Five nicotinic acetylcholine receptor (nAChR) mutations are currently linked to autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE). The similarity of their clinical symptoms suggests that a common functional anomaly of the mutations underlies ADNFLE seizures. To identify this anomaly, we constructed rat orthologues (S252F, +L264, S256L, V262L, V262M) of the human ADNFLE mutations, expressed them in Xenopus oocytes with the appropriate wild‐type (WT) subunit (α4 or β2), and studied the Ca2+ dependence of their ACh responses. All the mutations significantly reduced 2 mM Ca2+‐induced increases in the 30 μM ACh response (P < 0.05). Consistent with a dominant mode of inheritance, this reduction persisted in oocytes injected with a 1:1 mixture of mutant and WT cRNA. BAPTA injections showed that the reduction was not due to a decrease in the secondary activation of Ca2+‐activated Cl− currents. The S256L mutation also abolished 2 mM Ba2+ potentiation of the ACh response. The S256L, V262L and V262M mutations had complex effects on the ACh concentration‐response relationship but all three mutations shifted the concentration‐response relationship to the left at [ACh]⩾ 30 μM. Co‐expression of the V262M mutation with a mutation (E180Q) that abolished Ca2+ potentiation resulted in 2 mM Ca2+ block, rather than potentiation, of the 30 μM ACh response, suggesting that the ADNFLE mutations reduce Ca2+ potentiation by enhancing Ca2+ block of the α4β2 nAChR. Ca2+ modulation may prevent presynaptic α4β2 nAChRs from overstimulating glutamate release at central excitatory synapses during bouts of synchronous, repetitive activity. Reducing the Ca2+ dependence of the ACh response could trigger seizures by increasing α4β2‐mediated glutamate release during such bouts.

1 We studied the pharmacological properties of native rat brain and heterologously expressed rat a4b2 nicotinic receptors immunoprecipitated onto a ®xed substrate with the anti-a4 antibody mAb 299. 2 Immunodepletion with the anti-b2 antibody mAb 270 showed that 89% of the mAb-299-precipitated rat brain receptors contained b2.

RNA binding proteins play an important role in regulating alternative pre-mRNA splicing and in turn cellular gene expression. Many of these RNA binding proteins occur as gene families with members sharing a high degree of primary structure identity and domain organization yet have tissue-specific expression patterns and regulate different sets of target exons. How highly similar members in a gene family can exert different splicing outcomes is not well understood. We conducted mass spectrometry analysis of post-translational phosphorylation and acetylation modifications for two paralogs of the polypyrimidine tract binding protein family, PTBP1 and PTBP2, to discover modifications that occur in splicing reaction mixtures and to identify discrete modifications that may direct their different splicing activities. We find that PTBP1 and PTBP2 have many distinct phosphate modifications located in the unstructured N-terminal, linker 1, and linker 2 regions. We find that the two proteins have many overlapping acetate modifications in the RNA recognition motifs (RRMs) with a few distinct sites in PTBP1 RRM2 and RRM3. Our data also reveal that lysine residues in the nuclear localization sequence of PTBP2 are acetylated. Collectively, our results highlight important differences in post-translational modifications between the paralogs and suggest a role for them in the differential splicing activity of PTBP1 and PTBP2.

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The Polypyrimidine Tract Binding Protein 1 (PTBP1) is an RNA binding protein that controls the alternative splicing of many gene transcripts. PTBP1 belongs to a gene family with paralogs expressed in varying cell types and stages of cell growth. PTPB1 is expressed in most cell types but is absent in differentiating neurons and muscle. Paralog PTBP2 is highly expressed in differentiating neurons. The amino acid sequence of PTBP1 is 74% identical to PTBP2 and the two proteins have a similar domain organization with 4 RNA binding domains (RBDs) connected by 3 linker regions and an N‐terminal region. The two proteins have over‐lapping and non‐overlapping target exons; PTBP1 functions as a splicing repressor of many neuronal exons while PTBP2 does not. PTBP1 is expressed in neuronal progenitor cells, but is down‐regulated during neuronal differentiation while the level of PTBP2 is up‐regulated. The switch in the levels of the two proteins effects the splicing of many exons that are sensitive to PTBP1, thus causing a change in the neuronal splicing program that is critical for development and maturation of neurons. How the two proteins exert different splicing outcomes is not well understood. Recent studies using PTBP1‐PTBP2 chimeric proteins highlighted regions of PTBP1 that change the splicing activity of PTBP2, and revealed the two proteins have different protein‐protein interactions. Thus, we hypothesize that additional factors such as post‐translational modifications (PTMs) dictate PTBP splicing activity. To test this, we used in vitro splicing reaction conditions, in which PTBP1 and 2 showed different splicing activity. To capture splicing relevant PTM's bacterial expressed recombinant PTB proteins were incubated in splicing reaction mixtures containing HeLa nuclear extract, pull‐down using Ni2+‐NTA beads, boiled and separated on SDS‐PAGE. The corresponding PTBP gel bands were analyzed by mass spectrometry for PTM's. Our Results highlight that PTBP1 and 2 have many PTM's including phosphate, acetate and ubiquitin groups. The N‐terminal and linker 1 region are modified primarily by phosphate groups while the RRM domains are modified by acetate and ubiquitin groups. We also note that some residues contain more than one type of modification such as acetate and ubiquitin. Percent modified for each residue‐modification was calculated by counting the modified residue and the total number of peptide fragments containing that residue. Our results highlight that the type and degree of modification vary between the two proteins, in regions including the N‐terminal, linker 1 and linker 2. These regions were identified by the chimera study to alter PTBP2 activity. Currently, we are conducting a mutational analysis of these modified residues that are different between the two proteins, to determine the role of PTM's in PTBP splicing activity.Support or Funding InformationThis work was supported by a Maximizing Access to Research Careers grant to CSUF from the National Institutes of Health [5T34GM008612‐21]This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.