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Zašto su kalijevi kanali sporiji od natrijumovih?

Zašto su kalijevi kanali sporiji od natrijumovih?


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Ja sam relativno nov u predmetu biologija. Imam jaku matematičku pozadinu i da bih ušao u polje računske neuronauke, pokušavam steći neku biološku pozadinu.

Čitam o općem mehanizmu akcijskih potencijala u neuronima. Razumijem da akcijski potencijal počinje brzim ulaskom natrija u postsinaptičku ćeliju. Budući da su kalijevi kanali sporiji od natrijumskih, kalij počinje da teče iz ćelije tek oko vrha akcionog potencijala, čime se hiperpolarizuje membranski potencijal ćelije.

Koji je mehanizam koji uzrokuje da se kalijumovi kanali aktiviraju sporije od natrijumovih kanala? Uključuje li to razlike u ponašanju odgovarajućih neurotransmitera? Jer nisam uspeo da pronađem transmitere ili parove transmitera i receptora koji aktiviraju kalijumove kanale, ali ne i natrijumove kanale (ili natrijumske kanale a ne kalijumove kanale).


Kratak odgovor
Kinetika aktivacije Na+ kanali su brži od K+ kanala.

Pozadina
Upravljanje kanalima ovisnim o naponu u osnovi se odvija kroz tri moguća stanja kanala: otvoreno, zatvoreno i deaktivirano (slika 1).


Slika 1. Usklađivanje natrijumskog kanala aktiviranog naponom. Izvor: Institut Balseiro.

U osnovi, jonski kanali su proteinske pore u membrani. Nakon depolarizacije, pore se otvaraju, što je proces s brzom kinetikom u Na+ kanala i sporija kinetika u K+ kanala (Lacroix et al, 2013.). K+ aktivacija kanala je 6 puta sporija od Na+ aktivacija kanala. Sporija inaktivacija K+ kanala omogućava dovoljno Na+ da teče u ćeliju da bi se razvila faza depolarizacije akcionog potencijala (slika 2).


Slika 2. Akcijski potencijali i osnovni Na+ i K+ struje. Izvor: Studentske bilješke Dundee Med.

Nakon otvaranja, kanal se inaktivira, što se može dogoditi kroz fizički "proteinski čep" kako je prikazano na slici 1, ili kroz konformacijske promjene u porama. Ova inaktivacija je opet brza u Na+ kanala koji vode do prolaznog skoka akcijskog potencijala, a deaktivacija je sporija za K+ kanali (K+ kanali se ne deaktiviraju, već deaktiviraju). Ovaj korak inaktivacije rezultira refraktornim periodom neurona (tihi period nakon ispaljivanja). Kanal se zatim deaktivira i pretvara u zatvoreno stanje, nakon čega je kanal ponovo spreman za učešće u drugom krugu paljenja.

Imajte na umu da neurotransmiteri ne aktiviraju izravno kanale s naponom. Neurotransmiteri mogu aktivirati jonske kanale, ali to su ionski kanali vođeni ligandom, kao što su nikotinergički acetilkolinski receptor, AMPA receptor ili NMDA receptor (Purves et al., 2001).

Reference
- Lacroix et al, Neuron (2013); 79(4): 651-7
- Purves et al., Neuroznanost, 2nd ed. Sunderland (MA): Sinauer Associates (2001)


Morate razumjeti kako funkcioniraju integralni proteini. $ Na^+$ kanali su brzi dok su $ K^+$ kanali spori i dugotrajni u smislu provodljivosti. $ Na^+$ kanali su ograničeni naponom, preko S4 domene koju blokira $ Mg^{2+} $, dok provodljivost $ K^+$ većinu vremena nije ograničena naponom.


Akcijski potencijal

Akcijski potencijal je poruka u obliku električnog impulsa uzrokovanog brzom promjenom membranskog potencijala ćelije.

Kada stimulus dosegne prag na brežuljku aksona, stvara se akcioni potencijal.

Akcijski potencijal ovisi o mnogim proteinima. U neuronu, kanal za curenje kalija i natrij-kalijeva pumpa održavaju potencijal mirovanja. Natrijski kanali s naponom i kalijevi kanali s naponom su uključeni u napredovanje akcijskog potencijala duž membrane.

Napredovanje akcijskog potencijala može se podijeliti u nekoliko koraka

  1. Naponski kanali su zatvoreni, a kanal za curenje kalija (K +) i pumpa natrijuma (Na +) održavaju membranski potencijal u mirovanju od -70 mV. Natrijeva/kalijeva pumpa (ATPaza) odgovorna je za održavanje membranskog potencijala na -70mv, protein aktivno ispumpava tri natrijeva iona iz ćelije i ispumpava dva iona kalija u ćeliju.
  2. Neuron postaje stimulisan. Natrijevi kanali s naponom počinju se otvarati, a membranski potencijal počinje polako depolarizirati i natrij ulazi u ćeliju niz gradijent koncentracije. Svi naponski kanali s natrijem se otvaraju kada membranski potencijal dosegne oko -55 mV i dođe do velikog priljeva natrija, uzrokujući nagli porast napona. Kako se potencijal približava +30mV, brzina depolarizacije se usporava kako se natrijevi kanali s naponom zasićuju i inaktiviraju, sprječavajući ulazak daljnjih natrijevih iona u ćeliju. otvoren, a kalijum napušta ćeliju niz njen gradijent koncentracije. Depolarizacija ćelije prestaje i repolarizacija se može dogoditi kroz ove kalijeve kanale s naponom.
  3. Natrijevi kanali s naponom potpuno su deaktivirani i kalij izlazi kroz kalijeve kanale s naponom,
  4. Kalijevi kanali s naponom zatvaraju se sporo, pa dolazi do hiperpolarizacije. Ovdje membranski potencijal pada ispod potencijala odmora od -70 mV kako kalij nastavlja izlaziti.
  5. Kada se kalijumski kanali sa naponom zatvore, stanje mirovanja se može ponovo uspostaviti kroz kanal za curenje kalijuma i natrijumovu pumpu.

Akcioni potencijal putuje duž aksona neurona preko strujnih petlji kako bi stigao do terminala aksona.

Akcijski potencijal je prolazni, električni signal, koji je uzrokovan brzom promjenom membranskog potencijala u mirovanju (-70 mV). To se događa kada se dosegne granični potencijal (-55 mV), što uzrokuje brzo otvaranje u naponskim kanalima natrijuma, što dovodi do dotoka natrijevih iona u ćeliju. Prag potencijala također uzrokuje sporo otvaranje kalijevih kanala pod naponom što dovodi do istjecanja kalijevih iona iz ćelije. To uzrokuje depolarizaciju ćelije, što znači da je unutrašnjost ćelije sada pozitivnija u odnosu na vanjsku.

Akcijski potencijal počinje na brežuljku aksona jer ovdje postoji velika gustoća natrijumskih kanala s naponom, a također i tamo gdje stupnjevani potencijali trebaju doseći prag da izazovu akcijski potencijal. Ako gradirani potencijal ne dosegne nivo iznad granice, tada se akcioni potencijal ne pokreće i gradirani potencijal je poznat kao podprag [1] . Iznad praga, povećanje snage stimulusa neće povećati veličinu ili amplitudu odgovarajućeg akcijskog potencijala. Jačina stimulusa ili veličina stupnjevanog potencijala označena je učestalošću akcijskih potencijala koji putuju duž neurona.

Akcijski potencijal putuje kroz strujne petlje. Kod mijeliniziranih aksona skokovi s čvora ranviera na čvor Ranviera, ovo je proces poznat kao saltatorna provodljivost.

Dva su glavna faktora koji utječu na brzinu provođenja: mijelinizacija aksona i promjer aksona. Kako mijelinski omotač djeluje kao električni izolator, struja ne može proći kroz mijelinizirana područja i morat će skakati od čvora do čvora (slana provodljivost). S povećanjem promjera aksona, ima više prostora za lokalni protok struje, pa se unutrašnji otpor membrane smanjuje, a zauzvrat povećava brzina provođenja.

Primjer bolesti povezane s mijelinizacijom je multipla skleroza, koja uzrokuje da imunološki sistem napadne mijelinsku ovojnicu, što rezultira demijelinizacijom aksona. Kada je akson demijeliniziran, brzina provođenja će se drastično smanjiti, stoga akcioni potencijal putuje sporije do efektora (npr. mišića), što dovodi do gubitka pokreta.

