Neurochemistry Concepts

the blood brain barrier, the bony skull, and the cerebral spinal fluid, which provides cushioning for the brain

potassium leak channels, which allow potassium out of the cell with the concentration gradient and help maintain the membrane potential at -70 mV

calcium ion activated potassium channel, which opens up and lets out a flow of potassium ions and couples changes in intracellular calcium levels to repolarize the membrane and maintain the resting membrane potential

GABAA receptor binds GABA, an inhibitory NT, opening a Cl- ion channel and allowing for hyperpolarization of the cell, stopping an action potential

leak channels, voltage gated ion channels, ion-gated ion channels and ligand gated ion channels

Na/K-ATPase pump serves to restore the cell to RMP; a transmembrane integral enzyme that favors Na binding when not phosphorylated, and K binding when phosphorylated

1. Pump is open to the inside of the cell and conformation favors binding of 3 Na ions inside cell
2. ATP –> ADP when an aspartate residue on the pump is phosphorylated
3. Conformational change occurs that no longer favors binding of Na ions
4. Phosphorylated enzyme opens to extracellular space and releases 3 Na
5. Now, phosphorylated favors K binding and binds 2 K from outside of the cell
6.Hydrolysis of Asp-Pi bond causes return to original conformation, and now pump reopens to inside of cells, drops off K, and favors Na binding

voltage gated sodium channels

1 transmembrane alpha subunit, which contains 4 repeated domains, with 6 segments per domain; 2 integral beta subunits attached to either side of the 4 domains of the “square” alpha subunit

not necessary for function of Nav, instead help with conduction rates and ion selectivity

segments s1-s4 on each domain of the alpha subunit

the voltage sensor is segment 4 of each domain of the alpha subunit; it is made up of repeating positive and hydrophobic aas (H.H.Arg.H.H.+.H.H.Phe…); when the S4 rod moves toward the ECF, the link between S4-S5 subunits causes a chain reaction in the S5-S6 pore

S4 is a rod of positive charge b/c of + charged aas, and because on alpha subunit, it is transmembrane, so when + charge of action potential comes intracellularly, electrostatic repulsion causes the S4 rod to move upward toward the extracellular fluid, and S4-S5 linker causes opening of S5-S6 pore

the pore is made up of the central cavity, the selectivity filter, and the gate. it is made up of S5 and S6

the radius can fit hydrated Na ions, but K ions are too large and could only fit if completely dehydrated

the central cavity, aka “core pore,” is very hydrophobic and contains aa F, M, V, T, so a fully hydrated Na ion will fly through the hydrophobic core

when central cavity is closed, 4 lateral openings from 4 domains (fenestrations) contain lipid tails of the cell membrane’s fatty acids, and so benzocaine and lidocaine, which are unprotected at physiological pH, will bind here, but then in this hydrophobic area they will become protenated and get stuck in the core pore, blocking it

the selectivity filter, or “p loop,” is the extracellular linker between S5-S6; in mammals is a conserved AA sequence DEKA; outermost ring contains 4 acidic aa (usually EEDD or EEMD) called “P2” and is a funnel for cations

the gate of Nav is the the bottom portion of S6; when activated, open and Na ions flow through; when deactivated, closed and no Na ions flow through; when inactivated, blocked and no Na ions flow through

1. activation gate – the movement of S4 as a function of membrane potential opens the pore by c-type activation
2. inactivation gate – an intracellular loop between S3-S4 contains the hydrophobic residues I, F, M; will block the gate and the flow of Na ions, by N-type inactivation

c-type activation is activation of a gate when the pore opens based on the movement of the S4 voltage sensor; a slow activation

n-type inactivation is inactivation in which some portion of the channel will block the gate; for Nav, an intracellular loop between S3 and S4 containing hydrophobic residues I, F, M will block the flow of Na ions; a very fast inactivation

Kv will repolarize the cell membrane; they open when the cell reaches ~30 mV, and they allow K ions to flow out of the cell with their concentration gradient

contain 4 transmembrane alpha subunits made up of 6 segments and 4 attached intracellular beta subunits; attached to each alpha subunit there is also a T1 segment hanging off of S1

there is no inactivation; S1 has an attached T1 subunit

Kv has a T1 subunit hanging off of S1 of the alpha domain.

intracellular – T.V.G.Y.G – Extracellular

ion dipole interaction of carbonyl backbone of aa in S.F. and k ions; electro-negative carbonyl oxygen atoms of T.V.G.Y.G. aligned toward the center of the filter pore and form a square anti-prism similar to the water solvating shell around each potassium binding site; the distance between the carbonyl oxygens and potassium ions in the binding sites of the selectivity filter is the same as between water oxygens on the hydration shell of the K ion, so this provides an energetically favorable environment for de-solvation of the K ions

the T1 domain contains many negatively charged aas that draw up K ions toward the central cavity; T1 is cytoplasmic; tetramerization domain; not present in all Kv channels

P2 domain is part of the selectivity filter, it contains negatively charged aas (EEDD or EEMD) that draw up the positively charged Na ions

helps to modulate gating and stability of the K channel; NADPH dependent oxido-reductase; known purpose is deactivation

