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PubMed2004

Imaging the activity and localization of single voltage-gated Ca(2+) channels by total internal reflection fluorescence microscopy.

The patch-clamp technique has enabled functional studies of single ion channels, but suffers limitations including lack of spatial information and inability to independently monitor currents from more than one channel. Here, we describe the use of total internal reflection fluorescence microscopy as an alternative, noninvasive approach to optically monitor the activity and localization of multiple Ca(2+)-permeable channels in the plasma membrane. Images of near-membrane Ca(2+) signals were obtained from >100 N-type channels expressed within restricted areas (80 x 80 micro m) of Xenopus oocytes, thereby permitting simultaneous resolution of their gating kinetics, voltage dependence, and localization. Moreover, this technique provided information inaccessible by electrophysiological means, demonstrating that N-type channels are immobile in the membrane, show a patchy distribution, and display diverse gating kinetics even among closely adjacent channels. Total internal reflection fluorescence microscopy holds great promise for single-channel recording of diverse voltage- and ligand-gated Ca(2+)-permeable channels in the membrane of neurons and other isolated or cultured cells, and has potential for high-throughput functional analysis of single channels.

Animals↗
PubMed1991

Structure and function of voltage-gated sodium and calcium channels.

The primary structures of the Na+ channel alpha-subunits from several species have now been deduced from cDNA sequences, and complete primary structures of all of the subunits of skeletal muscle L-type Ca2+ channels have been defined. Current research on voltage-gated ion channels is focusing on defining the structural components responsible for specific aspects of channel function. Recent experiments have identified regions of these channels that are important for voltage-dependent activation and inactivation, ion conductance, regulation by protein phosphorylation, and modulation by drugs and neurotoxins using a combination of antibody mapping and site-directed mutagenesis approaches. The results form the outlines of a structural map of ion channel function.

Animals↗
PubMed2006

LGICdb: a manually curated sequence database after the genomes.

Ligand-gated ion channels form transmembrane ionic pores controlled by the binding of chemicals. The LGICdb aims to be a non-redundant, manually curated resource offering access to the large number of subunits composing extracellularly activated ligand-gated ion channels, such as nicotinic, ATP, GABA and glutamate ionotropic receptors. Composed of more than 500 human curated entries, the XML native database has been relocated in 2004 to the EBI. Its facilities have been enhanced with a new search system, customized multiple sequence alignments and manipulation of protein structures (http://www.ebi.ac.uk/compneur-srv/LGICdb/). Despite the vast improvement of general sequence resources, the LGICdb still provide sequences unavailable elsewhere.

Databases, Protein↗
PubMed2004

Unusual functional properties of homo- and heteromultimeric histamine-gated chloride channels of Drosophila melanogaster: spontaneous currents and dual gating by GABA and histamine.

Histamine acts as a neurotransmitter of photoreceptors in insects and other arthropods, where it directly activates a chloride channel and mediates rapid inhibitory responses. Homo- and heteromultimeric histamine-gated ion channels formed by HisCl-alpha2 or HisCl-alpha1 + alpha2 subunits from Drosophila melanogaster were characterized by two-electrode voltage-clamp measurements of functionally expressed ion channels in Xenopus laevis oocytes. The sensitivity of heteromultimeric histamine receptors with an EC(50) of 2.3 microM is lower than that of either homomultimeric receptor. They can be further distinguished from the homomultimeric channels by their reduced sensitivity to d-tubocurarine. Heteromultimeric channels generate a spontaneous current in the absence of any agonist. This spontaneous current can be blocked in the absence of an agonist by d-tubocurarine and the histamine antagonists cimetidine, thioperamide and pyrilamine. Homomultimeric HisCl-alpha2 channels are dually gated by histamine (IC(50)=9.4 microM) and GABA (IC(50)=1.0mM), both of which are full agonists. The action of both agonists can be blocked with comparable IC(50) values by the histamine antagonists cimetidine, thioperamide and pyrilamine but not by the GABA antagonist bicuculline. Picrotoxin blocked with an IC(50) of 403 microM. Our data show that histamine and GABA act on the same ion channel, which thus might function as a site of integration of the action of different neurotransmitters.

Animals↗
PubMed1994

Evidence that the S6 segment of the Shaker voltage-gated K+ channel comprises part of the pore.