Točka na kojoj se membrana aksona depolarizira uzrokuje postavljanje lokalnog kola između depolarizirane regije i regije s njezine strane. Ovo također uzrokuje depolarizaciju ostataka na obje strane. Na taj način akcijski potencijal prelazi duž aksona.

Vatrostalni period sprečava da se akcijski potencijal kreće unatrag. Postoje dvije vrste vatrostalnih perioda, apsolutni i vatrostalni period. Apsolutni refraktorni period je kada membrana ne može da generiše drugi akcioni potencijal, bez obzira na to koliko je veliki stimulans. To je zato što su natrijski ionski kanali pod naponom inaktivirani. Relativni refraktorni period je kada membrana može proizvesti drugi akcijski potencijal, ako je podražaj veći od normalnog. To je zato što su se neki od naponsko vođenih natrijumovih jonskih kanala oporavili, a naponsko vođeni kalijumovi jonski kanali su i dalje otvoreni. Relativni refraktorni period je period hiperpolarizacije nakon akcionog potencijala [2] .

Akcijski potencijali u neuronima poznati su i kao "nervni impulsi" ili "šiljci" [3] [4].


Potencijal akcije

Akcijski potencijal je kratki preokret membranskog potencijala gdje se membranski potencijal mijenja s -70mV na +30mV. Kad membranski potencijal aksonskog brda neurona dosegne prag, dolazi do brze promjene membranskog potencijala u obliku akcijskog potencijala.

Ova pokretna promjena membranskog potencijala ima tri faze. Prvo je depolarizacija, zatim repolarizacija i kratak period hiperpolarizacije. Ova tri događaja se dešavaju u samo nekoliko milisekundi.

Akcijski potencijal: A. Šematski i B. snimci stvarnih akcionih potencijala. Akcijski potencijal jasan je primjer kako promjene u membranskom potencijalu mogu djelovati kao signal.

  • Depolarizacija, koja se naziva i rastuća faza, nastaje kada pozitivno nabijeni ioni natrija (Na+) iznenada projure kroz otvorene natrijumske kanale s naponom u neuron. Kako dodatni natrij ulazi, membranski potencijal zapravo mijenja svoj polaritet. Tokom ove promjene polariteta membrana zapravo na trenutak razvija pozitivnu vrijednost (+40 milivolti).
  • Faza repolarizacije ili pada uzrokovana je sporim zatvaranjem natrijumovih kanala i otvaranjem kalijum kanala pod naponom. Kao rezultat toga, propusnost membrane za natrijum opada na nivo mirovanja. Kako opada unos natrijevih iona, sporo se otvaraju kalijevi kanali pod naponom, a kalijevi ioni izlaze iz ćelije. Ovo izbacivanje djeluje na vraćanje lokaliziranog negativnog membranskog potencijala stanice.
  • Hiperpolarizacija je faza u kojoj neki kalijevi kanali ostaju otvoreni, a natrijevi kanali se resetiraju. Period povećane propustljivosti kalija dovodi do prekomjernog istjecanja kalija prije zatvaranja kalijevih kanala. To dovodi do hiperpolarizacije koja se vidi u blagom padu nakon skoka.

Širenje akcionog potencijala je nezavisno od snage stimulusa, ali zavisi od refraktornih perioda. Period od otvaranja natrijumskih kanala do početka resetovanja natrijumovih kanala naziva se apsolutni refraktorni period. Tokom ovog perioda, neuron ne može odgovoriti na drugi stimulans, bez obzira koliko jak.


Kako kalijum može difundirati kroz membranu koju natrijum ne može?

U biologiji govorimo o akcionim potencijalima. Jedan dio toga je otvaranje proteina natrijum-kanala tako da može proći u akson. Ali naučeni smo da kalij može proći kroz staničnu membranu bez problema. I natrij i kalij su pozitivno nabijeni i natrij bi zapravo trebao biti malo manji jer ima manje elektrona koji se međusobno guraju, što znači da ako kalij može proći kroz nešto, natrij bi također trebao biti u mogućnosti. Dakle, moje pitanje je kao što je gore navedeno: kako kalij može difundirati kroz nešto ako natrij ne može 't.

P.S. Naučio sam sve ovo na njemačkom jeziku i googlao za prevode, pa se izvinite zbog pogrešnog vokabulara i slobodno me ispravite zbog svih grešaka koje sam napravio.

Edit: Hvala svima na odgovorima. Bili su mi od velike pomoći u razumijevanju onoga što učim u školi!

Nijedan ion ne može direktno proći kroz staničnu membranu. Međutim, postoje kanali koji jesu obično otvorene za propuštanje iona, poput kalija. To se prikladno nazivaju kanali curenja. Nisu naponski ograničeni, tako da ako pogledate njihove IV krive to je u suštini omski (pravolinijski) odnos. Kad ljudi kažu da je membranski potencijal u mirovanju postavljen kalijem, to je zato što je membrana propusnija za kalij. Ili na drugi način, postoji više kanala koji omogućuju istjecanje kalija iz ćelije, smanjenje neto pozitivnog naboja ćelije i usmjeravanje membranskog potencijala prema obrnutom potencijalu kalija.

Kada razmišljate o kanalima, manji ion ne znači uvijek da će proći ako može veći ion. Postoje filteri za selektivnost koji mogu učiniti svaki kanal specifičnim za određene ione. Ovi filtri za selektivnost ovise o interakcijama između aminokiselina u porama i iona dok prolaze, i/ili ako je električno povoljno odvojiti svoju hidracijsku ljusku na trenutak kako bi se omogućio prolaz. Zapravo je prilično komplikovano.

Ovaj odgovor je tačan i dopunit ću ga izvorom s posebno jednostavnim i lako razumljivim slikama i opisima.

Ovo! Jednostavno rečeno: ioni ne mogu difundirati kroz membranu i mogu proći samo pasivnim ili aktivnim transportom (putem kanala). I drugi paragraf je tako tačan. Kanali su vrlo specifični u pogledu onoga što će proći, pa natrij ne može proći kroz kalijeve kanale i obrnuto.

Šta obično mislite pod tim? Postoje li slučajevi zatvaranja kanala koji propuštaju? Kako rade u usporedbi s drugim kanalima s naponom?

do piggybacka, kako kanal dozvoljava samo K umjesto Na, iako je zapravo 10.000:1, svaki ion ima vodenu ljusku i ion mora odbaciti svoju vodenu ljusku na krajnjoj poru, proteinski kanal tačno odgovara vodenoj ljusci K.

Kalijevi kanali zapravo ne propuštaju ione samo poput cijevi. Jon uzrokuje konformacijsku promjenu, kalijev kanal koji mu omogućuje izlazak s druge strane membrane. Zamislite to kao okretnik podzemne željeznice koji se otključava samo kada uđe objekt odgovarajuće veličine. Natrijevi ioni mogu ući u otvor, ali nisu odgovarajuće veličine da izazovu konformacijsku promjenu, pa ne mogu proći

Ili na primjer stavljanje novčića u otvor za gumbaš. Ako stavite bilo šta drugo osim četvrtine, neće dobiti##2727 poteza.

Vjerujem da to nije tačno. Jon bi se morao vezati za kanal kako bi izazvao promjenu njegove konformacije, a kinetika toga bi bila prespora da bi omogućila brz protok jona kao u akcionim potencijalima (s obzirom da svaki pojedinačni ion mora uspostaviti kontakt sa kanalom). Možete li dati neke reference jer je vaša tvrdnja super zanimljiva ako je istinita. U kanalima s ograničenim naponom, konformacijska promjena dolazi od napona i ioni bi trebali teći niz gradijent nakon otvaranja kanala. Razlog zašto natrij (koji ima manji atomski radijus od K+) ne teče niz gradijent je zbog molekula vode koji su povezani s Na+ ionom. U suštini, budući da je manji, više molekula vode stvara slabe veze oko njega nego oko K+ iona. Dakle, kada Na+ teče niz kanal, ima veću H2O prtljagu od K+, tako da se ne uklapa.

Ovo je malo netačno. Filter selektivnosti nije formiran konformacionom promjenom. K+ ioni koji se kreću niz pore stvaraju veze sa hemijskim grupama u kanalskim porama (karboksilne grupe). K+ jon je prave veličine da zamijeni svoj sloj molekula vode (hidratacijski omotač) za veze s porama. Ali Na+ jon ima hidratantnu ljusku pogrešne veličine, stoga se ne vezuje za karboksilne grupe i ne prolazi kroz pore. Vidi Lehningerova načela biokemije, str. 410.

