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Local [Ca.sup.2+] signaling occurring within nanometers of voltage-gated [Ca.sup.2+] (Cav) channels is crucial for CNS function, yet the molecular composition of Cav channel nano-environments is largely unresolved. Here, we used a proteomic strategy combining knockout-controlled multiepitope affinity purifications with high-resolution quantitative MS for comprehensive analysis of the molecular nano-environments of the Cav2 channel family in the whole rodent brain. The analysis shows that Cav2 channels, composed of pore-forming [alpha]1 and auxiliary [beta] subunits, are embedded into protein networks that may be assembled from a pool of ~200 proteins with distinct abundance, stability of assembly, and preference for the three Cav2 subtypes. The majority of these proteins have not previously been linked to Cav channels; about two-thirds are dedicated to the control of intracellular [Ca.sup.2+] concentration, including G protein-coupled receptor-mediated signaling, to activity-dependent cytoskeleton remodeling or [Ca.sup.2+]-dependent effector systems that comprise a high portion of the priming and release machinery of synaptic vesicles. The identified protein networks reflect the cellular processes that can be initiated by Cav2 channel activity and define the molecular framework for organization and operation of local [Ca.sup.2+] signaling by Cav2 channels in the brain. calcium channel | [Ca.sup.2+] signaling | proteome | biochemistry | mass spectrometry doi/ 10.1073/pnas.1005940107

Rasmussen, Hanne Borger, Morten Morten, Hans-Gunther Knaus, Bo Skaaning Jensen, Soren-Peter Olesen, and Nanna Koschmieder Jorgensen. Subcellular localization of the delayed rectifier [K.sup.+] channels KCNQ1 and ERG1 in the rat heart. Am J Physiol Heart Cire Physiol 286:H1300 H1309, 2004. First published December 11,2003; 10.1152/ajpheart.00344.2003.--In the heart, several [K.sup.+] channels are responsible for the repolarization of the cardiac action potential, including transient outward and delayed rectifier [K.sup.+] currents. In the present study, the cellular and subcellular localization of the two delayed rectifier [K.sup.+] channels, KCNQI and ether-a-go-gorelated gene-1 (ERG1), was investigated in the adult rat heart. Confocal immunofluorescence microscopy of atrial and ventricular cells revealed that whereas KCNQI labeling was detected in both the peripheral sarcolemma and a structure transversing the myocytes, ERG1 immunoreactivity was confined to the latter, Immunoelectron microscopy of atrial and ventricular myocytes showed that the ERGI channel was primarily expressed in the transverse tubular system and its entrance, whereas KCNQI was detected in both the peripheral sarcolemma and in the T tubules. Thus, whereas ERGI displays a very restricted subcellular localization pattern, KCNQI is more widely distributed within the cardiac cells. The localization of these [K.sup.+] channels to the transverse tubular system close to the [Ca.sup.2+] channels renders them with maximal repolarizing effect. immunohistochemistry: ultrastructure; KVLQTI; human ether-a-gogo-related gene; long QT syndrome

Research was conducted to examine the quantification and distribution of Ca2+-activated maxi K+ channels in rabbit distal colon epithelium. Quantification was performed by the binding of iberiotoxin, a selective peptidyl ligand for maxi K+ channels. Results indicate that the maxi K+ channels are predominantly expressed in the Na+-absorbing surface cells as compared with the Cl--secreting crypt cells.

The regional expression and subunit composition of high-conductance Ca(super 2+)-activated potassium channels in rat brain are investigated using an IbTX analogue. The analogue, (((super 125)I)IbTX-D19Y/Y36F), selectively binds to neuronal calcium-activated potassium channels with high affinity. Results show that the beta-subunit associated with the neuronal channel is a novel protein. It is also shown that the channels display a restricted distribution in the brain and possess a subunit composition different from those of their smooth muscle congeners.

Transient expression of either [Alpha] or [Alpha]+[Beta] subunits of the high-conductance [Ca.sup.2+]-activated [K.sup.+] (maxi-K) channel has been achieved in COS-1 cells. Expression has been studied using charybdotoxin (ChTX), a peptidyl inhibitor that binds in the pore on the [Alpha] subunit. Although some properties of monoiodotyrosine-ChTX (125I-ChTX) binding to membranes derived from each type of transfected cells appear to be identical, other parameters of the binding reaction are markedly different. Under low ionic strength conditions, the affinity constant for 125I-ChTX measured under equilibrium binding conditions is increased ca. 50-fold in the presence of the [Beta] subunit. The rate constant for 125I-ChTX association is enhanced ca. 5-fold, whereas the dissociation rate constant is decreased more than 7-fold when the [Beta] subunit is present. These data indicate that functional coassembly of maxi-K channel subunits can be obtained in a transient expression system, and that the [Beta] subunit has profound effects on 125I-ChTX binding. We postulate that certain negatively charged residues in the large extracellular loop of [Beta] attract the positively charged 125I-ChTX to its binding site on [Alpha] through electrostatic interactions, and account for effects observed on ligand association kinetics. Moreover, another residue(s) in the loop of [Beta] must contribute to stabilization of the toxin-bound state, either by a direct interaction with toxin, or through an allosteric effect on the [Alpha] subunit. Certain regions in the extracellular loop of the [Beta] subunit may be in close proximity to the pore of the channel, and could play an important role in maxi-K channel function.

