Voltage Gated Ion channels Flashcards
what facet of a channel pore will determine the size of the pore and hence ion selectivity?
- Amino acid residue side/diameter
Selectivity filter for K channels (think about the AA residues in Pore loop)?
hint: two components
- carbonyl backbone groups of AAs
- TVGYG in P loop
Selectivity filter for Na channels (think AA residues)?
-DEKA side chains
which transmembrane segment acts as the voltage sensor that detects depolarization, and what properties does it have?
- segment 4
- has Arg residues (positively charged)
what is the effect of a positive membrane potential on the S4 helical structure of a voltage gated ion channel?
- positive residues of helix are repelled, leading to helix movement out of channel i.e. conformational change–> channel opening
what causes the preceding outward current during recording of Na ion movement during depolarization
- outward movement of positive charges within S4
How are V-gated Na/Cl channels different from K channels (think about peptides needed to form channel)?
- Na/Cl channels are formed from a single polypeptide
- K channels formed from 4 polypeptides joined together
Describe the structure of the Alpha subunits voltage gated ion channels
each alpha subunit has 6 transmembrane regions consisting of a loop between TM5-TM6
significance for IFM (isoleucine, phenylalanine, Methionine) peptide motif in NaV channels?
- hydrophobic triad that forms inactivation gate– acts as pore blocker
what is the result of having an Na channel:
- with (Wtype) IFM
- without (IFM–>QQQ mutation) IFM motif
IFM— keeps Na current brief during depolarization
IFM–>QQQ: no more inactivation– persisting Na cannel activation as long as there is stimulus
how long do NaV channels typically stay open for?
- what condition occurs after channel opens and repolarization begins?
- 1 milisecond
- channel opening is followed by closure/refractory period– no conductance
what single-current channel state is transiently favored by depolarization?
- Open State
what single-current channel state is favored by hyperpolarization?
- resting (closed) state
what single-current channel state is favored by maintained depolarization?
- inactivated state
what is a consideration when thinking about P0 (probability of channel opening) value when calculating Whole cell current (I)
- NOT all channels will be opend at x point in time
what kind of feedback loop drives Na entry upon activation Gate?
- positive feedback loop
describe the relationship between Na current and magnitude of depolarization
- amount of Na current (which is proportional to number of channels open) is positively dependent on magnitude of depolarization
At one point, depolarization causes Na current to decrease
- why is that so?
- depolarization eventually nears ENa— Na driving force into cell decreases
what do the activation/inactivation gate both have in common (think about relationship with voltage) ?
- they are both voltage dependent
what subunit(s) is responsible for modulating NaV channel gating?
- B1/3, b2/4 Immunoglobin-like proteins made of Beta auxiliary subunits
what condition is a mutation in beta1 subunit of Beta1/3 protein of NaV linked to ?
- epileptic seizures
of the 9 NaV Alpha subunits (SNC1A, SCN2A, Nac1.1-1.9) , which subunits are predominant in CNS?
- what common response do they have in relation to TTX toxin?
- NaV1.1,2,3
- all sensitive
what do Nav1.8,NaV.19 isoforms of peripheral NS (i.e. sensory dorsal root ganglion) have in common?
- resistant to TTX
effect of reduced NaV1.8 expression in mice?
- attenuated neuropathic pain
- mice lacking 1.8 are analgesic to noxious mechanical stimuli
describe NaV1.7 (as well as result of lack of 1.7) and its role in pain sensing
-what part of body (hint: nociceptive cells)
- NaV1.7 KO effect on inflammatory pain response?
- selectively found in dorsal root ganglion neurons (most in nociceptive cells)
- TTX sensitive
lack of 1.7 (mice)
- increased mechanical/thermal pain thresholds,
- REDUCED inflammatory pain responses
result of NaV1.7 gain of function mutation (think symptoms of erythromelalgia autosomal dominant disorder)?
loss of function of Nav1.7?
- severe burning sensation, redness in response to thermal stimuli
LOSS OF FUNCTION
- unable to feel pain, acute/chronic
what is the effect of scorpion toxin on grasshopper mouse in terms of pain perception? compared to house mouse?
- why is this so?