Ni kalij ni natrijum ne mogu sami difundirati kroz ćelijsku membranu. Neuroni zapravo imaju kanale za propuštanje kalija koji omogućuju izlazak kalija iz ćelije u mirovanju, održavajući potencijal odmora.

Čini mi se da mnogi odgovori ovdje ne idu dovoljno detaljno, pa ću pokušati. Molimo ispravite/dodajte sve što kažem.

Dvoslojevi membrane ne provode ione vrlo dobro inherentno. To je zbog hidrofobnog sloja u sredini koji se sastoji od repova hidrofobnih masnih kiselina, što ga čini nepovoljnim za ulazak nabijenih iona. Razlog zašto je to nepovoljno je taj što repovi ne stvaraju nikakve interakcije naboj-naboj s ionima (mislite na +ve i -ve privlačenje, alkan/alkenske grupe nisu u stanju da ih formiraju). To znači da K+ i Na+ prelaze membranu vrlo sporo.

Za povećanje brzine provođenja kroz membranu postoje ionski kanali. Oni naravno imaju pitanje specifičnosti - želite samo da se određeni ioni provode u određeno vrijeme, ne možete samo imati rupu u membrani. U slučaju akcijskih potencijala, posebno želite prvo provesti Na + iz ćelije niz gradijent koncentracije, a K + u ćeliju odmah nakon toga, također niz gradijent koncentracije.

Jedna stvar koju treba razlikovati je očito pozitivan naboj. Aminokiseline poput glutamata i aspartata imaju negativan naboj za interakciju s kationima. To znači da će prisutnost ovih aminokiselinskih ostataka u lumenu kanala razlikovati negativno nabijene ione poput Cl-

Naravno, tu je i veličina. Relativno je lako razlikovati, recimo, Ca2+ i Na+ - imaju uži lumen za Na+

Ovdje dolazi vaše pitanje - kako razlikovati K+, veći ion i Na+, manji ion koji će se sigurno uklopiti u veću rupu potrebnu za K+?

U ovom trenutku razlikujete se po razlici u energiji hidratacije. Svaki ion će upotrijebiti određeni broj molekula vode kako bi se rastvorio i formirao će donekle uređenu ljusku. Ove ljuske sadrže različit broj molekula vode, ovisno o veličini i naboju iona.

To koriste ionski kanali. Imaju dovoljno uski kanal u koji se solvatirani ion neće uklopiti - solvacijsku ljusku prvo treba izbaciti. Da bi bio energetski povoljan, kanal mora u potpunosti kompenzirati energiju solvacije, što se postiže naručenim negativnim statičkim nabojima u najužem području kanala. Uzmimo K+ kanal, čiji je model protein Kcsa. Na najužem dijelu, protein ima dotičnu TVGYG sekvencu. Ova sekvenca je fleksibilna zbog ostataka glicina, koji omogućavaju karbonilnim kiseonikima da se orijentišu prema lumenu. Ovi karbonilni kisikovi, koji imaju negativni dipol, savršeno su orijentirani na interakciju s dolaznim K+, kompenzirajući energiju solvacije. U slučaju Na+, manjeg jona, ovi karbonilni kiseoniki su previše udaljeni da bi svi mogli da deluju istovremeno sa dolaznim ionom, tako da je provođenje Na+ nepovoljno.

Stoga imate kanal koji razlikuje različite idole i dozvoljava samo K+. To se radi diferencijacijom naboja, okluzijom veličine i razlikom u otapanju vode.

Izvor: Trenutno studira biokemiju. Doduše, ovo mi nije na kraj pameti - pa molim vas javite mi ako sam nešto pogrešio.


Kanal za curenje kalija

Kanal za curenje kalija je jedna od glavnih komponenti koja održava membranski potencijal u životinjskim stanicama. Potencijal membrane nastaje razlikom u električnom naboju s obje strane membrane. Ova razlika u električnom potencijalu uzrokovana je natrij-kalijevom pumpom i difuzijom K + iona kroz kanal za curenje kalija. Kanal Na + -K + pumpa tri Na +  iz ćelije za svaka dva K+ jona koja upumpa unutra. Ovo stvara hemijski gradijent za K+, koji se može slobodno kretati nazad preko membrane preko kanala za curenje kalijuma . Povrh toga, na K + djeluje električni gradijent pri čemu je unutrašnjost ćelije negativnija od vanjske strane. K+ se stoga privlače nazad u ćeliju niz ovaj električni gradijent. Na kraju je postignuta ravnoteža između električnog i hemijskog gradijenta K+. Kada je membrana u ovom stanju poznata je kao membranski potencijal u mirovanju, a relativni negativni naboj unutar ćelije koji stvara kanal za propuštanje kalija i Na + -K + pumpa omogućuje stvaranje akcijskog potencijala [1]

Funkcije - U uzbudljivim stanicama poput neurona postavljaju membranski potencijal u mirovanju.


Zašto je stanična membrana propusnija za kalijeve ione ako je veća od natrija?

Razumijem da postoje kanali curenja i Na+ K+ ATP pumpa, ali zašto bi bilo korisno da odabere K+ ili Na+. Na+ je manji i manje se stvari šire brže od većih, pa zašto je membrana evoluirala za K+?

Pitanje je ionskih kanala.

Putem ionskih kanala, živčane stanice mogu obavljati svoje funkcije. Ovi kanali pumpaju K+ u nervne ćelije, izbacujući Na+, tako da, u suštini, rade jedan protiv drugog. Kad se kalijevi kanali otvore, kalij nadire, čineći ćeliju pozitivnijom, što zatim stimulira zatvaranje kalijevog kanala i otvaranje natrijevog kanala.

Ali, ćelije su, kao i ljudi, lijene. Žele koristiti manje energije pri ubrizgavanju i izlasku tih iona. Zapamtite da se jonski kanali otvaraju i zatvaraju svaki put kada kalijum ili natrijum žele da uđu u ćeliju? Desilo se da se na svaka dva uneta K+ iona unose ukupno tri Na+ iona. Stoga je za unos natrija potrebno više energije.

Dakle, da odgovorim na vaše pitanje:

tldr: Potrebno je manje energije za dovođenje kalija u ćeliju, za razliku od natrija, pa, iako su veće od natrija, ćelije preferiraju kalij.

Razumem sve što ste rekli i koncepte i mislim da nisam pravilno postavio svoje pitanje. Postavljam pitanje u vezi evolucije i zašto je evolucijski gledano, stanica razvila jonske kanale za kalij umjesto natrijum prije milijardi godina? U svakom slučaju, odgovor je valjda zato što evolucija nije savršena i upravo se to dogodilo.

OP, mislim da imam odgovor za tebe! Razumijem vaše pitanje: ovo sam opisao na drugoj godini studija. Kasnije mogu o tome više saznati, ali TLDR je da su kalijevi kanali fiksnog promjera, a unutrašnjost je sastavljena tako da uvijek postoji 6 atoma kisika, parovi para elektrona kurvi mogu stabilizirati kalijev ion dok prolazi. Natrij je mnogo manji od kalija, pa njegov elektronski oblak ne može "dohvatiti" pojedinačne parove ovih atoma kisika (možda 3 ili 4), što je nedovoljno za stabilizaciju iona. IIRC ovo dolazi do izražaja jer se ionima mora ukloniti ljuska otapala (ljuska molekula vode koja stabilizira ione u otopini) kako bi prošli kroz kanal, a to košta energiju. Stabilizacija jona dok su u kanalu trebala bi olakšati proces, stoga kalijum može proći, ali natrijum ne.


Rezultati

Ionska ovisnost brzine inaktivacije C-tipa u A463C

Zamjene na poziciji A463 u S6 od Shaker kalijev kanal utječu na brzinu inaktivacije C-tipa, proces kojim se upravlja zauzimanjem iona (Hoshi i sur., 1991. Lopez-Barneo i sur., 1993. Baukrowitz i Yellen, 1995.). Ispitivali smo efekte mutacije afiniteta kalijuma A463C na inaktivaciju C-tipa i njenu zavisnost od jonskih uslova. Kao što je prethodno opisano (Hoshi i sur., 1990. Lopez-Barneo i sur., 1993.), u divljem tipu Shaker B kanal s uklonjenom inaktivacijom N-tipa, C-tip se inaktivira duž jednog eksponencijalnog vremenskog toka s vremenskom konstantom od 1,4 s u fiziološkim uvjetima (slika 2 A) povećavajući vanjski kalij na 140 mM usporava ovu konstantu vremena inaktivacije na 2,6 s ( Slika 2 B). Baukrowitz i Yellen (1995) pokazali su da je čak i u nedostatku vanjskog kalija, odljev kalija kroz kanal dovoljan da zauzme mjesto koje značajno utječe na inaktivaciju C-tipa. Shodno tome, smanjenje unutrašnje koncentracije kalija smanjuje protok kroz kanal i rezultira bržim stopama inaktivacije C-tipa (Kd = ∼2 mM) (Baukrowitz i Yellen, 1995. Starkus et al., 1997.).

