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Neurotoxins targets the ion channel, how?
attacks the neuronal ion channel, deadly, and fast-acting
the genetic disease: channelopathies affect what?
- affects the voltage gated Na+ channel
- -toxins are used as experimental tools
varieties of toxins are.
batrachotoxins, veratridine, and aconitine
it blocks inactivation, causing channels to always open
gating involves what kind of changes
conformational change in the protein
where does conformation change occurs and what kind of mechanism is involved
- occurs in discrete area of channel, leading it to open; entire channel changes
- -follows a ball-and-chain mechanism
what causes the conformation change in the channel?
nt or hormone binding
How does nt/hormone binding cause the channel to open?
binding of the two causes 2nd messenger to activate a protein kinase that phosphorylates a channel that opens it.
why do channels open/close?
- 1. when there's changes in the membrane potential
- 2. mechanical deformation
- 3. selectivity by charge (positively lined allow anions thru; negatively lined allow cations thru)
passive transporters are...
- ions flow from high to low conc (down gradient)
- -cannot be coupled to energy source (no energy required/used)
- -move ions /H20 thru gradient, otherwise, ions wont flow thru
- -small highly selective pores in cell membrane
where are ion channels found?
everywhere; present in almost every cell
whats the function of ion channels
- transport of ions and H2O
- -regulate electrical potential across membrane
gating mechanisms exist in 2 discrete states.
- open (conducting) and closed (nonconducting)
- *part of channel structure /external particle blocks; otherwise, open channel
What is nongated ion channels?
means they are always open and cannot be closed
whats gated ion channel?
- can go through conformational change when...
- there is voltage across cell membrane,
- there's a ligand, when there's mechanical stimulus, heat
whats the determining factors toward the conformational shape
atomic, electric and hydrophobic forces
ligand gated channels have what type of receptors
- glutamate receptors
- nicotinic acetylcholine receptor,
- vanilloid receptor family (TRPV)
gated ion channels are gated by?
- gated by ligand, present on outside of cell, whihc are receptors
- -all are nonselctive cation channels
- -mediate effects of neurotransmitters
- have pentamer of protein subunits
- 2 binding sites for acetylcholine - when bound alters configuration and cause internal pore to open
- allows Na+ ions to flow down electrochemical gradient into cell
what can you say about gene families that encodes ion channel receptor?
- corresponds to sets of protein subunits of the same functional class
- gene families for each majoe a, b, and y
genes are scattered...
- are scattered over many chromosomes, there's occasional clusters
- eg. GABAa receptor a1, a6, b2 and y2 are all close together at q31-35 on chromosome5
introns and exons?
no consistency in lengths
transcriptional control over genes and gene expression pays attention to...
- timing gene expression during development
- mechanism of expression of gene expression of neurons and types of neurons
- regulation of receptor gene expression during synapse formation
**lilltle known aobut mechanism involving environmental singals
does all genes have TATA box?
no, not all; some use other initiator elements
what does multiprotein transcriptional complex consist of ?
RNA pol II and plethora of ancillary factors
what is needed since simple binding to TATA box is not sufficient to transcribe a gene to physio level of txn
need additiolnal seq-specific interactions of various txn factors with cis-acting enhancer and silencer elements
role of silencing in neurons acting at 2 levels:
- 1. global silencing of neuronal genes in non-neuronal cell types
- 2. silencing at fine-tuning level to restrict expression of neuronal genes to a subset of neurons
GluR subunits genes...
- have no TATA or CAAT start sites;
- GC-rich with multiple txn start sites within CpG island
- promotors ocntian overlapping Sp1 and GSG recog sites near major txn start sites and NSR silencer
- -NSR sequence in NR1 and GluR2 genes have small modulatory effect with respect to neuronal specificity of expression
what is GluR NSR sequence?
a site of mediation of stimulatory effects on gene expression of signaling pathways initiated by neurotrophic factor, GDNF and BDNF
what neurotrophic factor initiates the signaling pathways of GluR2 NSR sequence
GDNF and BDNF
ligand-switching-induce changes in gene expression ...
occurs a lot during development
with alternative splicing...
not very widespread among RNA transcripts of LG ion channel receptors
where does alternative splicing occur?
means more than just a difference in short aa sequence that may regualte post-translational modification processes, such as phosphorylation and glycosylation sites
what does alternative splicing produce?