- bonus: what is typical effect of venom on NaV1.7?
- Grasshopper mouse is unaffected where house mouse perceives the venom
- due to venom binding to NaV1.8 and inactivating it— diminished Na current
Venom leads to NaV1.7 activation– pain sensing
properties of Local anesthetics that allow NaV channel blockage (?
- LA´s contain aromatic group that link to basic side chain residue of NaV channel
name of bond formed in Na blockade between Local anesthetic aromatic group and basic side chain
Amide/ester bond
what is the blocking action of local LA´s dependent on (think properties of channel/things affecting NaV)?
- USE-DEPENDENT: Drug can only block from inside, hence channel must be in open state
- VOLTAGE DEPENDENT: depolarization enhances block
effect of Local anesthetics (LA) on Na inactivation process?
enhances it
do LA drugs take the hydrophilic or hydrophobic pathway to enter the Na channel?
(note: at phys. pH, LA´s are positively charged)
- trick question—can take BOTH pathways
HYDROPHOBIC– from extracell.
HYDROPHILIC– entering from intracell.
Deletethis card
TTX affects neuronal, skeletal, and cardiac NaVs to varying degrees
which channels are blocked by a nanomolar dose of TTX (sensitive) and which ones are blocked by a micromolar (less sensitive) dose?
- neuronal, skeletal NaV: nM
- cardiac NaV: mM
Residues in P-loop of NaV domains 1-3 contribute to TTX sensitivity
in cardiac cells, what happens when the residue (cystine) responsible for microM TTX sensitivity is mutated to tyrosine (Y)?
note: Y residue is present in P-loop of neuronal channels
in Cardiac NaV P-loop: E,C residues mediate TTX sensitivity
Tyrosine mutation greatly increases affinity for TTX
For NaV channels of peripheral NS, what residue is present in the P-loop (same position as Cysteine in neuronal channels) resulting in decreased TTX sensitivity?
Serine
what toxin is responsible for paralytic shellfish poisoning and blocks Na at same site as TTX?
Saxitoxin (STX)
what toxins, derived from molluscs, are a small positively charged peptide, with toxin version GIIIA selectively blocking NaVs in skeletal muscle (little effect on neuronal NaVs)
Micro-conotoxins
what toxins modulate Na channels so that they remain open longer?
- Batrachotoxin
- pyrethrins
- b-scorpion toxin
describe batrachotoxin mechanism (poison frogs, bird skin/feather) of action on NaV in terms of
- where it binds from
- effect on channel state
effect on activation voltage
- inhibits inactivation, shifts
- activation voltage shifted to more -ve potential– channels open longer
- enters the cell and binds internally
describe Pyrethrin (similar to DDT insecticide) and its mechanism of action in terms of
- where it is derived from
- what organisms it affects
- effect on activation/inactivation
- from plant, natural insecticides
- non-toxic for mammals, effects insects
- prolongs activation, inhibits inactivation
describe b-scorpion toxin and its effect on NaV channels in terms of:
- where it binds
- physiolog. requirements for bound toxin to induce effect
- effect on activation
- binds to outer side of IIS4 voltage sensor
- only becomes effective upon depolarization
- enhances activation via shifting voltage potential (activation at more -ive potentials)
Describe Sea anemone, a-scorpion toxins effect on NaV channels in terms of:
- where they bind
- effect on gated state and overall activation
- bind receptor site at extracell. end of IVS4 segment (which initiates fast inactivation)
- normal gating movement of IVS4 prevented— remains in activated/gated state
- uncouples inactivation form activation
Describe ventricular arrythmia (congenital long QT syndrome) and its characteristics:
- effect on Na current?
- what mutations give rise to these properties?
- prolonged AP duration
- persistent inward Na current causing abnormal repolarization
- alpha subunit mutations in NaV1.5
describe Inherited epilepsy syndrome characteristics and mutations that cause it?
- what NaV channels are mutations found in?
