Experimentally derived biomolecular structures were determined by Nuclear Magnetic Resonance (NMR). The properties of selected peptides and proteins in solution and in membrane mimicking micelles were observed by circular Dichroism (CD), mass spectrometry (MS), and other spectroscopic techniques. The mDpl(1-30) peptide (30 residues) of the mouse Doppel protein was found to be positioned as an α-helix in a DHPC micelle. The same peptide can disrupt and cause leakage in small unilamellar vesicles. Single D-amino acid isomers of Trp-cage (20 residues), the smallest peptide with a protein-like fold, were analyzed by CD spectroscopy and were found to have different secondary structures and melting temperatures. They were compared against MS measurements specially designed to reveal the secondary structure of proteins. We studied a novel protein in E. coli of unknown structure that is encoded by the putative transcription factor ORF: ygiT (131 residues). This protein comprises a helix-turn-helix (HTH) domain in the C-terminus and contains two CxxC motives in the N-terminal domain, which binds Zn. This protein was named 2CxxC. We succeeded in overexpressing and purifying 2CxxC in E. coli with enough yield for a 13C, 15N uniformly labeled NMR sample. The chemical shift assignment was completed and the NMR structure was calculated in reducing, slightly acidic conditions (1mM DTT, pH 5.5). The determined HTH domain shows good similarity with structures predicted by a homology search, while the N-terminal domain has no other homologous structure in the Protein Data Bank (PDB). The structure of the paddle region (27 residues) of the HsapBK(233-260) voltage and Ca+2 activated potassium channel, in DPC micelles, was determined by NMR. It shows a helix-turn-helix loop, which agrees well with the expected structure and could help to verify the proposed models of the voltage gating mechanism. The C-repressor (dimer of 99 residues) of bacteriophage P2 was analyzed by NMR. We assigned the chemical shifts and NMR structure determination is under way.

In the shark rectal gland (SRG), apical Cl[superscript]- secretion through CFTR channels is tightly coupled to a basolateral K[superscript]+ conductance. The identity of this K[superscript]+ conductive pathway is unknown. Studies were performed in the isolated perfused SRG with 16 K[superscript]+ channel inhibitors at their IC50 and with acidic perfusate. During maximal chloride secretion stimulated by forskolin and IBMX, secretion was inhibited >90% by barium chloride, a non-selective inhibitor of K[superscript]+ channels. Specific inhibitors of calcium sensitive, voltage sensitive, ATP sensitive, and inward rectifying K[superscript]+ channels had no effect on chloride secretion. The inhibitors quinidine, quinine, bupivicaine, anandamide, and low perfusate pH (6.0) abruptly and reversibly inhibited secretion by >90%, consistent with the presence of the Two-Pore-Domain (4TM 2P/KCNK/K2P) family of K+ channels. Degenerate primers were designed to regions of high amino acid homology in known mammal and teleost 4TM 2P K[superscript]+ channel subtypes: TWIK, THIK, TASK, TREK, and TRAAK. PCR with cDNA from several shark tissues identified a putative TASK-1 fragment (394 bp) in shark rectal gland, brain, gill, and kidney. 5and 3 RACE PCR was used to obtain the entire 3 sequence and partial 5 sequence of the shark gene. Genome walking was then used to obtain the remaining 5sequence, including 335 bp of untranslated region sequence upstream of the start codon. The full length clone (1282bp) had an open reading frame encoding a protein of 375 amino acids. This isoform was 80% identical at the amino acid level to the human TASK-1 protein (394 amino acids). Major structural features of the human protein were conserved in the shark ortholog, including the four transmembrane segments (M1-M4), the 2P domains (P1 and P2), short NH2- and long COOH-termini, and an extended extracellular loop between M1 and P1. Shark and human TASK-1 full-length clones were expressed in Xenopus oocytes and studied with two electrode voltage clamp (TEVC) techniques. Both the shark and human TASK-1 channel showed identical current voltage relationships (outward rectifying) with a reversal potential near -90 mV compared to water injected controls. The responses to the inhibitor quinine, and the TASK-1 inhibitor bupivacaine, were identical in shark and human TASK-1. However, shark TASK-1 differed from the human ortholog in two critical responses: response to pH and the metal zinc. The pKa for shark TASK-1 was 7.75 vs. 7.37 for human TASK-1, values that are exceedingly close to the arterial pH for each species, suggesting that TASK-1 channels are regulated closely by the ambient pH. An antibody specific to shark TASK-1 was generated and expression of TASK-1 protein in the rectal gland was confirmed by confocal immuno-fluorescent microscopy which revealed localization to the basolateral membrane, with some apical staining. Shark rectal gland TASK-1 appears to be the major K[superscript]+ channel coupled to secretion in the SRG, is the oldest 4TM 2P family member identified to date, and is the first TASK-1 channel identified to play an essential role in chloride secreting epithelia.