The binding of monoiodotyrosinyl margatoxin to plasma membranes prepared from either Jurka cells, a human leukemic T cell line, or CHO cells stably transfected with the Shaker-type voltage-gated K+ channel, has been determined. The results showed that the lymphocyte(125I)MgTX receptor is homomultimer of Kv1.3 subunits.

An iberiotoxin (IbTX) double mutant, IbTX-D19Y/Y36F, was synthetically made, cloned and characterized pharmacologically. The findings showed that this biologically active protein specifically and exclusively binds to the maxi-K channels in both the smooth muscle and brain. This initial finding can be the basis for a more detailed analysis of the channel's pharmacological profile in tissues with low channel abundance, especially when such tissues express a high density of voltage-gated K+ channels.

Sigma-ligands comprise several chemically unrelated drugs such as haloperidol, pentazocine, and ditolylguanidine, which bind to a family of low molecular mass proteins in the endoplasmic reticulum. These so-called sigma-receptors are believed to mediate various pharmacological effects of sigma-ligands by as yet unknown mechanisms. Based on their opposite enantioselectivity for benzomorphans and different molecular masses, two subtypes are differentiated. We purified the [sigma.sub.1]-binding site as a single 30-kDa protein from guinea pig liver employing the benzomorphan (+)[3H] pentazocine and the arylazide (-)[3H] azidopamil as specific probes. The purified (+)[3H]pentazocine-binding protein retained its high affinity for haloperidol, pentazocine, and ditolylguanidine. Partial amino acid sequence obtained after trypsinolysis revealed no homology to known proteins. Radiation inactivation of the pentazocine-labeled [sigma.sub.1]-binding site yielded a molecular mass of 24 [+ or -] 2 kda. The corresponding cDNA was cloned using degenerate oligonucleotides and cDNA library screening. Its open reading frame encoded a 25.3-kDa protein with at least one putative transmembrane segment. The protein expressed in yeast cells transformed with the cDNA showed the pharmacological characteristics of the brain and liver [sigma.sub.1]-binding site. The deduced amino acid sequence was structurally unrelated to known mammalian proteins but it shared homology with fungal proteins involved in sterol synthesis. Northern blots showed high densities of the [sigma.sub.1]-binding site mRNA in sterol-producing tissues. This is also in agreement with the known ability of [sigma.sub.1]-binding sites to interact with steroids, such as progesterone.

The large-conductance calcium-activated potassium (maxi-K) channel from bovine aortic smooth muscle consists of two subunits, alpha and beta with molecular weights of 62 and 31 kDa, respectively. Electrophysiological studies indicate the differences between maxi-K channels from different tissues. The beta subunit is involved in regulatory activities. A complex of alpha subunits constitutes the ionizing pore which has the elements necessary for voltage- and calcium-dependent channel opening.

A study of the binding characteristic of monoiodotyrosine margatoxin, ((125I)MgTX) in rat brain synaptic plasma membrane vesicles reveals that (125I)MgTX is a high affinity reversible radioligand and is suitable for the analysis of voltage-gated K+ ion channels. Charybdotoxin, agitoxins I and II and kaliotoxin mediate the binding affinity of (125I)MgTX and cations that are potential inhibitors of voltage-gated K+ channels prevent the binding of (125I)MgTX. Affinity labeling of binding sites in rat brain by (125I)MgTX induces toxins into the glycoprotein.

The study reports the identification of a amino acid in monoiodotyrosine charybdotoxin (ChTX) that is involved in the cross-linking reaction in the calcium-activated potassium channels. The presence of amino acids at positions 11 and 31 of ChTX yields wild-type cross linking patterns. All of the ChTX mutants display the same pharmacological profile similar to native ChTX in competition binding experiments.

Charybdotoxin (ChTX), a 37-amino acid peptide isolated from the venom of a scorpion provides an effective means to study K+ channels. The 1990s have seen considerable developments in the field of ion-channel study. A great deal of information has been collected regarding K+ channels. ChTX and the more selective K+ toxins that were subsequently discovered proved to be effective tools not only in studying the functional role of K+ channels but also in the development of molecular pharmacology of these channels.