- mutations in alpha subunit, some in beta subunit
- mutations occur in Nav1.1, NaV1.2 subunits
- MAY induce persistent Na currents
- mutations may alter voltage dependence of activation/inactivation
– ENHANCED excitability
describe function of CaV channels and cellular events in which they are involved in
Intracell. Ca conc. regulation via facilitating Ca entry into cells upon depolarization
CELLULAR EVENTS
- AP generation
- muscle contraction
-NT release
- cell differentiation/ gene expression
what are modulators of CaV channels?
- transmitters
- protein kinases
- toxins
what is the key trigger for fast-evoked transmitter release? (hint: its not K,Na ions)
- elevation of intracell. Ca
describe CaV structure
i.e. number of domains, TM segments
- residue present in Pore region
- any auxiliary SU´s?
4 repeat domains, 6 TM segments
- membrane associated loop betw/ TM5-TM6 (same as Na)
- Glutamic acid (E)
- Yes
Describe L-type (HVA) CaV channels in terms of:
- depolarization amplitude required for opening
- inactivation behavior
- functions
- blockers
- requires large depolarization to elicit opening
- variable inactivation (may inactivate, may not)
- E-C coupling, hormone sec., muscle contraction
- blocked by DHPs
Describe T-type (LVA) CaV channels in terms of:
- depolarization amplitude required for opening
- inactivation behavior
- functions
- blockers
- small depolarization (-60 mV) elicits opening
- exhibits rapid, voltage-dep inactivation (like in NaV)
- repetitive firing
- blocked by mibefradil
what do N,P/Q, R type CaV channels all have in common?
- compared to L,T type CaV?
- mostly found in neuronal cells (hence role in nt release) whereas L,T are widely expressed in various cells/tissue
describe CaV1.1-4 current type and location where it is typically found
- L type
- cardiac, skeletal muscle, neurons, endocrine cells
describe CaV2.1 current type and its location
- P (purkinje)/Q (cerebellar granule cells)
- nerve terminals, dendrites
CaV2.2 current type and location (hint: role in nt release)
-N type
- nerve terminals, dendrites
CaV2.3 current type and location
- R type (resistant component of neuronal current)
- nerve terminals, dendrites, cell bodies
CaV3.1-3 Current type and location
- T type
- cardiac, smooth muscle, neurons
differences between A1 CaV SU and Na channel SU?
- think in terms of domains, TM segments in each domain, linker loop
trick question: no difference, A1 subunit is structurally same as NaV one
describe beta subunit of CaV channel in terms of:
- location
- whether there is a specific number of b Su associated w/ A at a time
- intracellularly located
- there can be multiple b subunits associated w/ 1 A
describe glycosylated A2, Delta SU (component of CaV) in terms of:
- function
- location
- associations
- found extracellularly; A2 attached to membrane via Sulfide linkage w/ delta SU
– Delta SU anchors A2 w/ A1
Describe Gamma (Y9) glycoprotein in terms of:
- structure
- genetic makeup
- function
- 4 TM segments
- 8 genes encoding for it
- function unclear
L-type CaV channels display mode switching i.e multiple gating kinetics.
- describe the 3 different kinetic modes
Mode 0– no channel openings
Mode 1– brief channel opening state
Mode 2– prolonged channel opening
in what way do catecholamines modulate current of CaV1 channel family in the heart?
- consider consequences in terms of inotropic action (muscle contraction)
Catecholamines modulate CaV1´s via phosphorylation– enhance Ca current
– underlies positive inotropic action
in what way do beta-adrenergic agonists modulate CaV1 (L-type) channel activity?
- consider effect on AP amplitude (i.e. muscle contractility/rate)
- what kinetic mode is induced?
- CaV1 phosphorylation by agonist
- increased AP amplitude, contractility/rate
- mode 2 induced
what second messenger mimics the effect of beta-adrenergic stimulation of CaV1 channels through PKA activity?
PKA leads to phosphorylation of which residue (at what site?) of CaV1.2?
- cAMP
- Ser1928
effect of B2a SU on b-adrenergic agonist activation of CaV1.2?
- what residue site implicated?
- enhanced b-adrenergic agonist activation of CaV1.2
- phosphorylation of Ser478/479
Difference between CaV1 and CaV2(P/Q,N) regulation?
- CaV1 is regulated by kinases/phosphorylation
- CaV2 regulated by G-protein coupling
result of G protein coupling on CaV2 Ca current?
