Structure-guided Transformation of Charybdotoxin Yields an Analog That Selectively Targets Ca2+-activated over Voltage-gated K+ Channels

118

116

Membrane proteins of cytotoxic T cells specifically reorganize to form an immunological synapse (IS) on interaction with their specific target. In this paper, we investigated the redistribution of Kv1.3 channels, which are the dominant voltage-gated potassium channels, in the plasma membrane of allogen-activated human cytotoxic T lymphocytes (CTLs) on interacting with their specific target cells. Kv1.3 channels bearing a FLAG epitope were expressed in the CTLs and the cell-surface distribution of fluorescently labeled ion channels was determined from confocal laser-scanning microscopy images. FLAG epitope-tagged Kv1.3 channels showed a patchy distribution in CTLs not engaged with target cells, whereas the channels were accumulated in the IS formed between CTLs and specific target lymphocytes. Localization of Kv1.3 channels in the IS might open an unrevealed possibility in the regulation of ion channel activity by signaling molecules accumulated in the IS.

Section: Hla-a2-specific Killing Of Target Cells By Allogen-activatedsupporting

confidence: 67%

Kv1.3 potassium channels are localized in the immunological synapse formed between cytotoxic and target cells

Panyi

Vámosi

Bacsó

et al. 2004

118

116

“…ChTx is known to block Ca 2+ -activated KCa1.1 and KCa3.1 channels and specific Kv1.x channels (7,20). KCa3.1 channels are apamin-insensitive but have specific binding sites for ChTx and the clotrimazole-related compound TRAM-34 (9,21). TRAM-34 has been established as a selective KCa3.1 blocker with no effects on KCa1.1, KCa2.2, or a wide array of Kv channels (9,10) or Cav3 channels (Fig.…”

Section: Resultsmentioning

confidence: 99%

Intermediate conductance calcium-activated potassium channels modulate summation of parallel fiber input in cerebellar Purkinje cells

Engbers

Anderson²,

Asmara³

et al. 2012

100

Encoding sensory input requires the expression of postsynaptic ion channels to transform key features of afferent input to an appropriate pattern of spike output. Although Ca 2+ -activated K + channels are known to control spike frequency in central neurons, Ca 2+ -activated K + channels of intermediate conductance (KCa3.1) are believed to be restricted to peripheral neurons. We now report that cerebellar Purkinje cells express KCa3.1 channels, as evidenced through single-cell RT-PCR, immunocytochemistry, pharmacology, and single-channel recordings. Furthermore, KCa3.1 channels coimmunoprecipitate and interact with low voltage-activated Cav3.2 Ca 2+ channels at the nanodomain level to support a previously undescribed transient voltage-and Ca 2+ -dependent current. As a result, subthreshold parallel fiber excitatory postsynaptic potentials (EPSPs) activate Cav3 Ca 2+ influx to trigger a KCa3.1-mediated regulation of the EPSP and subsequent after-hyperpolarization. The Cav3-KCa3.1 complex provides powerful control over temporal summation of EPSPs, effectively suppressing low frequencies of parallel fiber input. KCa3.1 channels thus contribute to a high-pass filter that allows Purkinje cells to respond preferentially to high-frequency parallel fiber bursts characteristic of sensory input.C entral neurons receive an enormous number of spontaneously active synaptic inputs, but exhibit the capacity to differentiate features of sensory input from background noise. Cerebellar Purkinje cells are contacted by up to ∼150,000 parallel fibers from granule cells, of which only a subset will convey sensory information at any given time. The activation of a peripheral receptive field is transmitted to the cerebellar cortex by mossy fibers in the form of high-frequency spike bursts (1). The resulting temporal summation of excitatory postsynaptic potentials (EPSPs) generates a similar high-frequency burst in granule cells (2). Purkinje cells should then also possess the means to respond effectively to bursts of parallel fiber input that convey sensory information compared with background activity.Postsynaptic membrane excitability can be controlled by activation of K + channels. There are two established types of Ca 2+ -activated K + (KCa) channels in CNS neurons: small conductance (SK, KCa2.x) and big conductance (BK, KCa1.1) (3, 4). A third class of intermediate conductance (KCa3.1, SK4, IK1) KCa channel is thought to be expressed only in microglia and endothelial cells in the CNS (3, 5, 6). KCa3.1 channels are gated by calmodulin in a similar manner to KCa2.x channels but are insensitive to block by apamin and tetraethylammonium (TEA) (6-8). Instead, the KCa3.1 α-subunit, encoded by the gene KCNN4, has specific residues that bind charybdotoxin and 1-[(2-chlorophenyl) diphenylmethyl]-1H-pyrazole (TRAM-34) (5-7, 9, 10).In cerebellar Purkinje cells, KCa1.1 and KCa2.2 channels are activated during a spike by high voltage-activated (HVA) P-type Ca 2+ channels (11). In contrast, low voltage-activated (LVA) Cav3 (T-type) Ca 2+ channe...

