Rapid activation and desensitization by glutamate of excitatory, cation-selective channels in locust muscle

'~emos~wm¢ L ¢ t t ¢ ~ 8811988) 33 38 Llse',~er St.mnt~hc Publishers Ireland Ltd

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Rapid activatmn and desensmzatlon by glutamate of excitatory, cauon-selective channels in locust muscle Josef Dudel ~, Chnstmn Franke ~, Hanns Hatt ~, Robert L Ramsey e and P e t e r N R U s h e r w o o d -~ :Pin stolo~,ts~ hes In ~ltlttl ch r Te~ hnts~ ht n ( 'ntvers~tat Mun~ h~ n M u n u h ( F R G ~ and 2D~ pa~ tm~ nl o/ Zoolo~l

Untver~ttt o / N o t t m g h a m

~Votlm~ham t U h )

(Rccel~ed 11 Januar'¢ 1988 Accepted 1 Pebruary 1988) /x~ ~ ~otd~

L o c u s t muscle Outside-out patch Lxc~tator', membrane channel ~-Glutamatt. punt. Rap~d desenslllZatlon

Outside-out patdles ol membrane weru excised lrom uxtcnsor tibiae muscles of locusts l-(Jlutamalt. or its agom~ts ~ere apphed to such patcht.s m shorl pulses by means ot a llqmd hlamunt sWllch ( atlonselectp, e excltator,¢ channels ~ere at.tl~ated b', qulsqualate t-glutamate and aspartatc (m dot.teasing order ot effcctlxlt'~) but not bv ibotenate kamate ,~-meth~A-D-aspartate and gl,cclnc At high agonlM conucntrat~ons channul autl'~atlon reached a peak within 1 ms Two kmuhc t,~pes ot channels ha~c been ~dent~hod L-channels V,lth on a,.eragu relatp, dx long and S-channels V,lth short openings Both txpes ol d~annul openings shov, ud surpnslngl~ h,gh rates ofdesunslUzaUon channel actp, fl,~ dedmmg alt~.r the m~tlal surge to zero with trine constants of about 25 and ~ ms respecU',cl,~ The L-channels exh~Nt open ltmcs clo~c to those ol channels recorded m Mf2-seal stud~cs The S-channel has not bu~.n reported pre'~umsl',

In p a t c h e s o f e x t r a j u n c t l o n a l

muscle membrane

o f l o c u s t m u s c l e to w h i c h t h e

t r a n s m i t t e r L - g l u t a m a t e ts a p p l i e d c o n t i n u o u s l y , e x c i t a t o r y , c a t l o n - s e l e c t l ~ e c h a n n e l o p e n i n g s a r e r a r e l y o b s e r v e d [10, 11], u n l e s s c o n c a n a v a h n Concana~ahn

A is p r e s e n t [2, 9

13 14]

A ts k n o w n t o p r e v e n t d e s e n s i t i z a t i o n [17], b u t m a y h a ~ e f u r t h e r el-

fects o n c h a n n e l k i n e t i c s T h e r e f o r e , tt w a s t r i e d t o r e c o r d c h a n n e l o p e m n g s sence of lectm, before desensmzatton,

applying t-glutamate

m short pulses

in a b This

m o d e o f r e c o r d i n g is c l o s e t o t h e s t t u a t l o n m n a t u r a l s y n a p t t c t r a n s m i s s i o n a n d m a y l e a d to a b e t t e r u n d e r s t a n d i n g

of the generation of the excitatory postsynapttc cur-

rent Metathoraclc

extensor ttblae muscles from adult locusts (S~htwo~et~a

etevaJm)

w e r e s u p e r f u s e d w i t h 0 5 2 m g / m l c o l l a g e n a s e ( S i g m a I A ) m o r d e r to r e m o ~ e c o n nectl,Je t i s s u e [7]

Superfusmg

t h e m u s c l e w i t h p h y s i o l o g i c a l s a l i n e m a b s e n c e of

e n z y m e , o u t s i d e - o u t p a t c h e s w e r e o b t a i n e d w i t h f i r e - p o h s h e d p t p e t t e s c o a t e d vclth sylgard

Pulses of L-glutamate or related compounds

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were dtrected at such patches

! Dudel Phsslologlsches Instltut der TUM, Bmd~.rstemer Strasse 29 D-8000 Mum.hen

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using the hqmd filament switch [8], and the resulting channel actl',lty wa~ ret.orded on video-tape and analyzed off-hne by computer [5, 7] The locust sahne m the superfusion contained 180 mM Na('l 10 mM KC1, 2 mM CaCI2 and 10 mM Frls-m,tleatc buffer, the pH wa~ adjusted to 6 8; with N a O H [13] The temperature was held ,it 23 C The patch pipettes contained an mtracellular' solution with 150 mM potassium proplonate, 5 m M sodmm proplonate, 2 mM MgCI2 I mM C a C 6 and 10 mM