No description available.

KATP channels couple cell metabolic status to the membrane excitability by sensing the cytoplasmic ATP/ADP ratio. Present studies examined how conserved motifs (Walker A (WA), signature sequence (L), and Walker B (WB)) within each NBF of SUR2A bind to Syn-1A to affect its actions on cardiac KATP channels. In vitro binding experiments illustrated that Syn-1A binds cardiac SUR2A at WA and L of NBF-1 and WA, L, and WB of NBF-2. Electrophysiology experiments on stably expressing SUR2A/Kir6.2 cell-lines showed that only L and WB of NBF-1 and all three NBF-2 motifs could abrogate the inhibitory effect of Syn-1A on SUR2A/KATP channels. These results lead me to hypothesize that more independent motif in NBF-2 can bind and abrogate Syn-1A’s inhibition than NBF-1 on SUR2A/KATP channels. A corollary postulate is that Syn-1A acts as a scaffold to secure the NBF-1 and -2 in dimer conformation required for SUR2A to modulate Kir6.2 gating.

KATP channels couple cell metabolic status to the membrane excitability by sensing the cytoplasmic ATP/ADP ratio. Present studies examined how conserved motifs (Walker A (WA), signature sequence (L), and Walker B (WB)) within each NBF of SUR2A bind to Syn-1A to affect its actions on cardiac KATP channels. In vitro binding experiments illustrated that Syn-1A binds cardiac SUR2A at WA and L of NBF-1 and WA, L, and WB of NBF-2. Electrophysiology experiments on stably expressing SUR2A/Kir6.2 cell-lines showed that only L and WB of NBF-1 and all three NBF-2 motifs could abrogate the inhibitory effect of Syn-1A on SUR2A/KATP channels. These results lead me to hypothesize that more independent motif in NBF-2 can bind and abrogate Syn-1A’s inhibition than NBF-1 on SUR2A/KATP channels. A corollary postulate is that Syn-1A acts as a scaffold to secure the NBF-1 and -2 in dimer conformation required for SUR2A to modulate Kir6.2 gating.

PSD95 is the canonical member of the Membrane Associated Guanylate Kinase class of scaffold proteins. PSD95 is a five-domain major scaffolding protein abundant in the postsynaptic density (PSD) of the neuronal excitatory synapse. Within PSD95 three PDZ domains modulate protein-protein interactions by selectively binding to short peptide motifs of target proteins. Under the direction of the multivalent PDZ domain interactions, the interacting proteins tend to cluster at the PSD, a phenomenon that is critical for synaptic signalling regulation. Earlier studies have shown that the N-terminal PDZ domains of PSD95 are obligatory for the clustering to occur. This thesis focuses on the strong inwardly rectifying potassium channel, Kir2.1 as the PSD95 binding partner. Kir2.1 is known to maintain membrane resting potential and control cell excitability. Previous studies have reported that Kir2.1 clustered into ordered tetrad complexes upon association with PSD95.This study investigates the detailed clustering mechanisms of Kir2.1 by PDZ domains. To achieve this, components that are involved in the formation of a complex namely PSD95 sub-domains comprising single PDZ and the tandem N terminal PDZ double domain (PDZ1-2), and Kir2.1 cytoplasmic domains(Kir2.1NC) are studied in detail via different structural and biophysical approaches; 1) PDZ1-2 is examined in apo- and bound ligand form with a Kir2.1 Cterminal peptide in crystal and solution via X-ray crystallography and small angle X-ray scattering; 2) the tandem and the single PDZ domain interaction with ligand are measured thermodynamically via isothermal calorimetry (ITC); 3) the complex of full length PSD95 with Kir2.1NC is analyzed with electron microscopy (EM). The protein components are produced in high quality by protein expression and multiple-step protein purification techniques. PDZ1-2 crystallographic structures were solved at 2.02A and 2.19A in theapo- and the liganded forms respectively. The solution state analysis showed domain separation and structural extension of the tandem domain when incorporated with the ligand. The ITC experiment revealed PDZ1-2 to have greater affinity towards the peptide ligand relative to the single PDZ domains. These combinatorial outcomes lead to the conclusion that PSD95 clusters Kir2.1 by adopting an enhanced binding interaction which is associated with increased PDZ1-2 inter-domain separation. The preliminary analysis of PSD95-Kir2.1NC complex with cryo-EM showed the establishment of a tetrad and led to a reconstruction at 40A resolution. The work in obtaining a higher resolution complex structure is promising with further data collection required to allow the employment of more sophisticated model reconstruction processes.