Potassium channels are highly selective and allow the rapid flux of potassium ions through their pore. Several studies have implicated the H5 (P or SS1-SS2) segment as part of the pore in voltage-gated ion channels. The proposal that H5 spans at least 80% of the electric potential drop across the K+ channel pore is based on altered internal tetraethylammonium sensitivity arising from mutations of H5 residues that are 100% conserved among K+ channels having differing sensitivity to tetraethylammonium. Here we report that the S6 segment is also involved in K+ ion permeation and in governing the sensitivity to internal tetraethylammonium and barium. Transplanting the S6 segment of NGK2 into Shaker causes this S6 chimaera to adopt the single-channel conductance and sensitivity to internal tetraethylammonium and barium ions from the NGK2 channel. The differences between NGK2 and Shaker in external tetraethylammonium sensitivity, but not single-channel conductance, can be attributed to the differences in their H5 sequences. Three nonconserved S6 residues have been found to affect either single-channel conductance or internal tetraethylammonium sensitivity.

Amino Acid Sequence↗
PubMed2000

Contributions of the different extramembranous domains of the mechanosensitive ion channel MscL to its response to membrane tension.

MscL is a mechanosensitive channel that is gated by tension in the membrane bilayer alone. It is a homo-oligomer of a protein comprising two transmembrane segments connected by an external loop, with the NH(2) and COOH termini located in the cytoplasm. The contributions of the extramembranous domains of the channel to its activity were investigated by specific proteolysis during patch-clamp experiments. Limited proteolysis of the COOH terminus or the NH(2) terminus increased the mechanosensitivity of the channel without changing its conductance. Strikingly, after cleavage of the external loop of each monomer, the channel was still functional, and its mechanosensitivity was increased dramatically, indicating that the loop acts as a spring that resists the opening of the channel and promotes its closure when it is open. These results indicate that the integrity of most of the extramembranous domains is not essential for mechanosensitivity. They suggest that these domains counteract the movement of the transmembrane helices to which they are connected, thus setting the level of sensitivity of the channel to tension.

Amino Acid Sequence↗
PubMed1997

Differences in the distribution of responses to ATP and acetylcholine between outer hair cells of rat and guinea pig.

Adenosine 5' triphosphate (ATP) and acetylcholine (ACh) are neurotransmitters (ACh) and/or modulators (ATP) in the mammalian cochlea. In guinea pig, it appears that both neurotransmitters have a similar response distribution, with larger responses being evoked by the ligands in short hair cells compared to long hair cells (e.g., Chen et al., 1995b. Noise exposure alters the response of outer hair cells to ATP. Hear. Res. 88, 215-221.; Erostegui et al., 1994. In vitro pharmacologic characterization of a cholinergic receptor on outer hair cells. Hear. Res. 74, 135 147). The purpose of the present study was to test whether the distribution of responses to ACh and ATP in the OHCs of rat is the same as guinea pig. The ligand-induced current was monitored using the whole-cell configuration of the patch-clamp technique. Results show that in guinea pig OHCs, extracellular application of 100 microM ATP induced a current response in a majority of the same cells that responded to the application of 100 microM ACh. In contrast in rat OHCs, 100 microM ATP did not induce a current in the majority of cells that responded to the application of 100 microM ACh. N-methyl-glucamine (NMG+) substituted for K+ in the pipette solution failed to unmask an ATP-evoked inward current in rat OHCs. In addition, no response was produced in rat or guinea pig OHCs by adenosine, adenosine 5'-monophosphate (AMP) or adenosine 5'-diphosphate (ADP) at 100 microM. Results suggest that in guinea pig ACh-gated channels are present on most of the same OHCs that have ATP-gated ion channels, whereas in rat ACh-gated ion channels are present without ATP-gated channels on some OHCs.

Acetylcholine↗
PubMed1992

Potentiation of inositol trisphosphate-induced Ca2+ mobilization in Xenopus oocytes by cytosolic Ca2+.