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FJg 1 Recordings and evaluations of single channel currents from an outside-out patch of membrane stud~ed over a 2 h period Channels exhibmng long openings ArD~, results of 200 ms apphcatlons ol 10 4 M L-glutamate A_, D2, ofapphcat~ons of 10 ~ M L-glutamate A t and Az single traces, cahbratlon - 50 pA Bj averaged currents ehclted by 50 glutamate pulses, cahbratlon - 10 pA B2 averaged currents for 51 glutamate pulses, cahbraUon - 5 0 pA C~ distribution of open times, blnwldth 50 `us, loganthmtt. scale of events,'bm Distribution fitted by two exponentials with time constants r~ and r2 respectively D~ and D2 distributions ol current amplitudes held for at least 100 ,us [16] Bmwldth 0 I pA hnear scale ot events/bin Glutamate pulses repeated every 5 s Patch pipette potentml - 7 5 mV Recordings dsgltlzed at 25 kHz displayed with 8-pole Bessel filter at 5 kHz

E G T A to achmve a buffered Ca 2+ concentration o f 10 s M, and 10 m M Trls-maleate with pH adjusted to 7 2 with K O H , reaching a final p o t a s s m m concentration of 190 m M [7] When L-glutamate or its agomsts were applied m short pulses openings ol e\~.~tatory glutamate receptor (GluR) coupled catiomc channels (Figs I and 2) ,sere triggered on almost all patches examined Low-level activation (hke e g m l~g 1A) `sa~ eh~.lted b 3 pulses o f 50 ILM L-glutamate 10 llM qmsqualate and 10 mM aspartatc N o activation ,sas observed with I m M ibotenate or with 10 mM kamatc ,~-mcthbl1)-aspa! tate ( N M D A ) and glycme The channels thus correspond to the qmsqualat~t.x pe ot glutamate receptors found m vertebrate C N S [20] and exhibit thc same ph,nmacologlcal propertms as the extra junctional D-receptors o f locust leg muscle [1 1~)] i -Glutamate also e\cltes qmsqualate-type G l u R in crayfish muscle but act1\ ~t\ ol the crayfish G l u R channel is blocked m : e r o extracellular Ca 2+ [12], ,shereas the e\t.ltatol~ G l u R channel ot locust muscle ,sas found to be unaffected b~ Ca -~ ~emoxal (see also ref 15) T,so kinetic types of excitatory G l u R channel (L and S) were found ,slm.h ale c h a r a c t e n : e d m Figs 1 and 2, respectively Both types have the same single chanm.1 currcnt amphtude, and have reversal potentials near 0 mV In the patch ~llust~atcd m Fig 1 pulses o f 10 4 M glutamate (Fig IA~) ehclted a fe~ openmgs ol L-channels ~mme&ateh dur|ng agomst apphcatlon, although about 1 4 o f the glutamate pulses lh~lcd to ehclt openmgs Superpositions o f two channel opemngs ,serc rarel 5 sct.n m this p a t c h , s l t h 10 4 M glutamate, but 10 ~ M g l u t a m a t e ( F i g 1Ae) a c t l x a t c d m a n ' : mmall} S u p e r p o s m o n s of channels are manifest m the distributions ol current amphtudes obtained during the glutamate pulses (Fig 1DI 2) With 10 4 M glutamate and a patch pipette potentml o f 75 mV two levels, 0 and 15 pA ~erc pre\ alent and 30 pA a m p h t u d e were rare, while at 10 ~ M glutamate s u p e r p o s m o n s ol up to 7 unit currents each o f - 15 pA ,sele &scermble The umt current coHesponds to a channel conductance o f 150 pS (of ref 1S) If the fev~ superposltlons at 1() 4 M glutamate ,ire excluded channel open t~mes can be estimated The distribution o f channel open times m Fig 1 ( i ,shlch is for I() a M glutamate shoves tyro t.omponents eqtuvalent to mean open times of about 0 2 and 2 ms and possibly a longcr c o m p o n e n t Exaluatmn o f mterxals between openings would ymld equl\ocal results since man~ channels ~ere present m the patch, and re-openings o f one d~annel cannot be d~stmgmshed from openings o f other channels The tmle course o f the response to L-glutamate can be evaluated from the a \ e r a g e channel currents ~llustrated m F~g 1B~ _~ W~th l0 ~ M glutamate, the rise time (10 90% o f peak current) o f the averaged current was 0 6 ms W~th 10 4 M glutamate ~t was 2 7 ms The axerage channel currents declined `s~th time constants ol about 25 ms v~h~ch appeared to be the same at 10 a and 10 ~ M glutamate At the end of the 200 ms glutamate pulse channel openings `sere xer~r rare An interesting cxampie ~s shown m the third trace (Fig IA~) in which a burst" o f channel opemngs cont> nued alter remox al o f glutamate It seems that b o u n d glutamate can support repeated openings (compare ref 8) Data obtained from a patch containing the second type o f channel, the S-channel

