current CpAD
80
-40
40
V p C m V ]— 85 —
f o r th e means a re quoted i * th e standard d e v ia tio n . The slope conductance o f th e channels shown in f ig u r e 3 .6 was 57pS a t Vp=+10mV. The mean slope conductance a t +10mV, from s ix e xperim ents was 5 3.8db3.3p8 and th e mean channel a m p litud e a t t h is p o te n tia l was 2 .5 io . 3 p A . The term s 54pS and 14pS channels s h a ll be used to r e fe r to th e la rg e and s m a ll channels reco rd ed in c e ll-a tta c h e d patches o f th e Cl neurone.
The f a c t th a t th e I-V re la tio n s h ip s o f both sm a ll and la rg e u n ita r y outw ard c u rre n ts agreed w e ll w ith th e th e o r e tic a l r e la tio n s h ip , a c c o rd in g to th e GHK e q u a tio n f o r a K c u rre n t, suggested th a t th e charge c a rry in g sp e cie s o f these u n ita ry outw ard c u rre n ts was io n s . The GHK e q u a tio n i s based on th e co n sta n t f i e l d th e o ry f o r th e r e c t if ic a t i o n o f c u rre n t flo w th rou g h c e ll membranes (H odgkin and K atz, 1949) . In fig u r e s 3.5 and 3 .6 i t can be seen th a t th e I-V r e la tio n s h ip s o f both typ e s o f channels a re n o n -lin e a r. R eversal o f c u rre n t flo w th ro u g h these channels was never observed in c e ll-a tta c h e d p a tch e s. No u n ita r y c u rre n ts were observed a t h y p e rp o la ris e d patch p o te n tia ls .
The absence o f u n ita ry outw ard c u rre n ts a t h y p e rp o la ris e d p o te n tia ls c o u ld be due to a vo lta g e -d e p e n d e n t g a tin g o f th e io n ch an n els. A lt e r n a tiv e ly , i t co uld be e x p la in e d by a r e c t if ic a t i o n o f th e c u rre n t flo w through th e channels, such as th e r e c t if ic a t i o n d e s c rib e d by th e co n sta n t f i e l d th e o ry . T h is p re d ic ts th a t th e a m p litu d e o f th e K c u rre n t w i l l be much s m a lle r in th e h ype r p o la ris e d d ir e c t io n th a n i n th e d e p o la ris e d d ir e c tio n . S m all u n ita ry c u rre n ts may th e n be lo s t amongst th e background n o is e , th e re b y p re v e n tin g t h e ir
-
86
-d e te c tio n . U n ita ry c u rre n ts were g e n e ra lly n o t observed in c e ll-a tta c h e d patches a t p o te n tia ls le s s d e p o la ris e d th a n -30raV. The agreement o f th e u n ita ry c u rre n t data w ith th e c o n s ta n t f i e l d th e o ry f o r a K c u rre n t gave in d ir e c t evidence th a t io n s c a r r ie d th e charge throu g h these channels. T h is th e o ry was te s te d d ir e c t ly by exam ining th e dependence o f th e u n ita ry c u rre n ts on th e K c o n c e n tra tio n .
3 .3 INCREASING THE K CONCENTRATION OF THE PIPETTE
R ecordings were made from c e ll-a tta c h e d patches o f th e Cl neurone, u sin g a h ig h K s o lu tio n in th e patch p ip e tte . T h is s o lu tio n co n ta in e d 35mM K in s te a d o f th e usual 5mM K, and was made by s u b s titu tin g 30mM N a d w ith K C l. R a is in g th e K c o n c e n tra tio n on th e e x te rn a l s u rfa c e o f th e membrane w ould be expected to reduce th e d r iv in g fo rc e f o r the outw ard movement o f io n s , th e re b y re d u c in g a c u rre n t w hich was due to th e flo w o f io n s . In these expe rim en ts th e c e ll was bathed in normal s o lu tio n , so a v o id in g a d e p o la ris in g e ff e c t o f th e h i ^ K s o lu tio n . R e s u lts frcm these e xpe rim en ts suggested th a t th e u n ita ry c u rre n ts observed were c a rrie d by K*^ io n s .
Outward channel c u rre n ts , reco rd ed w ith h ig h K s o lu tio n in th e patch p ip e tte , were s m a lle r th a n c u rre n ts n o rm a lly re co rd e d in c o n tro l c o n d itio n s . F ig u re 3 .7 shows some u n ita ry outw ard c u rre n ts re corded frcan a c e ll- a tta c h e d p a tch , w ith 35mM io n s i n th e patch p ip e tte . T h is re c o rd in g was made a t a patch p o te n tia l o f +10mV. Three d if fe r e n t a m p litu d e s o f s in g le channel c u rre n ts appeared t o be p re se n t i n t h is re c o rd . However, i t i s p o s s ib le th a t th e la r g e s t a m p litu d e observed here was due to th e sim u lta ne o us opening o f th e o th e r ch an n els. The
F ig u re 3 .7
S in g le channel outw ard c u rre n ts recorded from a c e ll-a tta c h e d p atch o f th e Cl neurone, w ith a h ig h K s o lu tio n (35mM) in th e patch p ip e tte . The appearance o f th re e d if fe r e n t a m p litu d e s in t h is re c o rd i s marked on th e r ig h t o f th e tra c e s . Vp=+10mV. Records were f i l t e r e d a t 2.5KHZ.