- effect on synapt. transmission?
- effect on Ca entry at nerve terminal?
- inhibition via local membrane action
- decreased synaptic transmission
- reduced Ca entry
type of inhibition caused by G protein beta/gamma dimer?
- (bonus: where else do we see beta/gamma GP dimer inhibit Ca entry?)
- tonic inhibition
- presynaptic GABAb receptor
what mechanism reverse G-Protein tonic inhibition of Ca current?
- Depolarizing prepulse
issues related to A1 KO in CaV1 (L-type) family?
(hint: think about location in which they are found)
- blockers of CaV1?
- cardiac, skeletal, dysfunction– endocrine as well
phenotypes: cardiac arrythmia, deafness, weak muscle contraction
- DHPs
issues related to A1 KO in CaV2.1 (P/Q) family?
blocker?
- pain hyposensitivity
- agatoxin IVA
issues related to A1 KO in CaV2.2 (N) family?
(bonus: describe effect of baclofen on mutant N type CaVs)
- pain hyposensitivity
- resistant to baclofen induced seizures
issues related to A1 KO in CaV3 (T) family? (remember: T types expressed in cardiac, smooth muscle (Cav3.3 exception– in neural tissue))
blocker?
- compromised vascular function
- blockers include kurotoxin (3.2 is blocked by nickel)
Mechanism of Dihydropyridine (DHP) antagonists in L type CaV inhibition?
- what kinetic mode do they encourage?
- effect on gating behavior?
- allosteric modulators (gating behavior altering)
- stabilize mode 0
what is nifedipine and describe its effect on L type CaV channels
- i.e. what kinetic mode does it encourage?
- CaV1 agonist– stabilizes mode 2
what are the 3 major classes of L- type CaV channel antagonists
describe their clinical uses.
what happens if they bind the same site on channel (note: will still cause blocked Ca influx) ?
- DHps
- PAAs (verapamil)
- Benzothiazepines (diltiazem)
treatment of hypertension, cardiac arrythmia, ischaemic heart disease
- binding of same site will induce different effects/ mechanisms of blockage
location of binding site of DHPs
(hint: particularly TM segments in domains III-IV, I as well)
- S5,S6 TM segments of A1 SU
what region are these DHP sites found in?
- Y1485
- M1486
- I1493
- S6 TM segment of domain IV (aka IVS6)
describe phenylalkylamines (PAAs) L-type CaV blockage mechanism in terms of:
- location of binding
- conditions of effect (voltage/use dep/independent?)–
bonus: what antagonist of Na channel shares similar action i.e use dep
- intracellular binding— inner end of TM pore i.e domain III-IV S6
- Ca blockage is USE-DEPENDENT– open channel block
action of benzothiazepine antagonists on L-type CaV channels?
- location of binding
- act extracellularly
- bind residues of Domain IV S5-S6 linker
Toxin that interacts with N type CaV
- causes chronic neuropathic pain
Ziconotide
mechanism of gabapentin/pregabalin in treatment of epilepsy, chronic pain?
- interaction with A2delta subunit of CaVs
describe Type 1 hypokalemic periodic paralysis, a CaV1.x channelopathy, in terms of:
- what CaV channel (hint: L-type) it effects
- location of mutation (hint: voltage sensor segment)
- consequence of mutation
- CaV1.1 in skeletal muscle
- S4 segment
- reduced Ca current– muscle weakness
describe Timothy syndrome in terms of:
- what CaV channel (hint: L-type) harbors mutation
- consequence of mutation
- CaV1.2 mutation
- loss of inactivation (i.e. inc. Ca entry) —> cardiac dysregulation
describe Night blindness channelopathy in terms of:
- what CaV channel (hint: L-type) it effects
- consequence of mutation (hint: think NT release at retinal photoreceptor terminals)
- multiple mutations associated with loss of CaV1.4 function
–decreased NT release from retinal photoreceptor terminals
what CaV channel mutations are familial hemiplegic migraines linked to(hint: non L/T type,and affects nt release)?
- consequences of mutation?