KCNE β-subunits modulate KCNQ1 (Q1) voltage-gate K+channels providing the current diversity required for Q1 channels to function in a wide variety of cell types and tissues. In the present thesis, the stoichiometry of KCNE1 (E1) β-subunits in functioning Q1 channels is investigated, along with the formation of heteromeric channel complexes, complexes containing 2 different KCNE β-subunits. The chemical approaches used to answer these questions were then expanded to generate a novel labeling reagent. To determine the stoichiometry of the Q1/E1 complex, I devised an iterative subunit counting approach that relies on a chemically releasable K+channel blocking reagent. The extracellularly applied reagent irreversibly blocks charybdotoxin (CTX) sensitive Q1 channels by chemically modifying E1 peptides that contain an N-terminal cysteine residue. Chemical release of the inhibitor and subsequent iterative applications of the reagent reported that Q1 channels partner with two KCNE β-subunits. To determine whether heteromeric Q1-KCNE complexes form, I synthesized a similar, but non-cleavable, K+channel blocking reagent that detects specific KCNE peptides in functioning complexes by irreversible channel inhibition. Using this “KCNE sensor”, heteromeric Q1/E1/E3, Q1/E1/E4 and Q1/E3/E4 complexes were shown to form, traffic to the cell surface and function. Using mathematical subtraction to visualize the irreversibly blocked current, the currents and gating kinetics of the different heteromeric complexes were revealed and a hierarchy of KCNE subunit modulation of Q1 channels was determined: E3>E1>>E4. Building on this technology, a chemically releasable K+ channel blocking reagent was created to specifically label KCNE β-subunits with biotin. The reagent delivers biotin to CTX sensitive Q1 channels and labeling occurs through free thiols provided by either cysteine residues or thiol modified sugars. This preliminary data demonstrates a novel strategy for labeling endogenous K+ channels in native cells.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / The interest in organoselenium biochemistry and pharmacology has increased in the last two decades due to a variety of organoselenium compounds that possess biological activity. Accordingly, bis selenide is known as a safe drug when administered acutely to mice at doses that have antiinflammatory and antinociceptive activities. Therefore, the search for the mechanisms by which this compound exerts its effects is extremely important for the therapeutic application. Based on the considerations above, the aims of the present study were to evaluate the antinociceptive properties, as well as the possible mechanisms involved in this process. The oral administration of bis selenide caused significant inhibition of the biting behavior induced by intrathecal (i.t.) injection of glutamate, kainate, (±)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (trans-ACPD), capsaicin, substance P (SP), interleukin 1β (IL-1β) and tumor necrosis factor-α (TNF-α) but completely failed to affect the nociception induced by α-amino-3-hydroxy-5-mehtyl-4-isoxazolepropionic acid (AMPA) and N-methyl-D-aspartate (NMDA). In addition, the oral administration (p.o.) of bis selenide caused a significant increase in hot plate (55 °C) response latency. The antinociceptive effect caused by bis selenide in the hot plate test was reversed by i.t. injection of several K+ channel blockers tetraethylammonium (TEA, non-selective voltage-dependent K+ channel inhibitor) and glibenclamide (ATP-sensitive K+ channel inhibitor) but was not significantly reversed by pretreatment of animals with apamin and charybdotoxin (large- and smallconductance Ca2+- activated K+ channel inhibitors, respectively). These results suggest the participation of glutamatergic, peptidergic and vanilloid systems and potassium channel in the antinociceptive action caused by bis selenide in mice. / Nos últimos anos, os compostos orgânicos de selênio têm sido alvos de interesse em síntese orgânica em virtude da descoberta de suas aplicações sintéticas e de suas propriedades farmacológicas. O seleneto vinílico bis substituído (SVBS) é um composto orgânico de selênio que apresenta baixa toxicidade quando administrado pela via subcutânea em camundongos, nas doses em que exerce ação antinociceptiva e antiinflamatória. Assim, a pesquisa dos mecanismos pelos quais esse composto exerce os efeitos farmacológicos é importante para a sua aplicação terapêutica. Desta forma, no presente trabalho investigou-se as propriedades antinociceptivas do SVBS, bem como os possíveis mecanismos envolvidos em tal processo. A administração oral do SVBS preveniu a nocicepção induzida pela injeção intratecal (i.t.) de glutamato, cainato, ácido (±)-1- aminociclopentano-trans-1,3-dicarboxílico (trans-ACPD), capsaicina, substância P (SP), fator de necrose alfa (TNF-α) e interleucina 1β (IL-1β), mas não bloqueou significativamente a nocicepção causada pela injeção i.t. do ácido α- amino-3-hidroxi-5-metil-4-isoxazolopropionico (AMPA) e ácido N-metil-D-aspártico (NMDA). Além disso, a administração oral do SVBS foi capaz de prevenir a nocicepção térmica, no modelo da chapa quente a 55°C. A antinocicepção causada pela administração oral do SVBS no teste da chapa quente foi revertida pelo pré-tratamento com tetraetilamônio (TEA; bloqueador de diferentes tipos de canais de potássio, inclusive os dependentes de voltagem) e glibenclamida (bloqueador de canais de K+ dependente de ATP), mas não foi revertida pelo pré-tratamento dos animais com apamina (bloqueador de canais de potássio de baixa condutância ativados por cálcio) e caribidotoxina (bloqueador de canais de potássio de alta condutância ativados por cálcio). De acordo com o presente trabalho pode-se concluir que os mecanismos responsáveis pela ação antinociceptiva causada pelo SVBS em camundongos envolvem a participação dos sistemas glutamatérgicos, peptidérgicos e vanilóides e canais de potássio.

