gamma-Aminobutyric acid (GABA), the principal inhibitory neurotransmitter, activates a persistent low amplitude tonic current in several brain regions in addition to conventional synaptic currents. Here we demonstrate that GABA(A) receptors mediating the tonic current in hippocampal neurons exhibit functional and pharmacological properties different from those of quantal synaptic currents. Patch-clamp techniques were used to characterize miniature inhibitory postsynaptic currents (mIPSCs) and the tonic GABAergic current recorded in CA1 pyramidal neurons in rat hippocampal slices and in dissociated neurons grown in culture. The competitive GABA(A) receptor antagonists, bicuculline and picrotoxin, blocked both the mIPSCs and the tonic current. In contrast, mIPSCs but not the tonic current were inhibited by gabazine (SR-95531). Coapplication experiments and computer simulations revealed that gabazine bound to the receptors responsible for the tonic current but did not prevent channel activation. However, gabazine competitively inhibited bicuculline blockade. The unitary conductance of the GABA(A) receptors underlying the tonic current (approximately 6 pS) was less than the main conductance of channels activated during quantal synaptic transmission (approximately 15--30 pS). Furthermore, compounds that potentiate GABA(A) receptor function including the benzodiazepine, midazolam, and anesthetic, propofol, prolonged the duration of mIPSCs and increased tonic current amplitude in cultured neurons to different extents. Clinically-relevant concentrations of midazolam and propofol caused a greater increase in tonic current compared with mIPSCs, as measured by total charge transfer. In summary, the receptors underlying the tonic current are functionally and pharmacologically distinct from quantally activated synaptic receptors and these receptors represent a novel target for neurodepressive drugs.

In the hippocampus, two distinct forms of GABAergic inhibition have been identified, phasic inhibitory postsynaptic currents that are the consequence of the vesicular release of GABA and a tonic conductance that is activated by low ambient concentrations of extracellular GABA. It is not known what accounts for the distinct properties of receptors that mediate the phasic and tonic inhibitory conductances. Moreover, the physiological role of the tonic inhibitory conductance remains uncertain because pharmacological tools that clearly distinguish tonic and phasic receptors are lacking. Here, we demonstrate that GABA A receptors that generate a tonic conductance in cultured hippocampal neurons from embryonic mice have different pharmacological properties than those in cerebellar granule neurons or pyramidal neurons in the dentate gyrus. The tonic conductance in cultured hippocampal neurons is enhanced by the benzodiazepine, midazolam, and is insensitive to the inhibitory effects of the competitive antagonist, gabazine (Յ10 M). We also identify penicillin as an uncompetitive antagonist that selectively inhibits the synaptic but not tonic conductance. GABA was applied to hippocampal neurons to investigate the properties of synaptic and extrasynaptic receptors. GABA-evoked current was composed of two components: a rapidly desensitizing current that was blocked by penicillin and a nondesensitizing current that was insensitive to penicillin blockade. The potency of GABA was greater for the penicillin-insensitive nondesensitizing current. Single-channel studies show that the gabazine-insensitive GABA A receptors have a lower unitary conductance (12 pS) than that estimated for synaptic receptors. Thus, specialized GABA A receptors with an apparent higher affinity for GABA that do not readily desensitize mediate the persistent tonic conductance in hippocampal neurons. The receptors underlying tonic and phasic inhibitory conductances in hippocampal neurons are pharmacologically and biophysically distinct, suggesting that they serve different physiological roles.

channels have a higher K¤ permeability at hyperpolarizing than depolarizing membrane potentials. Activity of these K¤ channels is also controlled by several intra-and extracellular factors. One of these factors is the hydrogen ion (Coulter et al. 1995;Tsai et al. 1995;Doi et al. 1996;Fakler et al. 1996;Shieh et al. 1996;Choe et al. 1997;Sabirov et al. 1997). While H¤ concentration, or pH level, is maintained by intra-and extracellular buffer systems, there are conditions in which protons are overproduced during respiratory or metabolic acidosis. A decrease in intra-or extracellular pH has been shown to inhibit inward rectifier K¤ channels in

