Currents recorded through small areas of squid axon membrane with an internal virtual ground voltage clamp

BRIEF COMMUNICATION CURRENTS RECORDED THROUGH SMALL AREAS OF SQUID AXON MEMBRANE WITH AN INTERNAL VIRTUAL GROUND VOLTAGE CLAMP Jos LOPEZ-BARNEO, DONALD R. MAT ESON, AND CLAY M. ARMSTRONG, Depa men o Physiology, G4, Uni e si y o Pennsyl ania, School o Medicine, Philadelphia, Pennsyl ania 19104, and Ma ine Biological Labo a o y, Woods Hole, Massachuse s 02543 ABSTRACT A new ol age-clamp appa a us o he squid axon has been implemen ed o enable eco ding o cu en s h ough small a eas o axon memb ane. The pe o mance o his clamp was es ed by eco ding o al sodium cu en s om pe used axons (IQ,) and sodium cu en s om small memb ane pa ches (Ip Ch), which we e eco ded om inside he axon wi h an L-shaped pipe e. The Ip,,,h eco ds, al hough ou o de s o magni ude smalle han 1 o , we e s able and showed no mal kine ics and ol age dependence, and appea ed o e lec he ac i a ion o a small popula ion o no mal sodium channels. The size o he cu en eco ded om he pa ch was mainly a unc ion o he ip diame e o he L-shaped pipe e and o he shun esis ance be ween inside he pipe e and he axoplasm. In s udying cu en luc ua ions ela ed o ionic channels in exci able memb anes, i is ad an ageous o isola e and eco d om a e y small memb ane pa ch (Fishman, 1975; Nehe e al., 1978). Reducing he eco ding a ea enhances channel- ela ed luc ua ions ela i e o noise om o he sou ces. Se e al echniques ha e ecen ly been de eloped o elec ical isola ion o small pa ches. Success ul applica ion o hese echniques in squid gian axons will make i possible o apply he ensemble me hod o measu ing cu en luc ua ions (Sigwo h, 1977), and o eco d cu en om single memb ane channels (Con i and Nehe , 1980; Sigwo h and Nehe , 1980). We epo he e he gene al ea u es as well as he pe o mance o a ol age-clamp echnique o he squid gian axon which can be used o eco d cu en h ough small a eas o memb ane. 30-mm-long segmen s o axon we e placed in a chambe and in e nally pe used. The main ea u es o he ol age-clamp ci cui a e illus a ed in Fig. 1. In e nal ol age (VJi) was eco ded h ough a 0.6 M KCl- illed pipe e (1). Vin was ed back o an in e nal con ol ampli ie (ICA) which held Vi. a g ound po en ial by passing he app op ia e cu en h ough a pla inized-pla inum wi e (3) placed inside he axon. Ex e nal ol age (Vou ) was eco ded h ough a sil e -sil e chlo ide elec ode (2). V -Vin was ed o an ampli ie (ECA) which con olled memb ane ol age by a ying he ba h po en ial ela i e o in e nal g ound h ough wo la ge pla inum elec odes (4) placed longi udinally along he ou side o he axon. Vol age-clamp command pulses we e gene a ed om a PDP-8 compu e (Digi al Equipmen BIOPHYS. J. e Biophysical Socie y * 0006-3495/81/12/811/05 $1.00 811 Volume 36 Decembe 1981 811-816 Via FIGURE 1 Vol age clamp and cu en eco ding ci cui . The numbe s in he diag am e e o he ollowing elec odes: 1, in e nal ol age pipe e; 2, ex e nal ol age elec ode; 3, in e nal pla inized- pla inum axial cu en passing wi e; 4, ex e nal pla inized-pla inum elec odes; and 5, L-shaped pa ch pipe e. The la ge ex e nal elec odes (4) we e 15 mm long and ex ended he en i e leng h o he axon be ween he ai gaps, so I,.w was eco ded om his leng h o axon. Co p., Mayna d, Mass.) h ough a D/A con e e and ed o he summing junc ion o ECA. The cu en gene a ed by he whole axon (I o) was moni o ed by eco ding he ol age d op ac oss a 100 Q esis o connec ed in se ies wi h he ou pu o ECA. Cu en s h ough small pa ches o memb ane we e eco ded simul aneously wi h I w using a me hod simila o he one desc ibed by Con i and Nehe (1980). The me hod in ol es placing an L-shaped pipe e inside he axon and maneu e ing he ip o he inside su ace o he memb ane. The ben pa o he pipe e was 100-200 ,um in leng h and had an ou side diame e o 70-100 ,um. As he pipe e ip was i e polished he inside diame e dec eased o a inal diame e o 10-25 ,m. This pa ch pipe e (5) was connec ed o an ope a ional ampli ie wi ed as a cu en - o- ol age con e e o measu e cu en h ough he pipe e. The pa icula pa ch pipe e used in he expe imen s epo ed he e had an inside ip diame e o 20-25 