]
2p A
— 87 —
a m p litu d e s o f s in g le d ia n n e l c u rre n ts observed under these c o n d itio n s w ere 1 .2 5 , 0 ,7 5 and 0.35pA. These a m p litu d e s compare w ith v a lu e s o f 2 .5 and 0.73pA, f o r th e 54pS and l4pS channels r e s p e c tiv e ly , w ith norm al s o lu tio n in th e patch p ip e tte . However, due to th é n a tu re o f th e e xp e rim e n t, i t was n o t p o s s ib le to o b ta in c o n tro l re c o rd in g s frcxn th e same patch s in c e th e re was no f a c i l i t y f o r p e rfu s in g th e patch p ip e tte . The p e rm e a b ility v a lu e s o f th e u n ita ry c u rre n t re co rd e d in h ig h K were lo w e r than in c o n tro l c o n d itio n s . V alues o f Pp. were 1 .4 x1 0 "1 3 , 8.6x10“ *’ ^ and 4.0x10” *’ ^cra.^s“ ^ f o r th e 1 .2 5 , 0,75 and 0.35pA c u rre n ts r e s p e c tiv e ly . No I-V r e la tio n s h ip s f o r u n ita ry c u rre n ts w ith h i ^ K in th e patch p ip e tte were o b ta in e d . The presence o f d if f e r e n t ty p e s o f channels w ith s m a lle r u n ita r y c u rre n ts w ith h ig h K in th e patch p ip e tte , suggested th a t th e outw ard channel c u rre n ts observed on c e ll- a tta c h e d patches were c a rrie d by io n s . More c o n c lu s iv e evidence f o r t h is was o b ta in e d from e xp e rim e n ts on is o la te d o u t s id e -o u t membrane patches.
3 .4 PERFUSED OUTSIDE-OUT PATCHES
E xperim ents on is o la te d o u ts id e -o u t membrane patches a llo w e d th e o u te r s u rfa ce o f th e patch o f membrane to be p e rfu s e d w ith d if f e r e n t s o lu tio n s . P e rfu s io n o f is o la te d patches was a chieve d u s in g th e m ic ro p e rfu s io n chamber d e s c rib e d on page 75 o f th e methods s e c tio n . The p ip e tte c o n ta in in g th e is o la te d patch o f man b ra ne c o u ld be tra n s fe rre d to t h i s chamber to enable a ra p id exchange o f s o lu tio n in th e area o f th e p a tch . Out s i d e -out patches were form ed w ith a 96mM K s o lu tio n in th e p atch p ip e tte ( s o lu tio n 2 , Table 2 . 1 ) , R ecordings frcm is o la te d o u ts id e -o u t patches were made w ith norm al 5mM K s o lu tio n on
88
th e e x te rn a l su rfa ce or w ith 96mM K s o lu tio n o r 44mM K s o lu tio n ( s o lu tio n s 2 and 3> ta b le 2 .1 ) p e rfu s in g th e patch o f membrane. The 44mM K s o lu tio n was made by s u b s titu tio n w ith sucrose to m a in ta in th e o s m o la rity . F ig u re 3 .8 shows examples o f I-V re la tio n s h ip s f o r th e la rg e u n ita ry outward c u rre n ts w ith 5 and 96mM K o r 44 and 96mM K on th e e x te rn a l s u rfa c e . In gen e ra l th e la r g e r channels were ve ry a c tiv e in is o la te d patches but th e s m a lle r channels were n o t observed, o r were obscured by th e la r g e r channels.
A ttem pts to o b ta in re c o rd in g s from th e same patch in a l l th re e s o lu tio n s were not su cce ssfu l and th e re c o rd s shown a re from patches p e rfu se d w ith two o f th e s o lu tio n s . W ith 96mM K on th e in n e r su rfa ce and 5mM K on th e o u te r su rfa ce (c o n d itio n s re se m b lin g those in a c e ll-a tta c h e d p a tc h ), th e I-V curve o f th e channels d is p la y e d pronounced r e c t if ic a t io n . Under these c o n d itio n s no c le a r re v e rs a l o f th e s in g le channel c u rre n t was observed ( f ig u r e 3 .8 ,B ). As w ith re c o rd in g s from c e ll-a tta c h e d p a tch e s, th e I-V r e la tio n s h ip o f th e channels under these c o n d itio n s co u ld be f i t t e d by th e th e o r e tic a l r e la tio n s h ip o f th e GHK e q u a tio n f o r a K c u rre n t, a llo w in g th e re v e rs a l p o te n tia l to be e x tra p o la te d frcxn t h is f i t . In sym m etrica l 96/96mM K s o lu tio n th e I-V r e la tio n s h ip was lin e a r , re v e rs in g a t OmV. The conductance va lu e o f th e channels in sym m etrical s o lu tio n was between 130 and 170pS ( fig u r e 3 .8 ,A and B ). In 96/44mM K s o lu tio n s th e I-V curve d is p la y e d s lig h t r e c t if ic a t i o n . The mean re v e rs a l p o te n tia l o f th e u n ita ry c u rre n ts under these c o n d itio n s was -1 9 .6 ± 1.OmV in fo u r e xp e rim e n ts. In th e example in f ig u r e 3 .8 ,B th e conductance va lu e a t +10mV was about 125pS. In a l l th re e s o lu tio n s th e I-V r e la tio n s h ip s c o u ld be f i t t e d w e ll by th e GHK e q u a tio n and, th e re fo re th e re v e rs a l