1. The ability of cytosolic Ca2+ ions to modulate inositol 1,4,5-trisphosphate (Insp3)-induced Ca2+ liberation from intracellular stores was studied in Xenopus oocytes using light flash photolysis of caged InsP3. Changes in cytosolic free Ca2+ level were effected by inducing Ca2+ entry through ionophore and voltage-gated plasma membrane channels and by injection of Ca2+ through a micropipette. Their effects on Ca2+ liberation were monitored by video imaging of Fluo-3 fluorescence and by voltage clamp recording of Ca(2+)-activated membrane Cl- currents. 2. Treatment of oocytes with the Ca2+ ionophores A23187 and ionomycin caused a transient elevation of cytosolic Ca2+ level when cells were bathed in Ca(2+)-free solution, which probably arose because of release of Ca2+ from intracellular stores. 3. Membrane current and Fluo-3 Ca2+ signals evoked by photoreleased InsP3 in ionophore-treated oocytes were potentiated when the intracellular Ca2+ level was elevated by raising the Ca2+ level in the bathing solution. 4. Responses to photoreleased InsP3 were similarly potentiated following activation of Ca2+ entry through voltage-gated Ca2+ channels expressed in the plasma membrane. 5. Ca(2+)-activated membrane currents evoked by depolarization developed a delayed 'hump' component during sustained photorelease of InsP3, probably because Ca2+ ions entering through the membrane channels triggered liberation of Ca2+ from intracellular stores. 6. Ba2+ and Sr2+ ions were able to substitute for Ca2+ in potentiating InsP3-mediated Ca2+ liberation. 7. Gradual photorelease of InsP3 by weak photolysis light evoked Ca2+ liberation that began at particular foci and then propagated throughout, but not beyond that area of the oocyte exposed to the light. Local elevations of intracellular Ca2+ produced by microinjection of Ca2+ acted as new foci for the initiation of Ca2+ liberation by InsP3. 8. In resting oocytes, intracellular injections of Ca2+ resulted only in localized elevation of intracellular Ca2+, and did not evoke propagating waves. 9. The results show that cytosolic Ca2+ ions potentiate the ability of InsP3 to liberate Ca2+ from intracellular stores. This process may be important for the positive feedback mechanism underlying the generation of Ca2+ spikes and waves, and for interactions between the InsP3 pathway and Ca2+ ions entering cells through voltage- and ligand-gated channels.

Animals↗
PubMed1996

Altered ligand specificity by protonation in the ligand binding domain of cyclic nucleotide-gated channels.

Cyclic nucleotide-gated (CNG) ion channels are the critical mediators between the second messengers of sensory transduction and the cell's membrane potential. The photoreceptor CNG channels are activated by the direct binding of cGMP but can also be activated to a much lesser extent by cAMP. In rod CNG channels expressed in Xenopus oocytes, we demonstrate two types of potentiation by protons. One type potentiated cGMP-bound and cAMP-bound channels to the same extent, while another potentiated only cAMP-bound channels. Both types of potentiation could be described by a mechanism in which protons bound primarily to the channel open configuration. The potentiation specific to cAMP-bound channels could be accounted for by protonation of aspartic acid 604 (D604). It is the unfavorable electrostatic interaction between the carboxylate of D604 and the purine ring of cAMP that accounts for the normally poor activation of the channels by cAMP. Protonation at this site removed the unfavorable interaction and allowed cAMP to act as nearly a full agonist. Protonation of a second amino acid, H468, contributed to the nucleotide-nonspecific potentiation and is likely to be an element of the channel gating assembly. Protons potentiate native rod channels less than channels formed from subunit 1. In heteromultimeric channels formed by coexpressing subunit 1 with subunit 2, we found a similar attenuation of potentiation. The absence of protonatable amino acids in subunit 2 at positions corresponding to H468 and D604 can explain the reduced effects of pH on native channels.

Animals↗
PubMed1991

Exchange of conduction pathways between two related K+ channels.

The structure of the ion conduction pathway or pore of voltage-gated ion channels is unknown, although the linker between the membrane spanning segments S5 and S6 has been suggested to form part of the pore in potassium channels. To test whether this region controls potassium channel conduction, a 21-amino acid segment of the S5-S6 linker was transplanted from the voltage-activated potassium channel NGK2 to another potassium channel DRK1, which has very different pore properties. In the resulting chimeric channel, the single channel conductance and blockade by external and internal tetraethylammonium (TEA) ion were characteristic of the donor NGK2 channel. Thus, this 21-amino acid segment controls the essential biophysical properties of the pore and may form the conduction pathway of these potassium channels.