Mm and a DC esis ance o -0.5 MO. A loa ing 25-,um Diam pla inum wi e was placed inside he pipe e o dec ease impedance and he eby minimize se ies esis ance e o s caused by pa ch elec ode esis ance. The pa ch pipe es we e illed wi h a solu ion con aining 150 mM e ame hylammonium (TMA) glu ama e, 50 mM TMA luo ide, 20 mM sodium glu ama e, 10 mM T is and 450 mM suc ose. All eco ds we e ob ained a 80C. Linea leakage and capaci a i e cu en s we e sub ac ed ou elec onically using he P/4 echnique (A ms ong and Bezanilla, 1974). Fo ou pu poses he majo ad an age ha his pa icula ol age-clamp design o e s o e he mo e commonly used design (Cole and Moo e, 1960; Hodgkin e al., 1952) is he main enance o he in e io o he axon a i ual g ound po en ial, which elimina es capaci a i e cu en s ac oss he wall o he pa ch pipe e he eby imp o ing he equency esponse. Clamping he axoplasm o i ual g ound was also done in ol age-clamp echniques de eloped o he node o Ran ie (Dodge and F ankenhaeuse , 1958; Nonne , 1969) and sugges ed o he squid gian axon by Le is (1979). The pe o mance o his ol age-clamp was assessed by compa ing cu en s eco ded om BIOPHYSICAL JOURNAL VOLUME 36 1981 812 he whole memb ane (I o al) wi h cu en gene a ed by he pa ch (Ip, ch). Al hough he gene al cha ac e is ics o l a e simila o sodium cu en s eco ded wi h he mo e usual ol age- clamp con igu a ion, ou chambe lacked gua d elec odes; o al memb ane cu en s eco ded wi h his clamp a e he e o e no highly accu a e, since he ends o he p epa a ion a e no well con olled. To eco d accu a e o al cu en s, he measu ing egion could be es ic ed o a cen al egion and gua d elec odes in he pe iphe y could be d i en by a sepa a e con ol ampli ie . Fig. 2 A shows supe imposed sodium cu en s, eco ded as ' o al om 15 mm o axon, gene a ed by ol age-clamp s eps om -80 mV o he indica ed ol ages. The cu en - ol age ela ionship om hese eco ds is shown by he illed symbols in Fig. 2 C. Pa ch eco dings o sodium cu en s om he same axon a e shown in Fig. 2 B, and hei cu en - ol age ela ionship is plo ed as he open symbols in Fig. 2 C. The impo an poin o no e is ha he kine ics and ol age dependence o hese cu en s indica e ha hey we e gene a ed by a no mal popula ion o sodium channels, and he ampli ude o he cu en s sugges s ha he numbe o channels was small. P e ious a emp s o eco d pa ch cu en s om he inside o he axon upon s eps in memb ane ol age ha e ailed (Con i and Nehe , 1980). Con i and Nehe e e o he appea ance o la ge and slow a i ac s ha con amina e hei cu en eco ds. Al hough he gene al shape and posi ion o he I-V cu es a e simila he e a e se e al appa en di e ences be ween he se s o cu en s shown in Fig. 2. Fi s , he peak- o- s eady-s a e sodium cu en a io is no iceably smalle o I o al han o Ip ch. Second, he e e sal po en ial o he I o al cu en is - 12 mV less posi i e han o Ip' ch* We eel ha he pa ch eco ds a e a mo e accu a e ep esen a ion o he memb ane sodium cu en s o he 4 ! ' ' |^ i; . : d. FIGURE 2 A, Na cu en s (Iw) gene a ed by I 0-ms ol age-clamp pulses o he indica ed ol ages om a holding po en ial o - 80 mV. This axon was -440 um in diame e so ha he o al a ea eco ded om was app oxima ely 0.21 cm2. B, Na cu en s h ough a 25 Am (inside ip diame e ) pa ch elec ode gene a ed by 10-ms ol age clamp pulses o he indica ed ol ages om a holding po en ial o -80 mV. Solu ions o A and B we e he same. Ex e nal solu ion: 225 mM NaCl, 50 mM CaCl2 and 240 mM T is 7.0. In e nal solu ion: 150 mM TMA glu ama e, 50 mM TMA luo ide, 20 mM Na glu ama e, 10 mM T is 7.0, and 540 mM suc ose. C, Cu en - ol age ela ionships o peak cu en s om he eco ds shown in A (-) and B (O). LOPEZ-BARNEO ET AL. Cu en s Reco ded Th ough Squid Axon Memb ane 813 ollowing easons. Unco ec ed I,.,w eco ds we e ound o ha e a much highe leakage cu en han he pa ch eco ds and his leak p esumably lowed ou he cu ends o he axon. I his cu en inc eases nonlinea ly wi h ol age, due in some unknown way o he damaged ends o he axon, i could p oduce a la ge s eady-s a e cu en and an appa en ly lowe e e sal po en ial. The e e sal po en ial p edic ed by he Ne s equa ion is 58.8 mV, e y close o he e e sal po en ial o he pa ch eco