Voltage-gated K+ (Kv) channels are known to cause serious disease upon their malfunction. Kv channels desensitised to voltage show inability to fully repolarise the membrane in excitable cells, which can make the membrane hyperexcited and in turn cause seizures such as in epilepsy, periodic ataxia or heart arrhythmia. Therefore, enhancers of Kv channels could serve as potential drugs. Some of these enhancers are polyunsaturated fatty acids and resin-acids which bind at the proteinlipid surface and affect the movement of the voltage sensor in the channel by a mechanism called the lipoelectric effect. To explore the lipoelectric modulation mechanism, we have performed an extensive computational study including docking and molecular dynamics simulations on resin-acid derivatives added to a model potassium channel called Shaker. Four derivatives, Wu32 and Wu50 that excite the channel and thus induce repolarisation of the membrane, as well as Wu18 and Wu27, who were found to be non-potent in previous experimental studies, have helped to point out a novel binding site in Shaker. The site is located between the pore and voltage-sensing domain of the channel and is in direct contact with the first gating charge arginine, R1, and the residue W454. We hypothesize that it is possible for resinacid derivatives to directly bind to the voltage-sensor when it is in an activated state, prolonging the time Shaker stays open. Further experimental studies on Shaker and human homologs are now needed to test our hypothesis. Therefore, we suggest recording the sensitivity of Shaker towards potent derivatives in combination with mutations of W454. If our findings of the novel binding site are correct, the suitability of Shaker as a model system for human Kv channel modulation by lipoelectric modulators can be questioned as W454 is replaced by small hydrophobic side chains in mammalian Shaker homologs.

No description available.