- variatns of same receptors subunits
- eg: AMPA
ea. 4 AMPAR subunits occur in 2 alternatively spliced variants, what are these two 2 spliced variants?
- flip and flop
- correspond to alternative inclusion of either of 2 adjacent exons (exons 14 and 15 in GluR2 gene)
what is the functional difference between flip and flop?
- flip forms of most subunits desensitize more slowly and lesser degree than flop forms;
- easily displayed probed with various agents
RNA editing is carried out by 2 dsRNA A deaminases...
ADAR1 and ADAR 2
RNA editing causes...
- oxidative deaminated then inosine
- inosine bp like G causes change to codon, causing different aa in translated protein
ADAR1 and ADAR2 depends on?
- formation of ds structures involving intronic sequences, which bp with exonic sequences to be edited
- other protein factors may be involved cuz some cells express ADARS but cannot edit
RNA editing includes?
- GluR Q/R editing and
- GluR R/G editing
GluR Q/R Editing
- S RNA editing sites: GluR2, 5,6: leads to replacement of gln codon (CAG) by arg codon (CIG=CGG) and insertion of arg (R) to TM domain
- occurs in M2, has low Ca2+ permeability; low single channel conductance, linear rectifying properities
GluR R/G editing
- GluR2, 3, 4 undergo editing at R/G sites in exon 13; just N-term to flip flop regio nof alt splicing
- reduces sensitization and accel recover
how widespread translational control is among LG ion channel subunts?
- not exactly known;
- so shouldnt make inferences about levels of protein subunits from mRNAs
In many gluR subunits and NR subunits, what is removed?
removal of 5'UTR thats involved in putative stem-loop structure results in sig. disinhibition of translation
translational suppression of gluR2 mRNA is due largely to?
broad region containing a repeat sequence near 5' end of mRNA, which may affect various txn start sites differentially
- extensively phosophorylated and glycosylated
- -kinases known to phosphorylate LGICs
what are the LGICs?
PKA, CaMKII, PKC
phosphorylation of these kinases affect the function of LGICs
- PKA phos AMPARs causes increase channel open time or the P(O) state
- CaMKII phos AMPARs corre with increase synaptic resonses (synaptic plasticity)
- phosphorylation of receptors is corr. with activity
Phosphorylation and dephosphorylation efficiency dependent on...
- proximity to of LGICs to kinases and phosphatases in high [protein] milieu
- **refer to examples
5-10% of a subunit MW can be glycosylation
- oligomannosidic glycans + complex oligosaccharides
- increase efficiency of receptor asembly and cell-surface density of GABAaRs, but not essential for these processes
- strongly required for ER exit of assembled GlyR and nAchR
- appears that glycosylation - similar quality control over all receptors
- no clear role for glycosylation of LGICs
receptor assembly and trafficking.
- peptide synthesis
- post-translational modifications
- insertion to ER
- strict selectivity is required os that only certain combinations of the correct subunits are oligomerized and target to plasma membrane via Golgi
stoichiometry of subunits of nAchR and GlyRs
- nAchR: aabyS
- aby trimers form and recruit B and then 2nd a subunit
moving receptors from ER to plasma membrane involves what?
involves targeting correct sites on membrane
what does LGICs do at the synapse?
LGICs are held in hynamic relatinship (lateral diffusion) with a protein complex by multiple interactions with certain proteins playing key roles in receptor clustering and retention
gephyrin clusters what?
rapsyn clusters what?
Rapsyn associates with?
with intracellular M3-M4 loop of nAchR and mediates action of agrin-stimulated signaling pathway that drives nAchR into synapses
what does receptors do between clustering sites?
they alternate and assoicate with the clustering
receptors at synapses become?
- becomes extensively inolved with scaffolding proteins, cytoskeletal anchoring proteins, and signal transduction proteins
- C.f., PSD complex
- receptors are removed fr cell surface and from synapses in response to environmental signals as well for receptor production
- -signal adaptor proteins (eg. AP-2, arrestin, ubiquitin)
clathrin recruitment leaads to...
- membrane invagination, endocytosis --> early endosomes --> recycle to plasma membrane or delivered to late endosomes for sorting --> recycled via trans-Golgi to plasma membrane or to lysosomes for degradation
- -all events involve protein-protein interactions
to protect receptors from degradation...
- block ubiquitinization
- or shuttling them to recycling