channels, open at low potential, tend to close as the potential is i n c r e a s e d i n e i t h e r d i r e c t i o n .

(1) Maser, F. and BJat~mann A.p C. R. Acad. Sc i . P a r i s , 307, 679-684 (1988) (2) Mazet F . , and ~azet, J . - L . , Exper. C e l l . Res., 188, 312-315 (1990)

RAPID KINETICS OF THE CALCIUM-INDEPENDENT BLOCK OF GAP-3UNCTIONAL

CONDUCTANCE INDUCED BY HEPTANOL IN NEONATAL RAT VENTRICULAR CELLS. Bruno BASTIDE, 3ean

Claude HERVE, Laurent CRONIER and 3ean DELEZE. Laboratoire de Physioiogie Cellulaire, URA CNRS

n°290, 86022 Poitiers, France.

Cell-to-cell communication in pairs of neonatal rat ventricular myocytes maintained in culture for

two days is investigated by means of a double whole-cell patch clamp technique. Gap junctional conductance

is measured continuously by recording the current necessary to maintain one cell at a holding potential (-80

mV) while the other cell is clamped to about -90 mV for 4 to ~ minutes. Electrical uncoupling by a stream of

heptanol-containing (3 rnM) Tyrode solution directed on the cell pair is complete within 2-3 seconds, and

recovery to a steady level (90% to 100% of control) takes about 30 seconds, lntracellular calcium detected

by photometry of the fluorescent emission of fluo-3 (Minta et ai., 1989) does not increase during heptanol

exposure, and the uncoupling effect is not inhibited by the K+-channel blocker 4-aminopyridine as it is in

the crayfish septate axons (Peracchia, 1991). These resull:s are consistent with a direct action of heptanol on

the jt,nctional channel or on its lipid environment.

Mi,ta A., Kao P.P.Y. and Tsien R.Y., 3. Biol. Chem., 264, 8171-8178 (1989).

I~eracc'hia C., 3. Membrane Biol. 121, 67-78, (1991).

7a