1. activation gate – like Nav activation gate; movement of S4 causes closure of pore due to S4-S6 overlap; slow activation; c-type activation
2. inactivation gate – from the T1 domain; there is a ball and chain, and a peptide of the N-terminus of S1 called the inactivation peptide that is charged at physiological pH and competes for inactivation binding site; very fast process

closed at RMP, and activated/opened upon depolarization

-NT release
-hormone release
-synaptic transmission
-muscle contraction
-gene transcription

alpha 1, beta, gamma, alpha 2 and delta

they are similar to alpha subunit of Nav channels; they contain the VSD and the pore; sufficient for function, but when no other subunits present, abnormal kinetics and voltage dependence

the alpha 2 and delta subunits are connected; role in activation/inactivation kinetics

has a role in activation/inactivation kinetics and is a kinase, so phosphorylates stuff; connected/affiliated with alpha 1 subunit through the AID (alpha interaction domain)

Cav sequence -EEEE
Nav sequence -DEKA

1. selectivity filter sequence
2. there is an intracellular loop between the alpha 1 subunit of the s6 segment on domain 1 and the s1 segment of the alpha 1 subunit on domain 2

1. ca dependent inactivation – CDI
2. voltage dependent inactivation – VDI

in the c-terminal lobe there is a Ca ion sensing protein called calmodulin that is permanently attached to the inner mouth of the calcium channel; calmodulin will bind to four calcium ions and will create an allosteric effect by causing a conformation change that is relayed to the pore and causes S6 to close

CDI will halt

VDI – voltage dependent inactivation; this deals with the alpha interaction domain, a part of the linker which links beta and alpha 1 subunits of Cav together; there is a protein-protein interaction with the beta and alpha subunits that may reduce the mobility of the AID section of the loop, and so when conformational changes in the voltage sensing domain occur, protein-protein interactions at the AID are disrupted; freeing up the loop between the two domains to block the pore

1. action potential propagated down the nerve terminal
2. depolarization of the membrane at the axon terminal causes Cav to open and consequently Ca ions enter the cell and the intracellular concentration of Ca rises
3. the rise in Ca ions triggers Ca ion sensing proteins (SNARE protein synaptotagmin), leading to fusion of the synaptic vesicle to the presynaptic membrane
4. fusion causes release of the NT into the synaptic gap
5. the NT binds to receptor of the postsynaptic membrane, eliciting some type of response

when n-ethylmaleimide is present

Soluble N-ethylmaleimide sensitive factor Attachment protein Receptor

-synaptobrevin or VAMP
-synaptotagmin (Ca sensing protein)

-syntaxin (SNARE)
-SNAP-25 (SNARE)
-SM protein

docking is when the SV docks itself into the active zone, and the SV is loaded with NT; no Ca ions are present at this point

priming occurs when the SV is preparing for fusion; there are still no Ca ions present and the Cav are closed, but close to Cav; ATP drive partial assembly of SNARE proteins, and synaptobrevin, syntaxin and SNAP-25 form a loose 4 helix bundle called the trans complex, and synaptotagmin coordinates loosely with the trans helices complex

priming is driven by ATP via the ATPase NSF (N-ethylmaleimide sensitive fusion protein), and synaptobrevin, SNAP-25 and syntax in form a loose trans helices, and synaptotagmin coordinates loosely with the trans helices

Cav channels open with depolarization and the concentration of Ca ions increases; Ca ions bind to synaptotagmin, causing a conformational change that results in the formation of a tight complex of SNARE proteins, and a fusion pore is created through the membrane, and exocytosis occurs and NTs spill out into the synaptic cleft

SV docks but doesn’t completely fuse with membrane; NTs are released, SV reforms and the pore closes; SV goes back into presynaptic neuron and refills with NTS

SV docks but doesn’t completely fuse with membrane; NTs are released,SV is refilled without leaving the membrane

SV completely fuses with presynaptic membrane; followed by endocytosis machinery, then merges with early endocymes and then pinches off to form a new SV

protease that targets SNARE proteins of cholinergic neurons, so the neuron can no longer release ACh at the neuromuscular junction; specifically cleaves synaptobrevin, syntaxin, and SNAP 25; so now less muscle contraction and flaccid paralysis occurs

a protease that cleaves synaptobrevin in 1/2 in GABA and glycine receptors, so you end up with an increase in muscle contraction – and especially bad because tetanus travels up the CNS and affects spinal motor neurons

the pH of SVs is 5.5, and the SV has a V-ATPase pump, which pushes 3 H+ ions into the SV, and also as a NT transporters, so brings the NT into the vesicle; we also see a chloride channel that lets chloride ions out of the SV to balance the charge in the SV

they have a 7-transmembrane alpha helices structure; the N-terminus is on the extracellular side and the C-terminus is on the intracellular side; the extracellular side communicates with the ligand, and the intracellular side communicates with the g-protein; binding of an agonist causes an allosteric change on the intracellular side of the GPCR

-GTPases, so hydrolyze GTP –> GDP + Pi
-Heterotrimeric structure (TMR), so have alpha, beta and gamma subunits
-beta and gamma are often associated
-four main classes, but we focus on Gs, Gi and Gq

1. inactive
2. ligand binds to N-terminus, extracellular side of TMR
3. binding creates conformational change on intracellular side that disfavors binding of GDP but favors binding of GTP
4. GTP binds to G alpha
5. conformational change in G alpha due to binding, and G alpha dissociates from G[betagamma], so now they move downstream and can have an effect on downstream effects
6. hydrolysis of GTP –> GDP now favors re-association of G alpha and G[betagamma]
7. now, favors complexation of GPCR