Structure-function analyses of small-conductance, calcium-activated potassium channels

Ion channels are integral membrane proteins present in all cells. They are highly selective and assure a high rate for ions down their electrochemical gradient. In particular, small-conductance calcium-activated potassium channels (SK) are conducting potassium ions and are activated by binding of calcium ions to calmodulin, which is constitutively bound to the carboxy-terminus of each SK channel -subunit. Until now, only three SK channel subunits have been cloned, SK1, SK2 and SK3. Sequence alignment shows that the transmembrane and pore regions are highly conserved, while a high grade of divergence is observed in the amino- and carboxy-termini of the three subunits. In order to determine the expression of the different SK channel subtypes, pharmacological tols such as apamin and d-tubocurarine have been widely used. In this work, I show the characterization of a novel toxin, tamapin, isolated from the scorpion Mesobuths tamulus, which targets SK channels. Our experiments show that this toxin is more potent in blocking SK2 channels than apamin. Furthermore, tamapin only blocked the SK1 and SK3 channels at higher concentrations, with higher efficiency to block SK3 than SK1. Therefore, tamapin should be a good pharmacological tool to determine the molecular composition of native SK channels underlying calcium-activated potassium currents in various tissues. Secondly, I determined the molecular mechanism that prevents the formation of functional SK1 channels cloned from the rat brain (rSK1). Until now, little information was available on the rSK1 channels. rSK1 shows highly sequence identity (84%) with the human homologue, hSK1. hSK1 subunits form functional potassium channels that are blocked by apamin and d-tubocurarine. However, when I expressed rSK1 in HEK-293 cells no potassium currents above background were observed, although immunofluorescence experiments using a specific antibody against the rSK1 protein showed expression of the channel. I generated rSK1 core chimeras in which I exchanged the amino-and/carboxy-terminus with the same region of rSK2 or hSK1. Exchange of amino-and carboxy-terminus or only of the carboxy-terminus resulted in the formation of functional potassium channels. Furthermore, I used these functional chimeras to determine the toxin sensitivity of rSK1 for apamin and d-tubocurarine. Surprisingly, when these blockers wre applied, no sensitivity was observed, although hSK1 and rSK1 show a complete sequence identity in the pore region, which is suggested to contain the binding site for apamin. Finally, I characterized a novel splice variant of the calcium-activated potassium channel subunit rSK2, referred to as rSK2-860. The rSK2-860 cDNA codes for a protein which is 275 amino acids longer at the amino-terminus when compared with originally cloned rSK2 subunit. Transfection of rSK2-860 in different cell lines resulted in a surprising expression pattern of the protein. Th protein formed small clusters around the cell nucleus, but no membrane stain could be observed. This data shows that the additional 275 amino acid-long stretch at the amino-terminus is responsible for retention and clustering of rSK2-860 protein. In order to narrow down the region responsible for this phenotype, I generated truncated proteins. This resulted in the isolation of an 100 amino acid-long region that seems to be responsible for the retention and clustering of rSK2-860 channels. Further truncations and deletions could help us to find the exact signal which is responsible for this characteristic behavior of the rSK2-860 protein

Structure-function analyses of small-conductance, calcium-activated potassium channels