36

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t7 are illustrated m Fig 2 T w e n t y millisecond pulses of 10 ~ M g l u t a m a t e ehclted bursts of c h a n n e l openings which subsided very rapidly The single openings m Fig 2C were all briefer t h a n 0 5 ms The histogram of c h a n n e l open times illustrated m Fig 2C was fitted by a single e x p o n e n t i a l This had a time c o n s t a n t of a b o u t 50 lls which is at the hmlt of the recording resolution The short openings ymlded httle ,l~erage current (Fig 2B) c o r r e s p o n d i n g at peak to 0 03 open channels The ,l~eraged c u r r e n t declined to zero within less than 15 ms with a decay time c o n s t a n t of a b o u t 3 ms A p p l i c a t i o n of 10 4 M g l u t a m a t e to this patch elicited even less actlwt,,' (not illustrated) than 10 x M glutamate, b u t the averaged c u r r e n t still declined with a tmle c o n s t a n t of a b o u t 3 ms Interestingly, m some patches L-openings were observed m m a l l y with l0 4 M g l u t a m a t e (Fig 1) b u t within e g 30 m m groups of shorter openings appeared After I h the o p e n i n g s were all ot the brief type The glutamate-activated c h a n n e l s described here clearl~y c o r r e s p o n d to the cat~onselective e x t r a j u n c t l o n a l D - G l u R c h a n n e l s studmd extensively m the locust m presence of c o n c a n a v a h n A using cell-attached, M.Q-seal patches [9 11] lnterestmgl.~, G~Q-seal excised patches c a n n o t be p r o d u c e d m presence of the lectln, but lectm apphed later to excised patches inhibits desensitization The S-channel type has not been reported previously Similar c h a n n e l s with rapid a c t l v a t m n a n d m a c t l x a t l o n kinetics hax e been f o u n d m outside-out patches excised from crayfish muscle [6, 8] While activation of G l u R within I ms was to be expected in a n a l o g ) to the rise time ol synaptlc currents, the desensitization of th~s system w~th time c o n s t a n t s of "~ and 25 ms is a surprise G l u t a m a t e applied to a muscle fiber elicits a c u r r e n t which ~s m a c t ~ a t e d v~th a p p r o x i m a t e time c o n s t a n t s of 0 5 1 s the rate of d e s e n s m z , m o n increasing for larger g l u t a m a t e c o n c e n t r a t i o n s [3] The molecular desenslttZ,mon rates d e t e r m i n e d here are higher by a factor of at least 20 a n d do n o t seem to depend apprecmbl.~ on agonlst c o n c e n t r a t i o n A similar discrepancy between a p p a r e n t macroscopm and molecular desensitization rates was also f o u n d for G I u R of crayfish [4 8] The macroscopic desensltlZatmn rates thus m a y largely reflect i n h o m o g e n e m e s m access o f g l u t a m a t e The d e s e n s m z a t l o n rates observed here a p p r o a c h the rates ol decay of s>napt~c currents Therefore, ~t m a y not be necessary to remoxe released g l u t a m a t e at the synapse within 1 ms hke assumed for acetylchohne at the endplate [16] and rap~d desensltlZatmn may c o n t r i b u t e to the t e r m m a t m n of synapt~c currents Th~s work was supported by the Deutsche F o r s c h u n g s g e m e m s c h a f l (SFB 220) We want to thank Miss Bettlna Winch for technical assistance a n d Mrs M a r t l n a Grlessl for secretarial help

t lg 2 Recordings and e~aluatlons ol single channel currents from an outside-out patch containing than= nels exhibiting brmt openings only I0 ~M l-glutamate ,,~as apphed by hqmd hlament switch lor 20 ms A single traces digitized at 50 kHz filtered at It) kH7 Single channel amphtude IS pA B ~.urrcnl a~erage tot 187 traces ( distribution ol channel open times bm width 25 l*s Exponential scale ol e~ents bm as m Pig I( ~ The distribution is hired approxlmatel~ b'~ a single exponential v,llh llmt. conslant v = S0 Its although a second component may N. present