Zamjenom ostatka cisteina za alanin na položaju 463 smanjuje se afinitet unutarnjeg mjesta vezanja iona u porama (Ogielska i Aldrich, 1998), a mi smo pretpostavili da bi se možda kanali A463C brzo deaktivirali zbog smanjenja popunjenosti mesto pomoću kalijuma. Međutim, otkrili smo da se mutant A463C inaktivirao sporije od divljeg tipa u niskom vanjskom kalijumu (2 mM K, slika 2 C). Nadalje, povećanje vanjske koncentracije kalija nije dodatno usporilo inaktivaciju (140 mM K, slika 2 D) u A463C kao u kanalu divljeg tipa. Ovaj rezultat nije u skladu s jednostavnom idejom da A463C smanjuje popunjenost stranice koja izravno regulira inaktivaciju C-tipa. Moguće su dvije alternative: (a) mutacija A463C remeti konformacijsku promjenu povezanu s inaktivacijom C-tipa i kanal se ne može inaktivirati bez obzira na koncentraciju kalija, ili (b) mjesto inaktivacije C-tipa je već maksimalno zauzeto sa 2 mM vanjskog kalij, pa je stoga daljnje povećanje koncentracije nedjelotvorno. Kako bismo razlikovali ove dvije mogućnosti, ispitali smo stopu inaktivacije u uvjetima koji bi trebali smanjiti zauzetost kalija na vanjskom (C-tip) mjestu. Utvrdili smo da ako je inaktivacija spora u A463C zbog zasićenja mjesta tipa C, smanjenje zauzimanja kalija na tom mjestu bi trebalo dovesti do brže inaktivacije. Stopa inaktivacije se nije povećala kada je vanjski kalij zamijenjen nepropusnim ionskim NMG -om (slika 2 E), što ukazuje na to da je možda istjecanje kalija dovoljno za zasićenje vanjskog mjesta (vidi Baukrowitz i Yellen, 1995). Zaista, kada smo smanjili unutrašnju koncentraciju kalija sa 140 na 5 mM, smanjenje istjecanja kalija je dovelo do brže inaktivacije C-tipa (slika 2 F). Ovaj rezultat ukazuje da je mehanizam inaktivacije C-tipa prisutan u A463C i da se još uvijek može regulisati zauzetošću kalijem.

Zasićenje učinkom kalija čak i u odsutnosti vanjskog kalija ukazuje da izgleda da mutacija A463C povećava zauzetost kalija na mjestu koje upravlja inaktivacijom C-tipa u usporedbi s kanalom divljeg tipa pod istim ionskim uvjetima. Slijedeći ovo zaključivanje, pitali smo se bi li se povećala i zauzetost natrija i stoga bi li inaktivacija A463C tipa C izmjerena u simetričnim otopinama natrija bila sporija nego u kanalu divljeg tipa. Visok afinitet prema kaliju kod divljeg tipa Shaker kanal otežava proučavanje natrijumske provodljivosti (Starkus et al., 1997 Ogielska i Aldrich, 1998). Međutim, Starkus et al. (1997) otkrili su da je divlji tip Shaker kanal se brzo inaktivira sa natrijumom kao provodnim jonom (vidi takođe sliku 3 A). Ili natrij ne ometa inaktivaciju C-tipa tako učinkovito kao kalij u kanalu divljeg tipa, ili mjesto koje utječe na inaktivaciju C-tipa nije dovoljno zauzeto natrijem da bi značajno usporilo konformacijsku promjenu inaktivacije C-tipa. Smanjenje prividnog afiniteta kalija u A463C dozvoljava velike protoke natrijuma u odsustvu dodanog kalijuma (Ogielska i Aldrich, 1998). Međutim, ne primjećuje se nikakva inaktivacija tijekom impulsa od 400 ms u simetričnom natriju (slika 3 B), iako je evidentan spor pad s dužim trajanjem impulsa (slika 3 C). To nije za razliku od kanala divljeg tipa, koji se inaktivirao za <20 ms u identičnim otopinama natrija (slika 3 A). Visoka stabilna struja koja preostaje nakon inaktivacionog prijelaza pripisuje se provodljivosti natrijuma kroz C-tip-inaktivirano stanje Shaker kanal (Starkus et al., 1998). U skladu s tim, postoje dva moguća objašnjenja za sporu inaktivaciju uočenu u mutanta A463C: (a) natrij je bolje u stanju ometati inaktivaciju C-tipa u mutantu nego u kanalu divljeg tipa, ili (b) A463C se izuzetno brzo inaktivira u ovim rastvorima i uočena struja natrijuma je posljedica provođenja natrijuma kroz inaktivirano stanje kanala.

Ranije smo pokazali da se u prisutnosti vanjskog natrija smanjuje sposobnost unutarnjeg kalija da blokira struje natrija, što ukazuje na to da vanjski natrij može ući u pore i destabilizirati unutarnji blokirajući ion kalija u A463C (Ogielska i Aldrich, 1998). Utvrdili smo da bi možda vanjski natrij također mogao ometati inaktivaciju tipa C u mutantu A463C, predviđajući da će se u nedostatku vanjskog natrija kanal lako deaktivirati. Ako se eksperiment prikazan na slici 3 C ponovi s nepropusnim ionom NMG + kao primarnim vanjskim monovalentnim, tada se kanal lako inaktivira s vremenskom konstantom od 43 ± 8 ms (n = 19 Slika 3 D). Sa kalijumom kao provodnim jonom, tok kroz kanal je bio dovoljan da zasiti mesto inaktivacije C-tipa (slika 2 E). Iako natrij ometa inaktivaciju C-tipa u A463C, čini se da istjecanje natrija nije dovoljno da zasiti vanjsko mjesto (slika 3 D). Zapažanje da brzina inaktivacije varira ovisno o tome koja vrsta iona prodire kroz pore može odražavati inherentnu selektivnost kalija na mjestu inaktivacije C-tipa ili, alternativno, može biti rezultat manje popunjenosti Na + na C- tip mjesta zbog niže stope protoka Na + kroz kanal. Ako natrijum sprječava inaktivaciju C-tipa u mutantu A463C vezivanjem unutar pora, tada bi niske koncentracije vanjskog natrijuma trebale biti u stanju da uspore proces inaktivacije. Zaista, kada se vanjskom NMG + otopini doda 1 mM natrija, inaktivacija se usporava (slika 4 A). Uočeno smanjenje amplitude vrha može biti rezultat interakcije više jona u porama, ali to nije dalje istraženo. Radi lakšeg poređenja, stacionarna struja je oduzeta i amplitude struje su povećane (slika 4 A, dolje). Zapažanje da natrij izravno usporava inaktivaciju C-tipa osporava mogućnost da je očigledan nedostatak inaktivacije u simetričnim otopinama natrija posljedica toga što su kanali već inaktivirani i prolaze kroz inaktivirano stanje. Međutim, u nedostatku vanjskog natrijuma, kanali A463C vjerovatno vode Na + kroz otvoreno i inaktivirano stanje kako je ranije primijećeno u divljem tipu Shaker kanal (vidi Starkus et al., 1998). Iako je ideja da je struja u stabilnom stanju rezultat jona koji provode kroz stanje inaktivirano C-tipa intrigantna, njeno porijeklo od mutanta A463C je nejasno i izvan okvira ove studije.

Budući da je u prisustvu eksternog NMG+ i unutrašnjeg natrijuma, spoljašnje mesto češće prazno i ​​A463C se lako inaktivira, pitali smo da li bi dodavanje unutrašnjeg kalijuma moglo usporiti inaktivaciju C-tipa u ovim rastvorima. Zaključili smo da povećanjem interne koncentracije kalija trebamo povećati zauzetost kalijem na više eksternom (C-tip) mjestu i istovremeno usporiti inaktivaciju C-tipa. Na slici 4 B kontrolni trag je nastao u prisustvu spoljašnjeg NMG + i unutrašnjeg Na + i primećena je brza inaktivacija. Under these ionic conditions, the apparent potassium affinity of the internal site is 2.5 mM (Ogielska and Aldrich, 1998). The addition of 2.5 mM potassium to the internal solution resulted in the expected channel block (∼45%) and the remaining current inactivated with a slower time course than the control trace (Fig. 4 B). Subtracting the steady state current and scaling the amplitudes of the two traces facilitates the comparison (Fig. 4 B, bottom). The inactivation time constant increases roughly twofold from 43 ± 8 ms (n = 19) to 82 ± 11 ms (n = 5). These experiments demonstrate that as potassium appreciably occupies the channel pore it is able to interfere with C-type inactivation in A463C.

