2Active Transporters: The proteins that created and maintain ion gradients Ion channels : give rise to selective ion permeability changes
3ION CHANNELSIon channels are transmembrane proteins that contain a specialized structure, THE PORE that that allow particulars ions to cross the membrane.Some ion channels contain voltage sensor ( voltage gated channels) that open or close the channel in response to changes in voltage.Other gated channels are regulated by extracellular chemical signals such as neurotransmitter or by intracellular signals as a second messengers.
4ACTIVE TRANSPORTERSMembrane proteins that produce and maintain ion concentration gradients.For example the Na+ pump which utilizes ATP to regulate internal concentration of Na+ and K+.Transporters create the ionic gradient that drive ions through open channels, thus generating electric signals
5What is the mechanism for ion movement across the membrane? K+ and Na+ currents were distinct, suggesting distinct mechanismsMechanism is voltage dependent (must sense voltage)Voltage clamp recordings showed that ions move across membrane at high rates (~ 600,000 /s) – inconsistent with an ion pump mechanismIon selectivity of Na+ and K+ currents – size dependent permeability suggests pore of certain diameter.Armstrong (1965-6) – TEA block could be overcome by adding excess K+ to the extracellular fluid and stepping to hyperpolarized potentials (K+ comes into cell) suggesting that K+ ions dislodge TEA from poreAcetylcholinesterase – 5000 molecules/secIntroduction of TTX in 1964 – showed that Na current could be blocked while leaving voltage activated K current and leak K current intact. Indicates that distinct mechanisms are responsible for different currents. Similar but opposite effects of TEA (blocks voltage dependent K but not leak)Binding studies of labeled TTX (or STX) provided estimates of density of Na sites – knowing density and current, can estimate # of ions coming in from each siteTTX also only binds when added to extracellular surface rather than intracellular surface.
6Ion channels share several characteristics The flux of ions through the channel is passive .The kinetic properties of ion permeation are best described by the channel conductance (g) that is determinate by measuring the current flux (I) that flows through the channel in repose to a given electrochemical driving force. (Electrochemical driving force is determinate by difference in electric potential across membrane and gradient of concentration of ions) .At the single channel level, the gating transitions are stochastic. They can be predicted only in terms of probability.
7Ion channels share several characteristics In some channels the current flow varies linearly with the driving force ( channels behave as resistors)In other channels, current flow is a non-linear function of driving force ( Rectifiers)High conductance (γ)I (pA)V (mV)Low conductance (γ)Ohmic channelRectifying Channel( I=Vm/R)
8Ion channels share several characteristics The rate of ion flux (current) depends on the concentration of the ions in solution ( At low concentrations the current increases linearly with the concentration, at higher concentrations the current reach a saturation point ) .The ionic concentration at which current flow reaches half its maximum defines the dissociation constant for ion binding.Some ion channels are susceptible to occlusion by free ions or molecules
9The Opening and closing of channels involve conformational changes In all channel so far studied, the channel protein has two or more conformational states that are relatively stable. Each stable conformation represents a different functional state.. Each channel has an open state and one or two closed states. The transition between states is calling gating.
10The Opening and closing of channels involve conformational changes Three major regulatory mechanisms have evolved to control the amount of time that a channel remains open and active.Under the influence of these regulators ,channels enter one of three functional states: closed and activable (resting), open (active) or closed and nonactivable ( refractory).The signal that gate the channel also controls the rate of transition between states.
11The Opening and closing of channels involve conformational changes Ligand -gated and voltage gated channels enter refractory states through different process. Ligand-gated channels can enter refractory state when the exposure to ligand sis prolonged (desensitization)Voltage-gate channels enter a refractory state after activation. The process is called inactivation.Activation is the rapid process that opens Na+ channels during a depolarization.Inactivation is a process that closes Na+ channels during depolarization. The membrane needs to be hyperpolarized for many milliseconds to remove inactivation.
12The Opening and closing of channels involve conformational changes Exogenous factors such as drugs and toxins can affect the gating control sites.