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1 Boden P Bvcrolt BW ( h h a b l a S R (_hlplm J Crovdev P J Orant, R J King ~I I JM~.I), nald L Raflert~, P and Usherwood P N R, The action ol natural and s',nthctle I•omers ol qmsqua late on a well dehned glutamaterglc •vnapse, Brain Re,, 385 (1986) 205 21 l 2 ( ull-(and> S (, and Parkel I Rap~d klnt.tlcs ol •Ingle glutamate receptor channels Nattue (L~md 295(19821410 412 t Daoud M A R and U•her,aood P N R l)csensm:allon and potentiation during glutamate apphca tmn to locust skeletal muscle ( omp Bmchem Physml 59C 11978) 1(15 110 4 Dude] J Dose-response eurxe ol glutamatt, apphed b', superlusJon to crayhsh muscle s~n,tpa~.s Pflugers Arch ~,68119771 49 54 5 Dudel J and Franke ( h Single glutamate-gated svnaptlc channels at the crayhsh neuromuseulaJ lunclon 11 Dependence ol channel open time on glutamale conccntratmn Plfugers AIch 40g 1198"71 g07 314 6 Dudel J Franke Ch Halt tt and Usherwood P N R Glutamate pulse• raDdly actwatc and dL.•cnsmzc excitatory channels on membrane patches ol arthropod muscle Pflugers Arch 411 (1988) Suppl 1 R51 7 Irrankc (_h Halt H and Dudel J "lhe e×cHatory glutamate-actwated channel recorded m cell-at tached and e,tclscd patches from the membrane• of tall leg and stomach muscles of erax hsh J Comp Ph~sml 159(1986) 579 '~89 8 Franke ( h Halt H and Dudcl I LNmd hlamcnt sv, ltch lot ultra-last exchanges ol •olutmns al exmsed patches ofsynaptlc membrane ol crayfish muscle, Neuroscl Lett 77 (19871 199 204 9 Gratlon K A F Lambert, l J Ramse,¢ R L and Usherwood P N R , Non-random openings anti coneentrauon-dependent hteumcs of glutamate-gated channels m muscle membrane Nature ( L o n d ) 291 (1981)423 425 10 Gratlon K A F Lamb~-rt J I and Usherv, ood P N R ~ comparison of glutamate •mgle-channd actlvJt~ at desen>mzmg and non-desensmzmg sites J Physml ( L o n d ) 31011980) 49P I 1 Gratlon K A I Lambert, J J and Usherwood P N R Glutamate-actwated channels m locust mu•cles Ad~ Physml Scl,20119811t77 383 12 HaU, H Frankc, (_h and Dudel J Calcmm dependent gating of the L-glutamate actwated excltator~ ~,maptm channel on cra~fish muscle, Pflugers A r c h , 411 ( 19881 17 26 13 Kerry (_J K~ts K S , R a m s c v L R Sansom M S P a n d U s h e r w o o d , P N R Smglechannelkmetics ol a glutamate receptor, J Bmphys 51 11987) 1t7 144 14 Kerry C 1 Ramsc~ R L Sansom M S P and Usherwood P N R Glutamate activated channd kinetics the effect o1 glutamate conccntratkm Bmphy> J 5] 119881 39 52 15 Kits K S and Usher,~ood, P N R Amon selccwdt~ ot single glutamate activated channels m locu•l skeletal muscle J Exp BIol m press 16 Maglcb', K L and Stevens ( t ~ quantJtatlvedescnptmn ofendplatecurrents J PhysmI ( L o u d ) 233(19721 173 197 17 Mathels I) A and Usherwood, P N R ( oncanavahn A blocks desensltlTatmn of glutamate rcceptol • on insect muscl~, hbres Nalme ( Lond 1 259 ( 19761 409 411 18 Patlak J B Grallon k A I~ and Ushcrv~ood P N R Single glutamate-activated channels m locust mu•dc N a t u r e ( L o n d ) 2 7 8 (1979) 64t 645 19 Usherwood P N R In G Dl (_h~ara and G L Gessa, (Eds) Glutamate as a Neurotransm~tter Raven N e w h o r k 1988 pp 183 19t 20 Watklns J ( and Lvans R H E\c~tator3 amino acid transmitters Annu Re,, Pharmacol To,~col 21 (1981) 165 2114