Tremorgenic mycotoxins inhibit the high conductance of calcium-activated potassium channels in smooth muscles. Mycotoxins bind the potassium channels to (I-125)ChTX by positive or negative allosterism. Mycotoxins are the potent nonpeptidyl inhibitors of ion channels and can be used to specify new sites of drug interaction.

Intrapulmonary veins (PVs) contribute to pulmonary vascular resistance, but the mechanisms controlling PV tone are poorly understood. Although smooth muscle cell (SMC) [K.sup.+] channels regulate tone in most vascular beds, their role in PV tone is unknown. We show that voltage-gated (Kv) and inward rectifier ([K.sub.ir]) [K.sup.+] channels control resting PV tone in the rat. PVs have a coaxial structure, with layers of cardiomyocytes (CMs) arrayed externally around a subendothelial layer of typical SMCs, thus forming spinchterlike structures. PVCMs have both an inward current, inhibited by low-dose [Ba.sup.2+], and an outward current, inhibited by 4-aminopyridine. In contrast, PVSMCs lack inward currents, and their outward current is inhibited by tetraethylammonium (5 mM) and 4-aminopyridine. Several Kv, [K.sub.ir], and large-conductance [Ca.sup.2+]-sensitive [K.sup.+] channels are present in PVs. Immunohistochemistry showed that [K.sub.ir] channels are present in PVCMs and PV endothelial cells but not in PVSMCs. We conclude that [K.sup.+] channels are present and functionally important in rat PVs. PVCMs form sphincters rich in [K.sub.ir] channels, which may modulate venous return both physiologically and in disease states including pulmonary edema. inward rectifier potassium channels; voltage-gated potassium channels; venous tone; pulmonary circulation; pulmonary edema

SK channels are potassium-selective, voltage-independent, and activated by increases in the levels of intracellular [Ca.sup.2+], such as what occurs during an action potential (1, 2). We have characterized three mammalian [...], In excitable cells, small-conductance [Ca.sup.2+]-activated potassium channels (SK channels) are responsible for the slow after-hyperpolarization that often follows an action potential. Three SK channel subunits have been molecularly characterized. The SK3 gene was targeted by homologous recombination for the insertion of a gene switch that permitted experimental regulation of SK3 expression while retaining normal SK3 promoter function. An absence of SK3 did not present overt phenotypic consequences. However, SK3 overexpression induced abnormal respiratory responses to hypoxia and compromised parturition. Both conditions were corrected by silencing the gene. The results implicate SK3 channels as potential therapeutic targets for disorders such as sleep apnea or sudden infant death syndrome and for regulating uterine contractions during labor.

Research was conducted to determine the synthesis of (3H)-iminodihydroquinoline (WIN 17317-3) and its interaction with rat brain synaptic plasma membrane vesicles. It was shown that (3H)WIN 17317-3 binds selectively and with high affinity to voltage-gated sodium channels in brain. Results show that WIN 17317-3 represents a new high-affinity ligand for voltage-gated sodium channels that can differentiate members of the ion channel superfamily in native tissues.

Local Ca2+ signaling occurring within nanometers of voltage-gated Ca2+ (Cay) channels is crucial for CNS function, yet the molecular composition of Cay channel nano-environments is largely unresolved. Here, we used a proteomic strategy combining knockoutcontrolled multiepitope affinity purifications with high-resolution quantitative MS for comprehensive analysis of the molecular nano-environments of the Cav2 channel family in the whole rodent brain. The analysis shows that Cav2 channels, composed of poreforming al and auxiliary subunits, are embedded into protein networks that may be assembled from a pool of ∼200 proteins with distinct abundance, stability of assembly, and preference for the three Cav2 subtypes. The majority of these proteins have not previously been linked to Cay channels; about two-thirds are dedicated to the control of intracellular Ca2+ concentration, including G proteincoupled receptor-mediated signaling, to activity-dependent cytoskeleton remodeling or Ca2+-dependent effector systems that comprise a high portion of the priming and release machinery of synaptic vesicles. The identified protein networks reflect the cellular processes that can be initiated by Cav2 channel activity and define the molecular framework for organization and operation of local Ca2+ signaling by Cav2 channels in the brain. [ABSTRACT FROM AUTHOR]

Presents information on a study on the effect of conditional overexpression of the small conductance calcium-activated potassium channels (SK channels) on respiration and parturition. Characterization of SK channel subunits; Regional distribution of SK protein; Investigation of respiratory patterns under normal and hypoxic conditions; Results and discussion.