13Structure of Ion Channels Ion channels are composed of several subunits. They can be constructed as heterooligomers from distinct subunits, as homooligomers from a single type of subunit o from a single polypeptide chain organized into repeated motifs.In addition to one or more pore forming unties, which comprise a central core, some channels contain auxiliary subunits which modulate the characteristics of the central core
14Structure of voltage gated ion channels Repeated series of 6 TM a helicesS4 helix is voltage sensorLoop between S5 & S6 composes selectivity filter
18Voltage gated sodium channels A large alpha subunit that forms the core of the channel and its functional on its own. It can associate with beta subunitsBlocked by: TTX, STX, *cain local anesthetics
19Persistent (non-inactivating) Na+ currents are produced by an alternative channel gating mode
20Functions of voltage-gated Na+ channel alpha subunits Protein nameGeneExpression profileAssociated channelopathiesNaV 1.1SCN1ACentral and peripheral neurons and cardiac myocitesFebrile epilepsy, severe myclonic epilepsy of infancy, infantile spasms, intractable childhood epilepsy, familial autismsNav1.2SCN2ACentral and peripheral neuronsFebrile seizures and epilepsyNav1.4SCN4ASkeletal musclePeriodic paralysis, potassium agravated myotoniaNav1.5SCN5ACardiac myocites, skeletal muscle, central neuronsIdiopathic ventricular fibrillationNav1.7SCN9ADorsal root ganglia, peripheral neurons. Heart, gliaInsensitivity to pain.
21Voltage gated Ca2+ channels Gene Product Cav Cav2.1 Cav2.2 Cav2.3 CavTsien Type “L” “P/Q” “N” “R” “T”Characteristics High voltage Mod voltage High voltage Mod voltage Low voltageactivated, activated, activated, activated activatedslow inactivation moderate moderate fast fast(Ca2+ dependent) inactivation inactivation inactivation inactivationBlocked by dihydropiridines Agatoxin Conotoxin SNX 482 Mibefridil(nimodipine) IVA GVIA High Ni2+Form by different subunits:α1, α2δ,β and γ. The α1 subunit forms the pore, the other subunits modulate gating.
24Inwardly-rectifying and “leak” K+ channels 2 pore “leak”Inwardly-rectifying channels• subunits: Kir 1.X - 7.X• Rectifying character due to internal block by Mg2+ and polyamines• Roles:• Constitutively active resting K+ conductance (eg. Kir1, Kir2)• G-protein activated (Kir3)• ATP sensitive (Kir6)2 pore “leak” channels• many different subunits, nomenclature still argued• Outwardly rectifying due to unequal [K+] across the membrane• Constitutively active resting K+ conductance• pH sensing• Mechanosensitive• Thermosensitive• Second messenger sensitive (cAMP, PKC, arachadonic acid)
25Voltage gated K+ channels Gene Product Kv1.X (1-8) Kv2.X (1-2) Kv3.X (1-4) Kv4.X (1-4) Kv7.X (1-5)“D type” “Delayed “Delayed “A type” “M current”rectifier” rectifier”Characteristics Low voltage High voltage High voltage Low voltage Low voltageactivated (~50 mV), activated (0 mV), activated (-10 mV), activated (-60 mV) activated (-60 mV)fast activation mod activation fast activation fast activation slow activation(< 10 ms) (>20 ms) (10-20 ms) (10-20 ms) (>100 ms)slow inactivation very slow very slow fast noinactivation inactivation inactivation inactivationfast deactivationBlocked by 4-AP (100 µM) TEA (5-10 mM) TEA ( mM) 4-AP (5 mM) XE991dendrotoxin 4AP (1-5 mM) 4AP (0.5-1 mM)BDS (50 nM)Kv4(“A type”)Kv1(“D type”)Kv2(“DR type”)Kv3(“DR type”)
26Ca2+ activated K+ channels - role in repolarization following APs Spike frequency accommodationVoltage responsecurrents mediating AHPRole of IKCa in burst duration
27Ca2+ activated K+ channels Channel Type BK SK sAHP“maxi K, IC fAHP” “mAHP” “sAHP”Gene product slo 1-3 SK1-3 ????Voltage dep? Yes No No[Ca2+] to activate 1-10 µM µM µMCa2+ binding direct to subunit calmodulin hippocalcin?Single channel pS 5-20 pS 5-10 pSConductanceBlocked by charybdotoxin apamin TEA (> 20 mM)TEA (< 1 mM) TEA (> 20 mM)
28Many drugs and toxins act on voltage gated ion channels
29Effect of drugs and toxins Many toxins block ion channels directly either from the outer (TTX) or inner (lidocaine) surface of the channelOther toxins change the properties of the channel without blocking itDelaying inactivationShifting voltage dependenceFUGUTTXLA29
30Modulation of Ion Channels Example, enhancement of Ca2+ channels in cardiac myocytes by NE
31Dendritic ion channels participate in synaptic amplification and integration The answer to that question is that within the past decade or so there is a lot of evidence that has shown that Na+ channels, particularly dendritic Na+ channels, have a lot of other interesting roles in the way dendrites process information.