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Neurotoxins targets the ion channel, how?
attacks the neuronal ion channel, deadly, and fast-acting
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the genetic disease: channelopathies affect what?
- affects the voltage gated Na+ channel
- -toxins are used as experimental tools
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varieties of toxins are.
batrachotoxins, veratridine, and aconitine
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batrachotoxin does...
it blocks inactivation, causing channels to always open
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veratridine.
inactivates channel
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aconitine
inactivates channels
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gating involves what kind of changes
conformational change in the protein
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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
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what causes the conformation change in the channel?
nt or hormone binding
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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.
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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)
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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
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where are ion channels found?
everywhere; present in almost every cell
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whats the function of ion channels
- transport of ions and H2O
- -regulate electrical potential across membrane
- -signaling
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gating mechanisms exist in 2 discrete states.
- open (conducting) and closed (nonconducting)
- *part of channel structure /external particle blocks; otherwise, open channel
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What is nongated ion channels?
means they are always open and cannot be closed
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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
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whats the determining factors toward the conformational shape
atomic, electric and hydrophobic forces
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ligand gated channels have what type of receptors
- glutamate receptors
- nicotinic acetylcholine receptor,
- vanilloid receptor family (TRPV)
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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
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acetylcholine receptor
- 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
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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
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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
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introns and exons?
no consistency in lengths
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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
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does all genes have TATA box?
no, not all; some use other initiator elements
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what does multiprotein transcriptional complex consist of ?
RNA pol II and plethora of ancillary factors
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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
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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
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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
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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
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what neurotrophic factor initiates the signaling pathways of GluR2 NSR sequence
GDNF and BDNF
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ligand-switching-induce changes in gene expression ...
occurs a lot during development
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with alternative splicing...
not very widespread among RNA transcripts of LG ion channel receptors
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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
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what does alternative splicing produce?
- variatns of same receptors subunits
- eg: AMPA
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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)
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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
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RNA editing is carried out by 2 dsRNA A deaminases...
ADAR1 and ADAR 2
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RNA editing causes...
- oxidative deaminated then inosine
- inosine bp like G causes change to codon, causing different aa in translated protein
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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
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RNA editing includes?
- GluR Q/R editing and
- GluR R/G editing
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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
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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
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how widespread translational control is among LG ion channel subunts?
- not exactly known;
- so shouldnt make inferences about levels of protein subunits from mRNAs
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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
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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
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post-translational modification
- extensively phosophorylated and glycosylated
- -kinases known to phosphorylate LGICs
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what are the LGICs?
PKA, CaMKII, PKC
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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
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Phosphorylation and dephosphorylation efficiency dependent on...
- proximity to of LGICs to kinases and phosphatases in high [protein] milieu
- **refer to examples
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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
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receptor assembly and trafficking.
- peptide synthesis
- folding
- 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
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stoichiometry of subunits of nAchR and GlyRs
- nAchR: aabyS
- aby trimers form and recruit B and then 2nd a subunit
GlyRs, aaaBBB
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moving receptors from ER to plasma membrane involves what?
involves targeting correct sites on membrane
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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
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gephyrin clusters what?
BlyR
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rapsyn clusters what?
nAchR
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Rapsyn associates with?
with intracellular M3-M4 loop of nAchR and mediates action of agrin-stimulated signaling pathway that drives nAchR into synapses
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what does receptors do between clustering sites?
they alternate and assoicate with the clustering
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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)
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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
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to protect receptors from degradation...
- block ubiquitinization
- or shuttling them to recycling
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