Decreased Ion–Ion Interactions Result in Increased Occupancy of the C-Type Site

Previous work has shown that the A463C mutant decreases the apparent potassium affinity and, at subsaturating concentrations, the occupancy of an internal ion binding site in the pore (Ogielska and Aldrich, 1998). Present experiments, however, suggest that A463C also increases the occupancy of an external site as reflected by the slow rates of C-type inactivation in both sodium and potassium solutions. One possibility is that the A463C mutation alters the overall structure of the pore and therefore affects the properties of several ion binding sites. Alternatively, increased occupancy at the external (C-type) site may be a secondary effect of the decreased affinity at the more internal site. If ions at the internal and external sites can mutually destabilize one another, then decreasing the occupancy of the internal site should increase the occupancy of the external site by decreasing the electrostatic interactions among ions in the pore. The observation that external sodium ions can enter the pore and decrease the apparent affinity of a blocking potassium ion at the internal site in the A463C mutant is consistent with the hypothesis that ions interact with one another in the channel pore (Ogielska and Aldrich, 1998).

In the presence of external sodium, the external (C-type) site is occupied, preventing inactivation (Fig. 3, B and C). We reasoned that by increasing ion occupancy at the internal site we should increase repulsive ion interactions in the pore and destabilize the sodium ion bound at the external (C-type) site. The emptying of the external site would be reflected in an increased rate of C-type inactivation. Potassium binds with a higher affinity than sodium and therefore blocks the sodium current at low concentrations. In symmetrical sodium solutions, the apparent internal potassium affinity of A463C is ∼6 mM (Ogielska and Aldrich, 1998). Adding low (millimolar) concentrations of internal potassium should therefore increase the occupancy of the internal site and concomitantly increase the repulsive interactions among ions in the pore. Increased repulsive interactions should destabilize the sodium ion bound at the external site and increase the C-type inactivation rate of the channel (Fig. 5 A).

In symmetrical sodium solutions, the occupancy of the external (C-type) site by sodium is high, and no inactivation is observed (Fig. 5 B). When the occupancy of the internal site was increased by the addition of 1 mM potassium to the internal solution, significant inactivation was observed in addition to channel block (∼13%) (Fig. 5 B). Under these ionic conditions, A463C inactivates with a time constant of 198 ± 16 ms (n = 10), slower than in the absence of external sodium (43 ± 8 ms). These results are consistent with a decrease in the occupancy of sodium at the external site due to repulsive interactions with potassium at the internal site. Alternatively, the slow decline in current could be attributed to a slow potassium block at a second site in the pore. In the latter case, increasing the concentration of internal potassium should accelerate the blocking rate, making the current decline faster. Instead, the addition of 5 mM potassium further slowed the decline of the current (τ = 297 ± 10 ms n = 3 data not shown). We interpret this slowing to mean that at higher concentration potassium itself occupies the external site and slows C-type inactivation.

The increased occupancy at the external (C-type) site in A463C is therefore best explained by a secondary effect of the decreased potassium affinity of an internal ion binding site. Our results suggest that ions remain bound longer at the more external site in A463C because of decreased repulsive interactions in the pore, not because the intrinsic affinity at that site has been directly increased by the A463C mutation.

External K + Blocks Na + Currents without Affecting C-Type Inactivation

Having found that the A463C channel readily inactivates once the external site is depleted of ions, we wanted to further examine the interactions between potassium and the C-type inactivation gate. We used sodium as the permeant ion to measure the apparent potassium affinity of the external ion binding site. We reasoned that the apparent affinity of the external (C-type) site most likely cannot be determined in blocking studies using internal potassium. Once a potassium ion traverses the pore and reaches the external (C-type) site, it presumably rapidly equilibrates with the external solution, and occupancy of that site would therefore not be perceived as current block. Based on this reasoning, we expected that low concentrations of external potassium would block the sodium current by binding to the external (C-type) site and concomitantly slow the rate of C-type inactivation.

Currents were recorded from outside-out patches in the presence of external NMG + and internal Na + . Sweeps were 180 ms in duration to give the channels ample time to inactivate (Fig. 6 A). Micromolar concentrations of potassium were sufficient to block the observed sodium currents (Fig. 6 A). The fraction of unblocked currents (I/Imax) is plotted against external potassium concentration in Fig. 6 B (•). The data are well fitted with a single binding isotherm yielding an apparent potassium affinity of ∼100 μM. To compare the inactivation rates, the steady state current was subtracted and the control current was scaled with the current in the presence of 50 μM external potassium (Fig. 6 C). Contrary to our expectation, C-type inactivation progresses at the same rate regardless of the presence or absence of 50 μM external potassium (41 ± 7 ms, n = 4, and 43 ± 8 ms, n = 19, respectively). We interpret these data to mean that external potassium is binding to a high affinity site in the pore that is distinct from the external (C-type) site. This is unlike what was observed in a chimeric channel between Kv1.3 and Kv2.1. In that construct, external potassium both blocked the sodium currents with a high affinity and simultaneously slowed down the C-type inactivation process (Kiss and Korn, 1998).

To examine further the properties of the high affinity binding site that is accessible from the external solution, we asked whether external potassium could still block in the presence of 140 mM external sodium. The experiments were performed in symmetrical sodium solutions and we found that external potassium could still block the sodium current with a high affinity, Kd = ∼300 μM (Fig. 6 B, ○). The apparent potassium affinity is decreased in the presence of external sodium as compared with external NMG + (∼300 vs. ∼100 μM), presumably as a result of ion–ion interactions. Potassium and sodium are either competing for entry into the channel or else cannot occupy the pore simultaneously. External potassium blocks sodium currents at submillimolar concentrations independent of the occupancy of the external (C-type) site (compare Fig. 6 B, ○ and •).

Given the sensitivity of C-type inactivation to the presence of external ions (Lopez-Barneo et al., 1993 Baukrowitz and Yellen, 1995), the observation that it proceeds through a localized constriction of the outer mouth of the pore (Yellen et al., 1994 Liu et al., 1996), and the visualization of an ion bound at an external site in the crystal structure (Doyle et al., 1998), it is most likely that the site that controls C-type inactivation is the external site. Since the rate of C-type inactivation is unaffected when externally applied potassium is bound at its blocking site (Fig. 6 C), the high affinity site cannot be the external (C-type) site. External potassium presumably first binds to the C-type site but rapidly proceeds to a higher affinity blocking site, the occupancy of which does not interfere with C-type inactivation (Fig. 7, outlined scheme). The finding that external potassium still blocks sodium currents with a high affinity in the presence of external sodium indicates that the blocking ion presumably first displaces the bound sodium and subsequently binds at a higher affinity site in the pore. For example, sodium may bind and unbind rapidly at the C-type inactivation site while potassium enters deeper into the pore of the channel (Fig. 7, gray scheme). The rapid equilibration of the external (C-type) site is in agreement with the finding by Harris et al. (1998) that ions bound at the outermost site in the Shaker channel are in rapid equilibrium with the external solution.

It is not clear from the data whether the high affinity site accessible from the external solution is the site affected by the A463C mutation. The crystal structure of KcsA predicts that the A463C mutation alters the interaction between the side chain at position 463 in S6 and a conserved valine in the signature sequence (V443) that participates in the formation of an internal ion binding site in the pore (Fig. 1 Doyle et al., 1998). The structural alterations result in a decrease in the measured apparent internal potassium affinity (micromolar into the millimolar range) (Ogielska and Aldrich, 1998). However, present data indicates that externally presented potassium binds to a high affinity (micromolar) site deep within the pore of the A463C mutant channel. If potassium is binding to the same site, regardless of whether it entered the pore from the internal or external solution, then the measured apparent affinity difference must somehow depend on the pathway taken to reach that site, and specifically on the relative occupancy of the neighboring sites. Since potassium is a permeant blocker, the measured apparent affinities do not reflect equilibrium binding affinities and therefore need not be pathway independent. Alternatively, it is possible that the high affinity site is one of the two overlapping internal sites predicted by the KcsA structure. Perhaps the A463C mutation alters the conformation of the V443 backbone in such a way that a potassium ion can no longer rapidly equilibrate between the two internal sites and the different apparent affinities are reflective of the two possible positions a potassium ion can assume.

