Functional insights from genetic channelopathies Stephanie Schorge

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1 Functional Insights From Genetic Channelopathies Dr. 1 Royal Society University Research Fellow Department of Clinical and Experimental Epilepsy Aims of channelopathies lecture Describe channelopathies Overview of how genes are made into ion channels in 5 easy steps Specific examples of channelopathies 2 Channelopathies in the genomic era Any disease that results from a mutation in an ion channel Neurons are particular, not in that they have ion channels, but in that they have such a variety of them. Originally channelopathies were studied as changes in functioning of otherwise intact channels Now channelopathies are being recognized as often disrupting the product of an ion channel gene before it ever reaches the membrane Treatments of channelopathies are shifting (just beginning to shift) to acknowledge this recognition 3 1

2 What is an ion channel? Path of an ion into (or out of) a cell Lipid bilayer of a cell Protein: ion channel Ion channels provide a route for ions to cross cell membrane Different channels can select different ions Channels open (or close) in response to specific stimuli: 1. Binding an agonist 2. Voltage changes across membrane 4 3. Internal changes in cell metabolism (second messengers) Classical channelopathy: hyperekplexia Mutations in various glycine receptor subunits reduce negative chloride ions from entering the cells Decreases inhibition in spinal circuits Get an exaggerated startle response X 5 Overview: life and times of an ion channel in 5 steps Hurdles for an ion channel: 1. Transcription 2. Splicing (mrna processing) 3. Translation/folding/assembly Endosome 4. Trafficking/localization Golgi 5. Degradation Plasma membrane RER Nucleus 6 2

3 Step 1- transcription: some notes on our (eukaryotic, mammalian) genes To enhancers (many bases) Transcription starts Promoter TATA Exon Intron Exon Intron Exon 300 bases 7 Promoters and enhancers are very important, but not well understood, particularly for ion channels TATA box is necessary (core promoter) Up to 140 kb of pre-rna is transcribed to generate a 8 kb mrna It is a lot of work to make an mrna! Step 1: a theoretical image of how transcription factors get the attention of RNA polymerase II (polii to friends) 8 Scientific American image Step 1 example: a change in the NMDA subunit promoter for GRIN2A, may block activators from binding to enhancers, and may be associated with schizophrenia Pol II binding decreases NMDA Transcription decreases X Change in DNA Fewer NMDA receptors 9 Scientific American image NMDA gene Itokawa et al., (2006) Neurosci letts 3

4 Step 1 example: in experimental temporal lobe epilepsy (an acquired channelopathy) up regulation of a repressor, NRSF, reduce HCN1 expression NRSF NRSF NRSF NRSF Number of NRSF repressors increases NRSF binds to more NRSE sequences Pol II binding decreases Fewer HCN1 channels found Neurons with less HCN1 trigger seizures 10 Scientific American image HCN1 channel gene McClelland et al., 2011 Ann Neurology Step 2 - splicing: our mrna (and rats ) is heavily spliced Short exons (thick grey lines = protein coding sequences ~100 nt) are interspersed with: Larger introns (thin black lines = non-coding sequences, usually 500 nt-10 kb), which are spliced out during transcription Proteins bound to the splice junctions (= purple balls) seem to serve as markers for RNA quality Nascent mrna, with protective proteins 11 polii Genomic DNA Step 2 mrna processing: early stop codons trigger nonsense mediated decay (NMD) AA(~200) First ribosome removes all exon/exon markers 12 First ribosome stuck at premature stop codon: exon/exon markers signal decapping and tailing 4

5 Step 2 mrna processing: example of NMD in CLCN1/ClC-1 myotonia congentia Myotonia congenita episodes of tense muscles, inherited disease Side effect astonishing muscular physiques, pain There are several different ways of inheriting this dominant and recessive Individuals with nonsense mutations have fewer ClC-1 channels, but enough to get by - unaffected Individuals with mutations, can have dominant disease if the mutant subunits disrupt the normal ones. 13 Fiahlo et al., 2008 Brain, and unpublished Step 2 mrna processing: example of NMD in SCN1A/Nav1.1 sometimes half is not enough! GEFS+ (missense) vs. SMEI/Dravets (nonsense) GEFS+: generalized epilepsy with febrile seizures + SMEI: severe myoclonic epilepsy of infancy or Dravets syndrome Nav1.1 channels are monomers, so affected channels don t seem to disrupt normal ones. Haploinsufficiency (SMEI) Missense mutation (GEFS+) 14 Catterall et al., 2010 Jphysiol A digression: what is a missense mutation? Missense mutation (GEFS+) Polymorphisms >1% in controls Rare variants between 0.1 and 1% in controls (can be associated with increased likelihood of disease) Mutations generally <<1% - but not all mutations cause disease! 15 Bodmer and Bonilla 2008 Nat Genet 5