Ion channels are integral membrane proteins present in all cells. They are highly selective and assure a high rate for ions down their electrochemical gradient. In particular, small-conductance calcium-activated potassium channels (SK) are conducting potassium ions and are activated by binding of calcium ions to calmodulin, which is constitutively bound to the carboxy-terminus of each SK channel -subunit. Until now, only three SK channel subunits have been cloned, SK1, SK2 and SK3. Sequence alignment shows that the transmembrane and pore regions are highly conserved, while a high grade of divergence is observed in the amino- and carboxy-termini of the three subunits. In order to determine the expression of the different SK channel subtypes, pharmacological tols such as apamin and d-tubocurarine have been widely used. In this work, I show the characterization of a novel toxin, tamapin, isolated from the scorpion Mesobuths tamulus, which targets SK channels. Our experiments show that this toxin is more potent in blocking SK2 channels than apamin. Furthermore, tamapin only blocked the SK1 and SK3 channels at higher concentrations, with higher efficiency to block SK3 than SK1. Therefore, tamapin should be a good pharmacological tool to determine the molecular composition of native SK channels underlying calcium-activated potassium currents in various tissues. Secondly, I determined the molecular mechanism that prevents the formation of functional SK1 channels cloned from the rat brain (rSK1). Until now, little information was available on the rSK1 channels. rSK1 shows highly sequence identity (84%) with the human homologue, hSK1. hSK1 subunits form functional potassium channels that are blocked by apamin and d-tubocurarine. However, when I expressed rSK1 in HEK-293 cells no potassium currents above background were observed, although immunofluorescence experiments using a specific antibody against the rSK1 protein showed expression of the channel. I generated rSK1 core chimeras in which I exchanged the amino-and/carboxy-terminus with the same region of rSK2 or hSK1. Exchange of amino-and carboxy-terminus or only of the carboxy-terminus resulted in the formation of functional potassium channels. Furthermore, I used these functional chimeras to determine the toxin sensitivity of rSK1 for apamin and d-tubocurarine. Surprisingly, when these blockers wre applied, no sensitivity was observed, although hSK1 and rSK1 show a complete sequence identity in the pore region, which is suggested to contain the binding site for apamin. Finally, I characterized a novel splice variant of the calcium-activated potassium channel subunit rSK2, referred to as rSK2-860. The rSK2-860 cDNA codes for a protein which is 275 amino acids longer at the amino-terminus when compared with originally cloned rSK2 subunit. Transfection of rSK2-860 in different cell lines resulted in a surprising expression pattern of the protein. Th protein formed small clusters around the cell nucleus, but no membrane stain could be observed. This data shows that the additional 275 amino acid-long stretch at the amino-terminus is responsible for retention and clustering of rSK2-860 protein. In order to narrow down the region responsible for this phenotype, I generated truncated proteins. This resulted in the isolation of an 100 amino acid-long region that seems to be responsible for the retention and clustering of rSK2-860 channels. Further truncations and deletions could help us to find the exact signal which is responsible for this characteristic behavior of the rSK2-860 protein

Domain analysis of the calcium-activated potassium channel SK1 from rat brain - Functional expression and toxin sensitivity

Two small conductance, calcium-activated potassium channels (SK channels), SK2 and SK3, have been shown to contribute to the afterhyperpolarization (AHP) and to shape the firing behavior in neurons for example in the hippocampal formation, the dorsal vagal nucleus, the subthalamic nucleus, and the cerebellum. In heterologous expression systems, SK2 and SK3 currents are blocked by the bee venom toxin apamin, just as well as the corresponding neuronal AHP currents. However, the functional role and pharmacological profile of SK1 channels from rat brain (rSK1) is still largely unknown, as so far rSK1 homomeric channels could not be functionally expressed. We have performed a domain analysis to elucidate the pharmacological profile and the molecular determinants of rSK1 channel expression by using channel chimeras in combination with immunocytochemistry, immunoblot analysis, and electrophysiology. Our results reveal that the rSK1 subunit is synthesized in cells but does not form functional homomeric channels. Exchanging the carboxyl terminus of rSK1 for that of hSK1 or rSK2 is sufficient to rescue the functional expression of rSK1 channels. Additionally, transplantation of both amino and carboxyl termini of rSK1 onto hSK1 subunits, normally forming functional homomeric channel, hinders their functional expression, while hSK1 channels containing only the rSK1 carboxyl terminus are functional. These results suggest that the lack of functional expression of rSK1 channels is probably due to problems in their assembly and tetramerization but not in their calmodulin-dependent gating. Finally, we show that chimeric channels containing the core domain (S1 - S6) of rSK1, unlike hSK1, are apamin-insensitive

Transient receptor potential channels in sensory neurons are targets of the antimycotic agent clotrimazole