Since the channel inactivates at the same rate regardless of whether the high affinity site is occupied by potassium, the C-type inactivation gating conformational change must not involve a global alteration of the selectivity filter region of the pore. Our finding is in agreement with Harris et al. (1998), who showed that the C-type inactivation conformational change trapped barium at a high affinity potassium binding site in the Shaker channel pore. We have shown that when an internal site is loaded with K + , the Na + occupancy of the C-type site is decreased as a result of increased electrostatic interactions among ions in the pore (Fig. 5). Since a K + ion bound at a more internal site can repel Na + from the external (C-type) site, thus increasing the overall rate of inactivation, we wondered why the inactivation rate did not increase when externally applied K + is bound to a high affinity site deep within the pore (Fig. 6 C). We reasoned that perhaps, under these ionic conditions (NMG + outside and Na + inside), Na + occupied the C-type inactivation site so infrequently that the presence of the bound K + did not significantly affect the occupancy of the external (C-type) site. We expected that, under conditions when the C-type site is significantly occupied by Na + (symmetrical Na + solutions), the rate of inactivation would be affected when an externally applied K + ion is bound at a deeper high affinity site. However, we found that even in symmetrical sodium solutions the addition of 100 μM external K + blocked the channel without affecting the C-type inactivation rate (data not shown). This difference between the effects of internally and externally applied potassium ions on C-type inactivation is similar to the difference in apparent blocking affinities depending upon internal and external application. This most likely results from ion interactions and the differences in occupancy of the various binding sites depending upon the directions of Na + and K + movement in the channel. The complex behavior of permeant blockers illustrates the interdependence of binding interactions at the sites in the pore and the fact that the measured apparent affinities do not reflect true equilibrium binding constants.


Rezultati

In this section we test the validity of our hypothesis through simulations. The aim is to show that cation retention in thin processes leads to the formation of ionic microdomains at the PsC: Na + microdomains were experimentally observed [23] and our hypothesis may very well explain their origin. Specifically, we show that a K + microdomain formed at the PsC, provides the driving force for the return of K + to the extracellular space for uptake by the neurone, thereby preventing K + undershoot. We also show that our model can explain the slow decay of Na + at the PsC after a period of glutamate stimulation, which is in strong agreement with experimental observations [23]. Finally, we use our model to predict the dynamic behaviour of ions under more physiological conditions whereby we simulate neuronal co-release of K + and glutamate from the presynaptic terminal.

The simulation results presented in this section were obtained using Matlab 2015b 64 bit (Windows version) by Mathworks. The forward Euler method of integration was used for simulation with a fixed time step of Δt = 10μs.

ECS K + driven PsC K + microdomain formation

To explore how K + retention in the astrocyte process gives rise to a K + microdomain at the PsC and eliminates K + undershoot, several simulations were carried out with the presynaptic neurone stimulated using external currents to produce firing rates of 20Hz, 40Hz, 60Hz and 80Hz. These firing rates are all within physiological frequencies of most cortical pyramidal neurones and fast spiking neurones. The neural stimulus has a duration of

1 minute where the first 0.1 minute allows the model to reach a steady state condition and the stimulus ceases after 1min. Although this is a long period of time, it allowed an investigation into how extracellular and intracellular ionic concentrations would be affected during a sustained period of neural activity. For each simulation, PsECS [Glu] was held constant at the background level. Fig 4 describes [K + ] and [Na + ] dynamics for each of the 4 different stimuli where it can be seen that neuronal release of K + into the PsECS leads to an increase in the astrocyte membrane voltage (VA in Fig 4A) because of the change in ionic currents through the PsC membrane. It can also be seen that K + steadily increases within the PsECS ([K + ]PsECS in Fig 4B) and after a period of

0.8 minutes it approaches steady state at higher frequencies where the release rate of K + by the presynaptic neurone equates to the clearance rate by NKA and KB on both the PsC and the presynaptic terminal, and also K + lost into the GECS. It is also worth noting that as the concentration of K + increase in the PsC ([K + ]PsC in Fig 4C), the Na + concentration with the PsC decrease due to efflux by NKA at the PsC ([Na + ]PsC in Fig 4D). Note: the astrocyte membrane voltage VA, [K + ]PsECS and [K + ]PsC all increase with the presynaptic neurone firing rate while [Na + ]PsC smanjuje.

(A) Astrocyte membrane voltage (VA). (B) [K + ]PsECS. (C) [K + ]PsC. transient. (D) [Na + ]PsC transient.

During neural activity, the NKA and Kir channel currents are responsible for K + uptake while the background K + and KPF currents release K + from the PsC. These currents can be seen in Fig 5 where Fig 5B shows that, contrary to the current thinking [56], the NKA is the dominant driving force for K + uptake while Kir channel (Fig 5A) is much less so for K + clearance: furthermore, clearance by Kir diminishes over time because the changes in the associated reversal potential due to the [K + ]PsC microdomain. Fig 5C shows that IKPF is several orders of magnitude lower than IKir and therefore this slow leakage of K + away from the PsC appears to be a plausible explanation for the emergence of a K + microdomain. Note the saturation and subsequent fall off of IKB at higher frequencies is a direct result of the K + background reversal potential approaching VA. This is caused by the rapid build-up of K + in the PsECS and cradle. The high frequency oscillatory behaviour which appears as a thickening of Fig 5A–5D is due to the astrocytic response to the pulsed nature of presynaptic neuronal K + release. As the potassium in the PsECS fluctuates so does the astrocyte NKA pump and to a lesser extent the astrocyte membrane voltage. These fluctuations in the NKA and membrane voltage are also reflected in Na + and K + currents. Inserts in Fig 5A–5D, column 1, are used to show detail of astrocyte K + current dynamics in response to neurone K + release. Note: for clarity only the first column shows this detail as the dynamics for each current is similar for each of the stimulus frequencies.

(A) K + Kir struja. (B) K + NKA current. (C) K + current along the process. (D) Background K + current.

When the neurone stops releasing K + (

1min) it quickly flows from the PsECS into the ECS which reduces the K + gradient between the PsECS and PsC thereby reducing NKA pump rate, after which a net efflux of K + takes place from the stored K + in the associated microdomain. This points to a new theory whereby K + microdomain formation during neuronal excitation (due to ion retention in the astrocyte process) provides the driver for the return of K + to the PsECS, via background K + leak and Kir4.1 channels, for uptake by the neurone. Fig 6A shows the net transfer of K + across the perisynaptic membrane while Fig 6B shows the net current flow along the process (out of the perisynaptic cradle). During stimulation (0.1 min to 1min) it can be seen that there is a net transfer of K + into the perisynaptic cradle across the membrane (Fig 6A). Since the current flowing along the process to the soma (Fig 6B) is 3 orders of magnitude smaller than the currents entering the cradle, there is a net build-up of K + : essentially a K + microdomain forms because of the low conductance pathway from the cradle to the astrocyte soma. Furthermore, this microdomain allows the efflux of K + from the PsC into the PsECS after neurone stimulation ceases. This can be seen as a spike like current in Fig 6A after 1min and is more pronounced in the 80Hz simulation.

(A) The total K + current flowing across the perisynaptic membrane (IKPSC,mem). During neural activity (Start = 0.1 min) K + in the PsECS is removed by NKA and there is a net influx of K + . When neural activity stops (1min), K + is released back into the PsECS mediated by the background K + channel. This influx/efflux can be seen in A column 4. (B) K + current flowing along the process (IKPF).

Fig 7 shows the Na + currents for the four different stimulus frequencies. All Na + channels, except the NKA (Fig 7B) Na + current, result in Na + influx to the PsC. When the neurone stops firing there is a net influx of Na + into the PsC. The decrease in INaB (Fig 7A) can be explained as follows: Since INaB is dependent on the astrocyte membrane potential as well as Na + gradient there is a sharp decrease in the current due to the astrocyte membrane potential depolarising.

(A) Background Na + current. (B) NKA current. (C) Na + current along the process. During neural activity, the NKA pumps Na + from the cell to allow for K + uptake, therefore there is a net decrease in [Na + ]PsC. When the neurone stops firing, NKA slows down and there is a net uptake of Na + via the remaining channels.

