Protein Surface Topography as a tool to enhance the selective activity of a potassium channel blocker
Tk-hefu is an artificial peptide designed based on the α-hairpinin scaffold, which selectively blocks voltage-gated potassium channels Kv1.3. Here we present its spatial structure resolved by NMR spectroscopy and analyze its interaction with channels using computer modeling. We apply protein surface topography to suggest mutations and increase Tk-hefu affinity to the Kv1.3 channel isoform. We redesign the functional surface of Tk-hefu to better match the respective surface of the channel pore vestibule. The resulting peptide Tk-hefu-2 retains Kv1.3 selectivity and displays ∼15 times greater activity compared with Tk-hefu. We verify the mode of Tk-hefu-2 binding to the channel outer vestibule experimentally by site-directed mutagenesis. We argue that scaffold engineering aided by protein surface topography represents a reliable tool for design and optimization of specific ion channel ligands.
Regulation of the formation and rewiring of neural circuits by neuropeptides may require coordinated production of these signaling molecules and their receptors that may be established at the transcriptional level. Here, we address this hypothesis by comparing absolute expression levels of opioid peptides with their receptors, the largest neuropeptide family, and by characterizing coexpression (transcriptionally coordinated) patterns of these genes. We demonstrated that expression patterns of opioid genes highly correlate within and across functionally and anatomically different areas. Opioid peptide genes, compared with their receptor genes, are transcribed at much greater absolute levels, which suggests formation of a neuropeptide cloud that covers the receptor-expressed circuits. Surprisingly, we found that both expression levels and the proportion of opioid receptors are strongly lateralized in the spinal cord, interregional coexpression patterns are side-specific, and intraregional coexpression profiles are affected differently by left- and right-side unilateral body injury. We propose that opioid genes are regulated as interconnected components of the same molecular system distributed between distinct anatomic regions. The striking feature of this system is its asymmetric coexpression patterns, which suggest side-specific regulation of selective neural circuits by opioid neurohormones.
Scorpion venom is an unmatched source of selective high-affinity ligands of potassium channels. There is a high demand for such compounds to identify and manipulate the activity of particular channel isoforms. The objective of this study was to obtain and characterize a specific ligand of voltage-gated potassium channel KV1.2. As a result, we report the remarkable selectivity of the peptide MeKTx11-1 (α-KTx 1.16) from Mesobuthus eupeus scorpion venom to this channel isoform. MeKTx11-1 is a high-affinity blocker of KV1.2 (IC50 ∼0.2 nM), while its activity against KV1.1, KV1.3, and KV1.6 is 10 000, 330 and 45 000 fold lower, respectively, as measured using the voltage-clamp technique on mammalian channels expressed in Xenopus oocytes. Two substitutions, G9V and P37S, convert MeKTx11-1 to its natural analog MeKTx11-3 (α-KTx 1.17) having 15 times lower activity and reduced selectivity to KV1.2. We produced MeKTx11-1 and MeKTx11-3 as well as their mutants MeKTx11- 1(G9V) and MeKTx11-1(P37S) recombinantly and demonstrated that point mutations provide an intermediate effect on selectivity. Key structural elements that explain MeKTx11-1 specificity were identified by molecular modeling of the toxin–channel complexes. Confirming our molecular modeling predictions, site-directed transfer of these elements from the pore region of KV1.2 to KV1.3 resulted in the enhanced sensitivity of mutant KV1.3 channels to MeKTx11-1. We conclude that MeKTx11-1 may be used as a selective tool in neurobiology.
Acid-sensing ion channels (ASICs), members of the family of amiloride-sensitive degenerin/epithelial Na + -channels, are expressed in neurons of both the peripheral and central nervous system. Among six currently known isoforms of mammalian ASICs, two, namely ASIC1a and ASIC3 are widespread and have the largest physiological contribution such as synaptic plas- ticity, learning and memory as well as pain perception and inflammation development. We have previously shown that seva- nol, a new lignan isolated from Thymus armeniacus, inhibits ASIC1a and ASIC3 currents and exerts analgesic and anti- inflammatory effects when administered intravenously. Here we present a scheme for the synthesis of sevanol, which was devel- oped for the first time. In addition, sevanol analogues were syn- thesized, in which the basic core of the molecule of epiphyllic acid remained unchanged, while substituents for carboxyl groups in positions 9,10 were modified. These analogues demonstrate a clear correlation between the activity of the molecule and the number of free carboxyl groups in it. Using molecular modeling and analysis of the activity of sevanol in the presence of ASIC1a potentiator, an RF-amide peptide, we established a possible bind- ing site for sevanol on the channel. We also showed that with intranasal administration, sevanol can have the same effective analgesic effect as with intravenous administration. Such struc- tural and functional analysis demonstrates a correlation between the inhibitory effect value and the number of functional groups of the molecule, which may be important for the rational design of biologically active sevanol-based compounds.
Many environmental stimuli present a quasi-rhythmic structure at different timescales that the brain needs to decompose and integrate. Cortical oscillations have been proposed as instruments of sensory de-multiplexing, i.e., the parallel processing of different frequency streams in sensory signals. Yet their causal role in such a process has never been demonstrated. Here, we used a neural microcircuit model to address whether coupled theta–gamma oscillations, as observed in human auditory cortex, could underpin the multiscale sensory analysis of speech. We show that, in continuous speech, theta oscillations can flexibly track the syllabic rhythm and temporally organize the phoneme-level response of gamma neurons into a code that enables syllable identification. The tracking of slow speech fluctuations by theta oscillations, and its coupling to gamma-spiking activity both appeared as critical features for accurate speech encoding. These results demonstrate that cortical oscillations can be a key instrument of speech de-multiplexing, parsing, and encoding.
Hypoxia of trophoblast cells is an important regulator of normal development of the placenta. However, some pathological states associated with hypoxia, e.g. preeclampsia, impair the functions of placental cells. Oxyquinoline derivative inhibits HIF-prolyl hydroxylase by stabilizing HIF-1 transcription complex, thus modeling cell response to hypoxia. In human choriocarcinoma cells BeWo b30 (trophoblast model), oxyquinoline increased the expression of a core hypoxia response genes along with up-regulation of NOS3, PDK1, and BNIP3 genes and down-regulation of the PPARGC1B gene. These changes in the expression profile attest to activation of the metabolic cell reprogramming mechanisms aimed at reducing oxygen consumption by enabling the switch from aerobic to anaerobic glucose metabolism and the respective decrease in number of mitochondria. The possibility of practical use of the therapeutic properties of oxyquinoline derivatives is discussed.
In the internal medicine wide spectrum the gastroenterology is one of the chapters, less enlightened by the scientific evidence. It does not mean that the practice of the grasntroenterology may ot be improved by the systematic use of the approaches of the evidence based medicine