- CaV 2.1 (P/Q)
- increased channel activity, NT release
describe ATAXIA type 2 channelopathy
- what CaV channel mutated (hint:non L-type)
- consequences of mutation
- CaV2.1 gene disruption
- cannot form functional channels
describe epilepsy channelopathy and their effect on P/Q CaV function
- what SU´s mutated
- auxiliary subunit mutations
- Ca channel misfunction
describe autism channelopathy
- channels implicated
- bonus: what might parents of autistic child suffer from
- CaV3.2 (T type)
- parents might suffer from epilepsy– Ca channel misfunction
what V-gated ion channel is most diverse genetically and in terms of physiological function?
- Kv channels
Kv channel regulate what physiological functions
- cell volume control
- Vm/excitability control
- salt, hormone, NT secretion
intrinsic/extrinsic factors in which Kv channels are regulated?
- hormones/NTs
- kinases, phosphatases
- G proteins
- Ca/ATP conc. in cytoplasm
what K channels consist of 6 transmembrane segments?
- Kv channels
- hERG channels
- Ca activated K channels
- KCNQ channels
in the ball and chain model of ´A´ type Kv channels, what AA residues form ball/ chain that mediate fast inactivation?
BALL– first 20 amino acids form hydrophobic/charged surface domain
CHAIN– the following 50-60 AA´s
characteristic of ´A´ type Kv channels?
- rapid inactivation following opening of channel
by what interaction mechanism does “ball” component of Kv channel lead to inactivation?
hint: think of terminal
- N type/ N terminal structure involvement facilitates Kv inactivation
3 Ca activated Kv and their location
- Large conductance (maxi-K) – 250 pS– ubiquitously expressed (except in heart)
- Intermediate conductance channels– 60-100 pS
- Small conductance (SK)– <20 pS– function in neuron in AHP mediating
as Maxi K activation voltage is not fixed (dependent on intracell. Ca conc), what is the effect of increasing Ca conc. on Maxi-K channels?
- as intracell. Ca increases, electrical energy required for channel opening is reduced
given that Maxi-K alpha subunit is encoded for by single gene (slo) what is the reason for Maxi-K diversity?
- alternative splice exons in maxi-k gene
- accessory beta subunits interacting w/ alpha SU
- S0/N-terminal domains; needed for beta SU channel modulating
in what Kv channels is the Alpha subunit primary sequence found?
bonus: in what Kv channels is S0 terminal domain found?
- primary sequence is homol. to ALL Kv channels (trick question)
- S0 found only in maxi-k channels
describe the two independent sensing mechanisms of Maxi K channel (Voltage sensors, ligand gating mechanism)
- what segments make them up?
- Voltage sensor; made of S4 TM segment of alpha subunit
Ligand gating mechanism
- made of hydrophobic domain S7-S10
- S7-S8 is RCK (regulator of K conductance) domain
S8-S9 linker makes tail domain
effect of intracellular ring (RCK domain) on S4 (voltage sensing) upon Ca binding?
- Ca binding to intracellular RCK domain leads to expansion– acts on S4 gate, channel opens
what characteristic of A1 SU Tail domain makes it capable of being Ca sensitive?
- what would its mutation do to Ca sensing?
- D residue between S9-S10
- affect high affinity sensing
effect of increasing intracell. Ca conc. on RCK domain and overall channel gating?
- more Ca binding to RCK–> interacts with Ca bowl/tail –> greater force of channel opening
relationship between increased opening force on channel gates via RCK binding and S4 driven conformational change of Maxi-K channel?
i.e. which one depends on which?
- trick Q: they are INDEPENDENT of one another
describe physiological roles of Maxi-K
-cochlear hair frequency encoding
- opposes vasoconstriction by inducing hyperpol. in smooth muscle
- contributes to AHP i.e. refractory period
what class of K channels is most abundant
- hint: 4TM/2P family
ex. TWIK, TRAAK, TREK, TASK
K2p weak inward rectifiers
describe TREK1 (of K2p channel family) in terms of
- number of P loops
- state of channel at rest
- 2 P loops
- constitutively open at rest
what two ion channels mediate bursting pacemaker activity?
- CaV channels
- Ca activated K channels