Amino Acid Sequence↗
PubMed1994

Expression of cyclic nucleotide-gated cation channels in non-sensory tissues and cells.

Using a combination of PCR based cloning and Northern blot analysis we have investigated the tissue expression of cyclic nucleotide-gated (CNG) cation channels in a variety of non-excitable tissues and cells. Partial sequences of all three known CNG channels and of an auxiliary subunit of the rod photoreceptor channel were determined. The expression of CNG channel genes is both tissue and species specific. Southern blot analysis with human genomic DNA revealed specific patterns of hybridization with probes derived from the different CNG channel types indicating that they are encoded by distinct genes. Analysis of human genomic sequences showed that all three genes are derived from a common ancestral gene and might have a similar gene structure. We were not able to identify additional genes encoding CNG channels. The CNG3 channel, which was originally cloned from bovine kidney may be expressed also in bovine cone photoreceptor cells. These data suggest that some of the effects of cGMP in peripheral tissues and cells might involve the activation of CNG channels.

Animals↗
PubMed1994

A cGMP-gated current can control exocytosis at cone synapses.

The voltage-gated Ca2+ current in cone photoreceptors operates over only a small part of the physiological voltage range produced by light and, consequently, appears insufficient for controlling transmitter release. We have used a whole-cell voltage clamp to measure membrane current and the capacitance change produced by exocytosis in solitary cone and rod photoreceptors isolated from the salamander retina. In both types of photoreceptor, Ca2+ influx through voltage-gated Ca2+ channels initiated exocytosis. In addition, Ca2+ influx through a cGMP-gated channel in the inner segment and synaptic processes of cones also initiated exocytosis. The cGMP-gated current sustained exocytosis over the entire physiological voltage range.

Ambystoma↗
PubMed2004

Characterisation of hyperpolarization-activated currents (I(h)) in the medial septum/diagonal band complex in the mouse.

Hyperpolarization-activated cyclic nucleotide gated (HCN) channel subunits are distributed widely, but selectively, in the central nervous system, and underlie hyperpolarization-activated currents (I(h)) that contribute to rhythmicity in a variety of neurons. This study investigates, using current and voltage-clamp techniques in brain slices from young mice, the properties of I(h) currents in medial septum/diagonal band (MS/DB) neurons. Subsets of neurons in this complex, including GABAergic and cholinergic neurons, innervate the hippocampal formation, and play a role in modulating hippocampal theta rhythm. In support of a potential role for I(h) in regulating MS/DB firing properties and consequently hippocampal neuron rhythmicity, I(h) currents were present in around 60% of midline MS/DB complex neurons. The I(h) currents were sensitive to the selective blocker ZD7288 (10 microM). The I(h) current had a time constant of activation of around 220 ms (at -130 mV), and tail current analysis revealed a half-activation voltage of -98 mV. Notably, the amplitude and kinetics of I(h) currents in MS/DB neurons were insensitive to the cAMP membrane permeable analogue 8-bromo-cAMP (1 mM), and application of muscarine (100 microM). Immunofluoresence using antibodies against HCN1, 2 and 4 channel subunits revealed that all three HCN subunits are expressed in neurons in the MS/DB, including neurons that express the calcium binding protein parvalbumin (marker of fast spiking GABAergic septo-hippocampal projection neurons). The results demonstrate, for the first time, that specific HCN channel subunits are likely to be coexpressed in subsets of MS/DB neurons, and that the resultant I(h) currents show both similarities, and differences, to previously described I(h) currents in other CNS neurons.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1997

Single-channel properties of ionic channels gated by cyclic nucleotides.

This paper presents an extensive analysis of single-channel properties of cyclic nucleotide gated (CNG) channels, obtained by injecting into Xenopus laevis oocytes the mRNA encoding for the alpha and beta subunits from bovine rods. When the alpha and beta subunits of the CNG channel are coexpressed, at least three types of channels with different properties are observed. One type of channel has well-resolved, multiple conductive levels at negative voltages, but not at positive voltages. The other two types of channel are characterized by flickering openings, but are distinguished because they have a low and a high conductance. The alpha subunit of CNG channels has a well-defined conductance of about 28 pS, but multiple conductive levels are observed in mutant channels E363D and T364M. The conductance of these open states is modulated by protons and the membrane voltage, and has an activation energy around 44 kJ/mol. The relative probability of occupying any of these open states is independent of the cGMP concentration, but depends on extracellular protons. The open probability in the presence of saturating cGMP was 0.78, 0.47, 0.5, and 0.007 in the w.t. and mutants E363D, T364M, and E363G, and its dependence on temperature indicates that the thermodynamics of the transition between the closed and open state is also affected by mutations in the pore region. These results suggest that CNG channels have different conductive levels, leading to the existence of multiple open states in homomeric channels and to the flickering behavior in heteromeric channels, and that the pore is an essential part of the gating of CNG channels.