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The prominent role of voltage-gated sodium channel 1.7 (Nav1.7) in nociception was revealed by remarkable human clinical and genetic evidence. Development of potent and subtypeselective inhibitors of this ion channel is crucial for obtaining therapeutically useful analgesic compounds. Microproteins isolated from animal venoms have been identified as promising therapeutic leads for ion channels, because they naturally evolved to be potent ion channel blockers. Here, we report the engineering of highly potent and selective inhibitors of the Nav1.7 channel based on tarantula ceratotoxin-1 (CcoTx1). We utilized a combination of directed evolution, saturation mutagenesis, chemical modification, and rational drug design to obtain higher potency and selectivity to the Nav1.7 channel. The resulting microproteins are highly potent (IC 50 to Nav1.7 of 2.5 nM) and selective. We achieved 80-and 20-fold selectivity over the closely related Nav1.2 and Nav1.6 channels, respectively, and the IC 50 on skeletal (Nav1.4) and cardiac (Nav1.5) sodium channels is above 3000 nM. The lead molecules have the potential for future clinical development as novel therapeutics in the treatment of pain.

Protons are involved in gating Kir2.3. To identify the molecular motif in the Kir2.3 channel protein that is responsible for this process, experiments were performed using wild-type and mutated Kir2.3 and Kir2.1. CO 2 and low pH i strongly inhibited wild-type Kir2.3 but not Kir2.1 in whole cell voltage clamp and excised inside-out patches. This CO 2 /pH sensitivity was completely eliminated in a mutant Kir2.3 in which the N terminus was substituted with that in Kir2.1, whereas a similar replacement of its C terminus had no effect. Site-specific mutations of all titratable residues in the N terminus, however, did not change the CO 2 /pH sensitivity. Using several chimeras generated systematically in the N terminus, a 10-residue motif near the M1 region was identified in which only three amino acids are different between Kir2.3 and Kir2.1. Mutations of these residues, especially Thr 53 , dramatically reduced the pH sensitivity of Kir2.3. Introducing these residues or even a single threonine to the corresponding positions of Kir2.1 made the mutant channel pH-sensitive. Thus, a critical motif responsible for gating Kir2.3 by protons was identified in the N terminus, which contained about 10 residues centered by Thr 53 .The modulation of membrane excitability is a significant cellular property, which not only provides cells with an important strategy for responding to their external stimuli but also enables them to monitor their internal environment and intermediary metabolism (1). Inward rectifier K ϩ channels play a part in this process in which several intracellular molecules such as nucleotides and protons are involved (2, 3). Whereas the concentration of protons is normally controlled by several buffering and feedback-controlling systems, pH can fall beyond its normal levels under certain pathophysiological conditions. For instance, CO 2 retention, or hypercapnia, can cause a reduction in intra-and extracellular pH leading to respiratory acidosis (4).It is known that CO 2 sensing, which plays an important role in the feedback regulation of CO 2 and pH homeostasis in mammalian systems, is carried out by chemoreceptors, especially the central chemoreceptors (5-8). Through these chemoreceptors, a high PCO 2 level stimulates respiratory neuronal networks in the brain stem and enhances respiratory motor output. During this process, the information of PCO 2 levels received by these chemoreceptor cells may first be conveyed to the change in their membrane excitability and then passed to the respiratory neuronal networks through chemical or electrical synaptic transmissions (9, 10). Hence, the change in membrane excitability constitutes an important step in the CO 2 sensing.The alteration in membrane excitability with hypercapnia is known to be mediated by specific ion channels. CO 2 has been shown to enhance the firing activity of locus ceruleus neurons by suppressing a proton-and polyamine-sensitive inward rectifying K ϩ current (11). Our previous studies have shown that CO 2 induces a depolarization in Lymnaea snail ...