“…This value, selected from other pairwise experiments (22), is 2-fold higher than the minimal threshold value characterizing interacting residues separated by 4-7 Å (18). Table 3, which is published as supporting information on the PNAS web site, www.pnas.org, and the histogram in Fig 3 show ⌬⌬G int values derived from our pairwise experiments.…”

Section: Double-mutant Cyclementioning

confidence: 99%

Experimentally based model of a complex between a snake toxin and the α7 nicotinic receptor

Fruchart-Gaillard

Gilquin

Antil-Delbeke

et al. 2002

120

150

To understand how snake neurotoxins interact with nicotinic acetylcholine receptors, we have elaborated an experimentally based model of the ␣-cobratoxin-␣7 receptor complex. This model was achieved by using (i) a three-dimensional model of the ␣7 extracellular domain derived from the crystallographic structure of the homologous acetylcholine-binding protein, (ii) the previously solved x-ray structure of the toxin, and (iii) nine pairs of residues identified by cycle-mutant experiments to make contacts between the ␣-cobratoxin and ␣7 receptor. Because the receptor loop F occludes entrance of the toxin binding pocket, we submitted this loop to a dynamics simulation and selected a conformation that allowed the toxin to reach its binding site. T he ␣-neurotoxins from snake venom are potent antagonists that block nicotinic acetylcholine receptors (AChRs) and hence affect synaptic transmission (1-3). Despite many studies (reviewed in ref. 4), the molecular process associated with this efficient blockage remains unclear. To approach this question, we previously studied ␣-cobratoxin (␣-Cbtx), an ␣͞K neurotoxin that binds to both muscular and homopentameric neuronal receptors (␣7 and ␣8) with high affinities (4). This toxin, similar to other snake neurotoxins, is folded into three adjacent loops rich in ␤-sheet that emerge from a small globular core in which four disulfide bonds are located (5). By mutational analyses, the residues by which ␣-Cbtx interacts with the muscular-type or neuronal ␣7 receptors were identified previously (6, 7). The present study shows how functional residues account for the antagonistic properties of the toxin toward the ␣7 neuronal receptor. The ␣7 AChR possesses five identical ␣7 subunits (8) that offer five ligand-binding sites located at the interface of two subunits (9). These sites include residues located on the different functional loops described previously on the principal ␣7 (ϩ) face, loops A, B, and C and on the complementary ␣7 (Ϫ) face, loops D, E, and F (refs. 10-13; see Fig. 1). Until now, the residues of the ␣7 receptor involved in snake toxin binding have remained unknown.The aim of the present paper is fourfold. First, by an extensive mutational study we have identified ␣7 receptor residues involved in the interaction with ␣-Cbtx. Second, by using a double-mutant cycle approach we have disclosed several pairs of interacting residues in the toxin-receptor complex. Third, by using the three-dimensional (3D) structure of an AChBP that is similar functionally and structurally to the N-terminal domain of an AChR ␣-subunit (14), we used a 3D model for the ␣7 subunit extracellular region obtained by comparative modeling [see accompanying paper on page 3210 (15)]. Fourth, by using this model, a molecular dynamics simulation of the loop F region, and the constraints derived from our pairwise analysis, we propose an experimentally based 3D model of the complex between the ␣-Cbtx and ␣7 receptor, which explains the antagonistic properties of the snake toxin toward the neuronal recepto...