Glutamate driven PsC Na + microdomain formation

As well as K + buffering, astrocytes also provide a critical role in glutamate uptake and recycling via the glutamate-glutamine cycle (GGC) [57]. In this simulation, the role of glutamate transport via EAAT1/2 is investigated and results show that the slow leakage of Na + ions in the astrocyte process causes Na + to increase in the PsC before being returned to the PsECS via the NKA. These results support previously published experimental work [23]: there is no neuronal excitation and therefore the concentration of K + in the PsECS is held constant. The concentration of glutamate in the PsECS was modulated using a Gaussian function as shown in Fig 8A. Fig 8B–8D presents the results of the PsC ionic [K + ]PsC and [Na + ]PsC concentrations and membrane voltage, VA, for this simulation.

(A) Glutamate is injected into the PsECS with a Gaussian distribution for

10s with a maximum concentration of 1000μM. (B) PsC membrane voltage depolarises with ionic changes in the PsC. (C) [Glu]PsECS increase causes EAAT1/2 activation and a thereby removing K + and (D) the uptake of Na + .

From Fig 8C and 8D we clearly see that the [K + ]PsC decreases while [Na + ]PsC increases, this is the opposite dynamics to that observed in Fig 4C and 4D. This is because K + in the PsECS is now held constant at 3 mM and therefore all K + channels except the NKA and slow leakage through the astrocyte process remove K + from the PsC (Fig 9) resulting in a net K + efflux. The main driving force behind Na + uptake by the PsC is the EAAT1/2 transporter which is also responsible for the removal of glutamate from the PsECS (Fig 10).

(A) K + EAAT1/2 current. (B) K + Kir struja. (C) K + NKA current. (D) K + current along the process. (E) K + background current.

(A) Na + EAAT1/2 current. (B) Background Na + current. (C) Na + NKA current. (D) Na + current in the astrocyte process. With the increase of [Glu]PsECS, EAAT1/2 transport rate is increased to remove glutamate from the PsECS, in turn Na + is taken up. Furthermore, as [Na + ]PsC increases, the rate of Na + influx from the background channel decreases. All other channels remove Na + until [Na + ]PsECS reaches steady state conditions once again.

During [Glu]PsECS injection, the EAAT1/2 and Kir release K + at an accelerated rate. This is opposed by NKA and the transport of K + from the astrocyte soma to the PsC. When glutamate falls to baseline levels, the EAAT1/2 and Kir channels quickly revert to their initial rates. NKA and transport of K + from the astrocyte soma is then able to establish baseline ionic concentrations at the PsC.

As in the previous simulation, retention of Na + ions as they flow within the astrocyte process substantially limits the transport rate of these ions away from the PsC. In this case, Na + is restricted and therefore a Na + microdomain forms at the PsC. Note: similar to the results presented in [23] there is a long decay (

80s) transient of Na + which far outlasts the glutamate signal decrease (Fig 8D) and we propose that this is due to the slow removal of Na + by the NKA. These observations could explain previously observed experimental results [23].

ECS K + and Glu driven PsC microdomain formation

The previous two simulations have shown that K + or Na + microdomains form in the PsC when the system is stimulated with PsECS changes in K + or Glu respectively. However, while these simulations show that our hypothesis could potentially explain experimental observations, we now wish to use our model to predict ionic dynamics at the PsC under physiological conditions where both K + and Glu are released at the presynaptic terminal. In this case K + is released by the neurone as before and a 100 μM puff of Glu is released into the PsECS, with each spike event. Presynaptic neurone firing rates are 20Hz, 40Hz, 60Hz and 80Hz, for a period of 0.1min to 1min. The results presented in Fig 11 show that the overall behaviour of the model, i.e. microdomain formation of K + in the PsC, occurs. However, the astrocyte membrane voltage VA oscillates (

7mV amplitude) (Fig 11A) caused by the periodic reversal of the Kir channel (See Fig 12A). This reversal is caused by the efflux of K + via the EAAT1/2 (Fig 12E) channel. Moreover, the dynamic behaviour of the reversal potential of the Kir i VA continuously cause reversal of the overall polarity (Fig 13), thus causing the Kir channel to periodically reverse direction resulting in an efflux of K + into the ECS this can be seen as oscillations in [K + ]PsECS. It can be observed in Fig 11C and 11D that a K + microdomain is formed in the PsC and its magnitude increases with frequency while the magnitude of Na + reduces. This is due to the behaviour of the K + uptake by NKA dominating over the K + efflux pathways (See Fig 12).

(A) Astrocyte membrane voltage (VA). (B) [K + ]PsECS. (C) [K + ]PsC. transient. (D) [Na + ]PsC transient.

(A) K + Kir struja. (B) K + NKA current. (C) K + current along the process. (D) Background K + current. (E) K + EAAT current. Note: similar to the first simulation, as the neurone firing rate increases the magnitude of all currents also increase.

(A) The astrocyte membrane voltage (blue) and the Kir reversal potential continually cross over during neurone stimulation. This results in periodic reversal of the Kir kanal. (B) Magnification of A for the last few hundred milliseconds of stimulation.

Fig 14 shows the Na + currents for the four different stimulus frequencies. As expected all Na + channels on the PsC membrane, except the NKA (Fig 14B) result in Na + influx to the PsC. INaEAAT has a large peak amplitude for a short duration (few milliseconds) due to the EAAT channel slowing down after removal of Glu from PsECS.

(A) Background Na + current. (B) NKA current. (C) Na + current along the process. (D) EAAT Na + current During neural activity, the NKA pumps Na + from the cell to allow for K + uptake, therefore there is a net decrease in [Na + ]PsC. When the neurone stops firing, NKA slows down and there is a net uptake of Na + via the remaining channels.

Parameter sensitivity

Having analysed the formation of microdomains and model behaviour in the previous three simulations we now explore the sensitivity of the model to model parameters. These parameters are PsC surface area, the maximum NKA pump rate, Pmax, and the potential barrier to ion flow along the process, φw. In these simulations a neuronal firing rate of 40Hz was chosen.

Microdomain Sensitivity to PsC Surface Area (SA). Three different values of PsC SA were chosen for this simulation PsC SA × 0.75, PsC SA × 1 and PsC SA × 1.25. The results of these simulations are shown in Fig 15 where it can clearly be seen that the amplitude of the K + microdomain increased with PsC SA with a corresponding drop in the concentration of Na + . Also, the K + and Na + currents efflux/influx also increased with PsC SA (See Supplementary S1 Fig for the changes in K + currents).

(A) PsC K + concentration. (B) PsC Na + concentration. As the PsC surface area increases so the amplitude of the K + microdomain increases and the Na + microdomain amplitude decreases.

Microdomain Sensitivity to Pmax. Four different values of Pmax were chosen for this simulation Pmax × 0.2, Pmax × 0.5, Pmax × 1 and Pmax × 5. The results of these simulations are shown in Fig 16 where it can clearly be seen that [K + ]PsC and [Na + ]PsC is strongly dependent on Pmax. Using the Pmax x 0.2 value causes [K + ]PsC to decrease and [Na + ]PsC to increases and as Pmax increases, [K + ]PsC begins to form a microdomain with [Na + ]PsC steadily decreasing. From these simulations we can conclude that when the NKA pump rate is low it is no longer the dominant co-transporter and both the EAAT co-transporter and Kir channel dictate [K + ]PsC and [Na + ]PsC dinamika. The opposite is true when the pump rate is large.

Microdomain formation for different values of NKA maximum pump rate: (A) [K + ]PsC as a function of Pmax and (B) [Na + ]PsC as a function of Pmax.

Microdomain Sensitivity to φw. In this simulation φw was varied from 4 kBT to 15 kBT. Fig 17 shows the peak K + current along the process for the different values of φw. As φw is decreased, the peak current along the process increases exponentially. Therefore, with decreasing φw the formation of a microdomain becomes less likely as IKPF,max is increasing and eventually IKPF,max approaches an electro-diffusion limited model with no likelihood of a microdomain forming at the PsC.

From these simulations it is clear that the mechanism responsible for the formation of microdomains is the well formation along the process which effectively semi-isolate the PsC from the astrocyte soma when φw is 10 kBT or greater. It is also clear that the PsC SA can limit the maximum amplitude of the microdomain concentration. This is due to the increase/decrease of ion channel densities on the membrane of the PsC. Moreover, the NKA maximum pump rate also has an important role in the formation of microdomains whereby if the pump rate is low then K + clearance by NKA weakens effectively these to ion transporters compete to move K + and Na + ion across the membrane but in opposite directions.