ROMK channels are inhibited by intracellular acidification. This pH sensitivity is related to several amino acid residues in the channel proteins such as Lys-61, Thr-51, and His-206 (in ROMK2). Unlike all other amino acids, histidine is titratable at pH 6 -7 carrying a positive charge below pH 6. To test the hypothesis that certain histidine residues are engaged in CO 2 and pH sensing of ROMK1, we performed experiments by systematic mutations of all histidine residues in the channel using the site-directed mutagenesis. There are two histidine residues in the N terminus. Mutations of His-23, His-31, or both together did not affect channel sensitivity to CO 2 . Six histidine residues are located in the C terminus. His-225, His-274, His-342, and His-354 were critical in CO 2 and pH sensing. Mutation of either of them reduced CO 2 and pH sensitivities by 20 -50% and ϳ0.2 pH units, respectively. Simultaneous mutations of all of them eliminated the CO 2 sensitivity and caused this mutant channel to respond to only extremely acidic pH. Similar mutations of His-280 had no effect. The role of His-270 in CO 2 and pH sensing is unclear, because substitutions of this residue with either a neutral, negative, or positive amino acid did not produce any functional channel. These results therefore indicate that histidine residues contribute to the sensitivity of the ROMK1 channel to hypercapnia and intracellular acidosis.ROMK channels (Kir1.1 and Kir1.2), members in the inward rectifier K ϩ channel family, were first cloned in the kidney and have later been found in several organs including the central nervous system (1, 2). These K ϩ channels are believed to control K ϩ secretion in the renal tubular cells and membrane potential in excitable cells (3,4). ROMK channels have a relatively weak inward rectification allowing significant K ϩ currents in the outward direction and are subject to extensive modulations by second messengers, protein kinases, phospholipids, ATP, and other nucleotides (5). Another important modulator of the ROMK channels is proton. A decrease in intracellular pH strongly inhibits these channels (6 -11).Proton sensing in ROMK channels requires multiple sites or residues in the channel protein to interact with protons. A lysine residue (Lys-80 in ROMK1 or Lys-61 in ROMK2) in the N terminus of channel proteins plays a critical part in the channel sensitivity to intracellular pH. Mutation of this residue to methionine greatly reduces the pH sensitivity (8, 10). However, lysine is not titratable at a physiological pH range. Thus, how this lysine residue works in pH sensing is not fully understood. A nontitratable threonine residue at position 51 of ROMK2 is also involved in pH sensing (8). Mutation of this threonine to a negatively charged residue enhances pH sensitivity, whereas switching it to a positive amino acid decreases the pH sensitivity (8). Another residue related to pH sensing is histidine 206. Mutation of this residue to glycine enhances channel sensitivity to pH (10). Supporting multiple interaction...

Human CLDN18.2 is highly expressed in a significant proportion of gastric and pancreatic adenocarcinomas, while normal tissue expression is limited to the epithelium of the stomach. The restricted expression makes it a potential drug target for the treatment of gastric and pancreatic adenocarcinoma, as evidenced by efforts to target CLDN18.2 via naked antibody and CAR-T modalities. Herein we describe CLDN18.2-targeting via a CD3-bispecific and an antibody drug conjugate and the characterization of these potential therapeutic molecules in efficacy and preliminary toxicity studies. Anti-hCLDN18.2 ADC, CD3-bispecific and diabody, targeting a protein sequence conserved in rat, mouse and monkey, exhibited in vitro cytotoxicity in BxPC3/hCLDN18.2 (IC 50 = 1.52, 2.03, and 0.86 nM) and KATO-III/hCLDN18.2 (IC 50 = 1.60, 0.71, and 0.07 nM) respectively and inhibited tumor growth of pancreatic and gastric patient-derived xenograft tumors. In a rat exploratory toxicity study, the ADC was tolerated up to 10 mg/kg. In a preliminary assessment of tolerability, the anti-CLDN18.2 diabody (0.34 mg/kg) did not produce obvious signs of toxicity in the stomach of NSG mice 4 weeks after dosing. Taken together, our data indicate that targeting CLDN18.2 with an ADC or bispecific modality could be a valid therapeutic approach for the treatment of gastric and pancreatic cancer.