ds. The pa ch cu en s shown in Fig. 2 B we e eco ded wi h a 25-,um ip-Diam pipe e ha was ouching ligh ly agains he memb ane. Wi h his la ge diame e pipe e i is impossible o ob ain he high shun esis ances (RSh) which can be ob ained wi h smalle diame e pipe es (c . Nehe e al., 1978). The e o e, he Ip, ch eco ds a e no an accu a e e lec ion o he o al cu en gene a ed by he pa ch, since much o his cu en appa en ly lows h ough Rh. The Ip,, ch cu en gene a ed a 0 mV ep esen s a cu en densi y o -3.7 mA/cm2 assuming he a ea eco ded om was 490 ,um2 (i.e. a ci cle o 25 ,um Diam). Since he Ih.,w cu en a 0 mV was -0.42 mA/cm2, he Ipa ch eco ds mus ha e been eco ded om a much la ge a ea han he 25-,um Diam ci cle we assumed. An imp o emen in he isola ion o he memb ane pa ch can be ob ained unde he app op ia e condi ions and Fig. 3 illus a es he echnique we ha e used. The ou cu en s illus a ed a e pa ch eco dings o ol age s eps o 0 mV om a holding po en ial o -80 mV. I was ob ained wi h he pipe e placed in he cen e o he axon and he ip -100 Am away om he memb ane. The pipe e was hen mo ed un il i jus ouched he memb ane and he cu en was ound o inc ease app eciably (2). An e en highe cu en ampli ude could be ob ained by p essing he pipe e ip mo e i mly agains he memb ane (3), which p esumably inc eases R,h. The in e nal pe usion o he axon was hen swi ched o a low ionic s eng h solu ion (wi hou changing he solu ion in he cu en eco ding pipe e) and he e was a d ama ic inc ease in cu en ampli ude (4), al hough R,h was only inc eased o -1 Mg. S ill highe alues o R,h could mos likely be ob ained by using smalle diame e pa ch pipe es and by cleaning he inside su ace o he memb ane wi h p onase as was done by Con i and Nehe (1980). Since we do no ha e pe ec isola ion o he memb ane pa ch, some o he low ionic s eng h suc ose solu ion may each he memb ane a ea unde he pa ch elec ode. This could explain he ela i ely la ge s eady-s a e cu en seen in suc ose (4 in Fig. 3) since i has p e iously been shown ha pe usion wi h low ionic s eng h solu ion shi s he s eady-s a e FIGURE 3 Na cu en s h ough a 25-,um Diam pa ch elec ode as he shun esis ance om pipe e in e io o axon in e io inc eased. Numbe s e e o successi e 10-ms ol age s eps o 0 mV om a holding po en ial o -80 mV. Reco ds 1-3 we e eco ded wi h he same solu ions as in Fig. 2 and he pipe e in he ollowing posi ions: 1, cen e o he axon; 2, jus ouching he axon memb ane; and 3, p essing agains he memb ane. 4 was eco ded a e swi ching o an in e nal solu ion o he ollowing composi ion: 850 mM suc ose, 50 mM TMA luo ide, and 10 mM T is 7.0. BIOPHYSICAL JOURNAL VOLUME 36 1981 814 inac i a ion cu e o mo e posi i e ol ages (Chandle e al., 1965; Moo e e al., 1964). The e o e, in he suc ose solu ion inac i a ion may be less comple e a 0 mV, esul ing in mo e s eady-s a e sodium cu en . A second possibili y is ha he e is a change in he junc ion po en ial a he ip o he in e nal ol age elec ode when he in e nal pe usion is swi ched o low ionic s eng h solu ion. The e o e, he ue memb ane po en ial in his solu ion may be a a ol age whe e inac i a ion is less comple e. The majo poin o his communica ion is ha he sodium cu en eco ds p esen ed illus a e he abili y o ou i ual g ound ol age-clamp con igu a ion o p oduce s able sodium cu en s whose ampli ude is limi ed mainly by he size o he pipe e ip and by he alue o he shun esis ance. Since he in e nal i ual g ound ci cui e ec i ely elimina es he s ay capaci ance ac oss he wall o he pa ch pipe e, he equency esponse o he eco ding sys em is imp o ed. This may be why we ha e no seen he long las ing a i ac s obse ed by Con i and Nehe (1980) upon s ep changes in memb ane ol age. By using smalle diame e pa ch pipe es, we should be able o achie e accep able alues o RSh so ha his ol age-clamp con igu a ion will be use ul o eco ding memb ane cu en luc ua ions and es ima ing single channel conduc ance (Sigwo h, 1977). In addi ion, i should be possible o eco d he cu en h ough single memb ane channels. Recei ed o publica ion 19 Decembe 1980 and in e ised o m 22 Augus 1981. REFERENCES A ms ong, C. M., and F. 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