6 16 A digression (2): how do you know a missense mutation is causing a disease? Several missense mutations in SCN1A (type 1 sodium channels) cause GEFS+ In families one mutation may show segregation, but reduced penetrance or different clinical manifestations can obscure a relationship Functionally characterising a mutation can help if it causes an obvious change in function in your reporter system With sporadic mutations, it is sometimes not possible to show a functional effect, or segregation but does that mean it causes a disease? 3. Translation folding AA(~200) 17 Initiation of translation is a major hurdle for making a protein Individual mrnas can be translated up to 1000 times We are not polycystronic, but related mrnas may be translated near each other to give a similar effect In spite of this picture, ion channels are translated directly on the rough ER Step 3 - protein processing: introducing the rough endoplasmic reticulum, a quality control checkpoint for proteins Subunits retained until assembled Accessory subunits often help transport Misfolded subunits are degraded Many misfolded subunits trigger UPR Cytosol ER lumen Wait for accessory subunits To transport vesicle Re-folding or degradation Unfolded protein response 18 6

7 Step 3 protein processing: example episodic ataxia type 1 Misfolded subunits associate with normal subunits and hold everything in ER Cytosol ER lumen Eunson 2000 Ann Neurol 19 Manganas 2001 Biol Chem Re-folding or degradation Kv1.1 gene, KCNA1, has only one exon so it doesn t trigger NMD Triggered episodes of movement disorders Truncated proteins are often thought to be dominant-negatives Dominant-negative, means less functional than if the protein is just gone (NMD) Step 4 - localisation: accessory subunits can hold a channel in place β I II III I Gβγ II III IV Gα IV In neurons we do not know what subunits are present in functional channels, but virtually all channels have multiple subunits Accessory subunits can be required for normal channel behaviour and trafficking. The first GEFS+ epilepsy was not a mutation in the pore-forming subunit, but in the accessory beta subunit. It turns out these beta subunits bind to proteins outside the neuron and help anchor the sodium channel in place Non-neuronal expression systems cells can not recapitulate the whole anatomy of NaVs or their regulation 20 Step 4 localization of channels: example GABA A receptors in epilepsy (GEFS+) Normal location Plasma membrane The receptors behave normally if they make it to the cell surface GABA A receptors are a mix of subunits only one of which (gamma2) is mutated in these cases of epilepsy Endosome Golgi RER Receptors held in ER Nucleus 21 7

8 Step 4 localization of channels: loss-of-function GABA A receptor mutation in GEFS+ means all subunits are lost in ER After 30 min at 40 o C where are the subunits? α1 γ2 Membrane Overlap? wt α1 wt γ2 When the three fluorescent markers overlap, the membrane turns yellow wt α1 mut γ2 22 Adapted from Kang et al., 2006 J Neurosci The membrane remains red, but the only the RER inside is yellow Step 5 - degradation: once a receptor is in place, how long does it last? 23 It depends Anchored in synapses, nachrs are thought to last for weeks Ca channels in cultured neurons lasted days Extrasynaptic receptors can turnover quite quickly (hours) Receptor decay is very much a developing field A word about ubiquitin Ub 24 Ubiquitin is an extremely highly conserved marker that signals endocytosis and possibly degradation of proteins it binds A single ubiquitin is not fatal, as they are sometimes markers for cycling in vesicles A chain of four or more ubiquitins is usually the end Cytosolic proteins are sent to 26S proteosome, but membrane proteins seem to be degraded primarily in lysosome. 8

9 Step 5 - degradation of channels: example SCA6 and Ca 2+ channels Spinocerebellar ataxia type 6, CACNA1A Repeats in the Ca channel gene of the sequence CAG are expanded These sequences are very difficult for the cell to degrade The accumulated Ca channel bits can activate intracellular signalling channels Additional affects arise from changes in behaviour of the intact channels More than one step is usually affected by a channelopathy 25 Schorge (2010) TINS Ub Ub Ub CAG-bits resist degradation and accumulate in cell Mutant channel bound to ubiquitin Ub Ub Ub The future of channelopathies Accessory proteins Non-synonymous variants Mouse human differences Why are so many channelopathies episodic? 26 Final word Channelopathies are not just about ion channels! 27 9

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