Clotrimazole (CLT) is a widely used drug for the topical treatment of yeast infections of skin, vagina, and mouth. Common side effects of topical CLT application include irritation and burning pain of the skin and mucous membranes. Here, we provide evidence that transient receptor potential (TRP) channels in primary sensory neurons underlie these unwanted effects of CLT. We found that clinically relevant CLT concentrations activate heterologously expressed TRPV1 and TRPA1, two TRP channels that act as receptors of irritant chemical and/or thermal stimuli in nociceptive neurons. In line herewith, CLT stimulated a subset of capsaicin-sensitive and mustard oil-sensitive trigeminal neurons, and evoked nocifensive behavior and thermal hypersensitivity with intraplantar injection in mice. Notably, CLT-induced pain behavior was suppressed by the TRPV1-antagonist BCTC [(N-(-4-tertiarybutylphenyl)-4-(3- cholorpyridin-2-yl)tetrahydropyrazine-1(2H)-carboxamide)] and absent in TRPV1-deficient mice. In addition, CLT inhibited the cold and menthol receptor TRPM8, and blocked menthol-induced responses in capsaicin- and mustard oil-insensitive trigeminal neurons. The concentration for 50% inhibition (IC50) of inward TRPM8 current was ∼200 nM, making CLT the most potent known TRPM8 antagonist and a useful tool to discriminate between TRPM8- and TRPA1-mediated responses. Together, our results identify TRP channels in sensory neurons as molecular targets of CLT, and offer means to develop novel CLT preparations with fewer unwanted sensory side effects. Copyright © 2008 Society for Neuroscience.This work was supported by grants from the Human Frontiers Science Program (RGP32/2004), the Belgian Federal Government (IUAP P5/05), the Research Foundation-Flanders (G.0172.03; G.0565.07), the Research Council of the KU Leuven (GOA 2004/07; EF/95/010), the Spanish Minsitry of Education (SAF2004-01011), the Generalitat Valenciana Predoctoral Fellowship Program (CTBPRB/2003/151), and the Fundación Marcelino Botín.Peer Reviewe

Tamapin, a venom peptide from the Indian red scorpion (mesobuthus tamulus) that targets small conductance Ca2+-activated K+ channels and afterhyperpolarization currents in central neurons

The biophysical properties of small conductance Ca2+-activated K+ (SK) channels are well suited to underlie afterhyperpolarizations (AHPs) shaping the firing patterns of a conspicuous number of central and peripheral neurons. We have identified a new scorpion toxin (tamapin) that binds to SK channels with high affinity and inhibits SK channel-mediated currents in pyramidal neurons of the hippocampus as well as in cell lines expressing distinct SK channel subunits. This toxin distinguished between the SK channels underlying the apamin-sensitive IAHP and the Ca2+-activated K+ channels mediating the slow IAHP (sIAHP) in hippocampal neurons. Compared with related scorpion toxins, tamapin displayed a unique, remarkable selectivity for SK2 versus SK1 (~1750-fold) and SK3 (~70-fold) channels and is the most potent SK2 channel blocker characterized so far (IC50 for SK2 channels = 24 pM). Tamapin will facilitate the characterization of the subunit composition of native SK channels and help determine their involvement in electrical and biochemical signaling.</p

NMR Structure and Action on Nicotinic Acetylcholine Receptors of Water-soluble Domain of Human LYNX1*

Discovery of proteins expressed in the central nervous system sharing the three-finger structure with snake α-neurotoxins provoked much interest to their role in brain functions. Prototoxin LYNX1, having homology both to Ly6 proteins and three-finger neurotoxins, is the first identified member of this family membrane-tethered by a GPI anchor, which considerably complicates in vitro studies. We report for the first time the NMR spatial structure for the water-soluble domain of human LYNX1 lacking a GPI anchor (ws-LYNX1) and its concentration-dependent activity on nicotinic acetylcholine receptors (nAChRs). At 5–30 μm, ws-LYNX1 competed with 125I-α-bungarotoxin for binding to the acetylcholine-binding proteins (AChBPs) and to Torpedo nAChR. Exposure of Xenopus oocytes expressing α7 nAChRs to 1 μm ws-LYNX1 enhanced the response to acetylcholine, but no effect was detected on α4β2 and α3β2 nAChRs. Increasing ws-LYNX1 concentration to 10 μm caused a modest inhibition of these three nAChR subtypes. A common feature for ws-LYNX1 and LYNX1 is a decrease of nAChR sensitivity to high concentrations of acetylcholine. NMR and functional analysis both demonstrate that ws-LYNX1 is an appropriate model to shed light on the mechanism of LYNX1 action. Computer modeling, based on ws-LYNX1 NMR structure and AChBP x-ray structure, revealed a possible mode of ws-LYNX1 binding