Amino Acid Sequence↗
PubMed2005

Modifications to the tetracaine scaffold produce cyclic nucleotide-gated channel blockers with widely varying efficacies.

Five new tetracaine analogues were synthesized and evaluated for potency of blockade of cyclic nucleotide-gated channels relative to a multiply charged tetracaine analogue described previously. Increased positive charge at the tertiary amine end of tetracaine results in higher potency and voltage dependence of block. Modifications that reduce the hydrophobic character at the butyl tail are deleterious to block. The tetracaine analogues described here have apparent affinities for CNGA1 channels that vary over nearly 8 orders of magnitude.

Animals↗
PubMed1997

A cGMP-gated cation channel in depolarizing photoreceptors of the lizard parietal eye.

Rods and cones of the two vertebrate lateral eyes hyperpolarize when illuminated, a response generated by a cyclic GMP cascade leading to cGMP hydrolysis and consequently the closure of cGMP-gated, non-selective cation channels that are open in darkness. Lizards and other lower vertebrates also have a parietal (third) eye, which contains ciliary photoreceptors that under dark-adapted conditions depolarize to light instead. Depolarizing light responses are characteristic of most invertebrate rhabdomeric photoreceptors, and are thought to involve a phosphoinositide signalling pathway (see, for example, refs 7-9). Surprisingly, we have found in excised membrane patches a cGMP-gated channel that is selectively present at high density on the outer segment (the presumptive light-sensitive part) of the parietal eye photoreceptor. Like the light-activated channel of the cell, it is non-selective among cations. Inositol trisphosphate (InsP3) had no effect on the same membrane patches. These findings suggest that the photoreceptors of the parietal eye, like rods and cones, use a cGMP cascade and not an InsP3-mediated pathway for phototransduction, but in this case light increases cGMP. A unifying principle of evolutionary significance emerges: that phototransductions in various ciliary photoreceptors, whether hyperpolarizing or depolarizing, uniformly use a cGMP cascade and a cGMP-gated channel to generate the light response, although there are rich variations in the details.

Animals↗
PubMed1994

Subunit 2 (or beta) of retinal rod cGMP-gated cation channel is a component of the 240-kDa channel-associated protein and mediates Ca(2+)-calmodulin modulation.

The cGMP-gated cation channel mediating visual transduction in retinal rods was recently found to comprise at least two subunits, 1 and 2 (or alpha and beta). SDS gels of the purified channel show, in addition to a 63-kDa protein band (subunit 1), a 240-kDa protein band that binds Ca(2+)-calmodulin, a modulator of the channel. To examine any connection between subunit 2 and the 240-kDa protein, cGMP-gated channels formed from the expressed cloned subunits in human embryonic kidney (HEK) 293 cells were tested for Ca(2+)-calmodulin effect. Homooligomeric channels formed by subunit 1 alone showed no sensitivity to Ca(2+)-calmodulin, and neither did heterooligomeric channels formed by subunit 1 and the short alternatively spliced form of subunit 2 (2a). By contrast, the cGMP half-activation constant (K1/2) for heterooligomeric channels formed from subunit 1 and the long form of subunit 2 (2b) was increased 1.5- to 2-fold by Ca(2+)-calmodulin, similar to the increase observed for the native channel. In Western blots of rod outer segment membranes, a subunit 2-specific antibody also recognized the 240-kDa protein. Finally, amino acid sequences derived from peptide fragments of the bovine 240-kDa protein showed approximately 80% identity to regions of subunit 2b of the human channel. These results together suggest that subunit 2b of the rod channel is a component of the 240-kDa protein and that it mediates the Ca(2+)-calmodulin modulation of the channel.

Amino Acid Sequence↗