A.M.-C., L.P., M.C.F. and M.A. designed research P.K. performed manual patch-clamp experiments M.C.F., L.P. and A.V.T. performed photolabeling-coupled electrophysiology experiments H.T. performed molecular docking experiments A.M.-C., L.P. and K.Z. contributed to the methodology and provided resources M.C.F., P.K., L.P., H.T. and A.M. analysed data M.C.F., L.P., H.T. and A.M. wrote the manuscript all authors have read and approved the manuscript.

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Why are potassium channels slower than sodium channels? - Biologija

Nervous System: Neurons

A. Neurons - conduct nerve impulses

  • dendrites – short, highly branched, receive signal and carry it toward cell body
  • axons – long, carry signal away from cell body terminal branches
  • cell body – large section, contains the nucleus and other goodies
  • myelin – lipid coating derived from membrane of Schwann cells acts as a kind of insulation
  • Nodes of Ranvier – gaps between Schwann cell
  • synaptic terminal – end of the axon, junction with another neuron or effector
  • neuron form/function related - see diagram/overhead of different neurons

B. Supporting, or glial, cells.
The function of these cells is to structurally reinforce, protect, insulate or otherwise assist the neurons. Nervous system housekeeper cells. There are lots of glial cells in fact, in the brain there are 10-50x more glial cells than neurons. Glial cells include:

  1. Astrocytes (star-shaped they have many functions including - line capillaries, part of the blood brain barrier which serves to keep toxins from the brain, phagocytosis)
  2. Microglia - phagocytic cells, remove cellular debris
  3. Oligodendrocytes (wrap and insulate nerves).
  4. Schwann cells – wrap around neuron like a "jelly roll". Have a myelin (fatty substance) sheath which is like insulation on a wire. Vertebrates outside CNS in peripheral nervous system

C. Construction.
Neurons are bundled together to form nerves. Ganglia - clumps of cell bodies

II. Membrane Potentials

A. Charge distribution.
At rest, neurons (and most other cells, too) exhibit an electrical charge difference across the membrane. This difference, or membrane potential, can be measured with tiny electrodes connected to a voltmeter or an oscilloscope. At rest, the membrane potential is -60 millivolts (mV), which means that the inside of the cell is negative in comparison to the outside. This is ultimately due to an unequal distribution of ions, especially sodium and potassium, between the outside and inside of the cell (see Table 1).

B. Nernst Equation.
Relates the ion distribution to the membrane potential when the cell is at rest. See equation in text. Thus, if the ion distribution is know, the membrane potential can be calculated.


C. Why the unequal charge distribution?
The membrane is permeable to both potassium and sodium (recall that ions move through membranes slowly). These ions diffuse through protein channels in the membrane. There are separate "leakage channels" for both potassium and sodium and they are always "open", thereby permitting the diffusion of sodium and potassium down their concentration gradient. Thus, potassium diffuses from inside to outside and sodium diffuses from outside to inside.

The membrane is much more permeable to potassium so there is a net flux (leakage) of potassium, and hence positively-charged particles, out of the cell. This is the result of more potassium leakage channels and a steeper concentration gradient in potassium between inside and outside of the cell. Since the cell is permeable to both sodium and potassium, over time, you would expect an equilibrium state would be reached at which point the concentration of sodium and potassium would be approximately equal inside and outside of the cell, and hence no membrane potential.

So, what maintains the unequal charge distribution? The sodium-potassium pump! The pump is another integral protein complex that requires ATP and actively transports sodium out of the cell and potassium into the cell. For every three sodium that are moved out, two potassium are moved in. Thus, the sodium-potassium pump maintains the ion gradient across the membrane. It is an active (energy-requiring process) because it is moving the ions against their concentration gradient.

III. Excitability.
Some cells are excitable, that is, they can change their membrane potential. This occurs when the membrane depolarizuje (switches polarity - the inside becomes positive relative to the outside) or hyperpolarizes (becomes more negative inside relative to outside). A nerve impulse is simply a reapid change (reversal) neuronal membrane permeability.

A. Special Ion Channels (Voltage-Gated Ion Channels Voltage-Activated Ion Channels)
In the membrane there are proteins that respond to changes in membrane potential/voltage. In response, these gates permit the passage of sodium or potassium in/out of the cell. Let's examine the voltage-activated gates of sodium and potassium.

  • Gate 1 (Activation Gate) - closed at rest depolarization causes it to open rapidly
  • Gate 2 (Inactivation Gate) - open at rest, depolarization causes it to close slowly.
  • At rest, sodium cannot pass through these gates because gate 1 is closed.
  • closed at rest
  • depolarization causes it to open slowly.

B. Opening the gates. Consider what happens when.

1. . the potassium gate opens:

potassium gate open potassium diffuses out inside becomes more negatively charged, outside more positive hyperpolarization (membrane potential becomes more negative).

2. . the sodium activation gate opens:

sodium gate open sodium diffuses in cell interior becomes positive, outside more negative depolarization. If the depolarization reaches ca. -50 to -55 mV it will trigger an action potential, which is an all-or-nothing response.


IV. Akcijski potencijal
A nerve impulse is rapid (lasts 1-2 msec) transient depolarization of the membrane. It sweeps down the neuron like a wave (at a rate of about 100 m sec -1 ).

A. Phases.
An action potential can be dissected into the following phases:

    Resting phase.
    Membrane potential -60 mV potassium gate closed sodium gate 1 closed sodium gate 2 open. No "excess" movement of sodium or potassium across membrane

B. Refractory period.
Time period following an action potential during which the cell gates are returning to their original positions. The cell cannot respond to another nerve impulse during this period.

C. Nerve impulse.
Wave of action potential spreads along the neuron. It doesn’t turn back on itself because of the refractory period.

D. Rate of conduction.
In unmyelinated nerves, speed is directly related to the diameter of the axon. Rates vary from about 1 – 10 m s -1 . Saltatory conduction occurs when action potentials jump between nodes of Ranvier. These occur much faster. Voltage channels just in node area.

V. Communication between adjacent nerve cells.
A few neurons are hard-wired (tj., are directly connected to one another, as in gap junctions). Oni se zovu electrical synapses and important for really fast responses. However, most neurons are not in contact with one another, rather there is a gap (ca. 25 nm), called the sinapsa or synaptic cleft, between the adjacent cells. So, how does the nerve impulse get across the gap in these hemijske sinapse?? Chemical signals = neurotransmitters.

Action potential arrives at synaptic terminal stimulates voltage-gated calcium channels to open uptake of calcium stimulates migration of vesicles containing neurotransmitter to the membrane surface vesicles fuse with the membrane releasing their contents into the gap diffuses across gap binds to appropriate receptor opens gates for sodium and potassium ili stimulates a second messenger system that opens sodium and potassium gates causes membrane depolarization (or hyperpolarization) threshold potential action potential.

A. Various neurotransmitters have been identified.
Many known. Acetylcholine is a common one involved in muscle contractions and other responses. Norepinephrine, serotonin, and dopamine are involved in mood, mental state, ADD, schizophrenia. Gamma-amino butyric acid (GABA) is in the spinal cord and brain. The opiate types, endorphins and enkephalins are involved in pain perception.

B. Too much neurotransmitters is a bad thing.
In other words, the neurotransmitters must be removed after they do their job. Many are broken down by enzymes. For example, acetylcholinesterase breaks down acetylcholine. Others like serotonin are reabsorbed by the axon. Some drugs work by the inhibition of the reuptake of the neurotransmitter. For example, cocaine prevents the reuptake of serotonin.

C. Secondary messenger system.
As mentioned, the action of the neurotransmitter may be mediated by a secondary messenger system. In this case: neurotransmitter binds to the receptor protein activates G proteins adenyl cyclase converts ATP to cyclic AMP (cAMP) activates kinases phosphorylates proteins closes potassium channel depolarization

VI. Excitatory & Inhibitory Impulses
Some neurons cause depolarization of membrane (i.e, motor neuron and skeletal muscle). These are called excitatory. Other neurons cause hyperpolarization (inhibitory). GABA and glycine act in this manner. Ultimately, the final response of a nerve is a sum of the excitatory and inhibitory responses received - typically at the axon hillock (at base of axon, not insulated by glial cells, many voltage gated channels)

VII. Chemicals and Nerves
These can exert their effect in a variety of places. For example, DDT interferes with the sodium/potassium pump the anesthetics cocaine, lidocaine and procaine block sodium ion channels. Some poisons mimic the effect of the naturally-occurring neurotransmitter (i.e., hallucinogenic drugs, muscarine)

VII. Video - "The Neuron Suite" by James Burke. Available at Alcuin Library - QP 376.B8.


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