Showing papers on "Ion channel published in 1995"

Single-channel recording

Abstract: A Practical Guide to Patch Clamping R. Penner. Tightseal Wholecell Recording A. Marty, E. Neher. Guide to Data Acquisition and Analysis S.H. Heinemann. Electronic Design of the Patch Clamp F.J. Sigworth. Low Noise Recording K. Benndorf. Voltage Offsets in Patch Clamp Experiments E. Neher. Techniques for Membrane Capacitance Measurements K.D. Gillis. Patch Pipette Recordings from the Soma, Dendrites and Axon of Neurons in Brain Slices B. Sakmann, G. Stuart. Patchclamp and Calcium Imaging in Brain Slices J. Eilers, et al. Fast Application of Agonists to Isolated Membrane Patches P. Jonas. Electrochemical Detection of Secretion from Single Cells R.H. Chow, L. von Ruden. Technical Approaches to Studying Specific Properties of Ion Channels in Plants R. Hedrich. The Giant Membrane Patch D.W. Hilgemann. A Fast Pressureclamp Technique for Studying Mechanogated Channels D.W. McBride, O.P. Hamill. Electrophysiological Recordings from Xenopus oocytes W. Stuhmer, A.B. Parekh. PCR Analysis of Ion Channel Expression in Single Neurons of Brain Slices H. Monyer, P. Jonas. 3 additional articles. Index.

Clustering of Shaker-type K + channels by interaction with a family of membrane-associated guanylate kinases

Abstract: ANCHORING of ion channels at specific subcellular sites is critical for neuronal signalling, but the mechanisms underlying channel localization and clustering are largely unknown (reviewed in ref. 1). Voltage-gated K+ channels are concentrated in various neuronal domains, including presynaptic terminals, nodes of Ranvier and dendrites, where they regulate local membrane excitability. Here we present functional and biochemical evidence that cell-surface clustering of Shaker-subfamily K+ channels is mediated by the PSD-95 family of membrane-associated putative guanylate kinases, as a result of direct binding of the carboxy-terminal cyto-plasmic tails of the K+ channel subunits to two PDZ (also known as GLGF or DHR) domains in the PSD-95 protein2. The ability of PDZ domains to function as independent modules for protein–protein interaction, and their presence in other junction-associated molecules (such as ZO-1 (ref. 3) and syntrophin4), suggest that PDZ-domain-containing polypeptides may be widely involved in the organization of proteins at sites of membrane specialization.

TRPC1, a human homolog of a Drosophila store-operated channel

Abstract: In many vertebrate and invertebrate cells, inositol 1,4,5-trisphospate production induces a biphasic Ca2+ signal. Mobilization of Ca2+ from internal stores drives the initial burst. The second phase, referred to as store-operated Ca2+ entry (formerly capacitative Ca2+ entry), occurs when depletion of intracellular Ca2+ pools activates a non-voltage-sensitive plasma membrane Ca2+ conductance. Despite the prevalence of store-operated Ca2+ entry, no vertebrate channel responsible for store-operated Ca2+ entry has been reported. trp (transient receptor potential), a Drosophila gene required in phototransduction, encodes the only known candidate for such a channel throughout phylogeny. In this report, we describe the molecular characterization of a human homolog of trp, TRPC1. TRPC1 (transient receptor potential channel-related protein 1) was 40% identical to Drosophila TRP over most of the protein and lacked the charged residues in the S4 transmembrane region proposed to be required for the voltage sensor in many voltage-gated ion channels. TRPC1 was expressed at the highest levels in the fetal brain and in the adult heart, brain, testis, and ovaries. Evidence is also presented that TRPC1 represents the archetype of a family of related human proteins.

Ion channel regulation by G proteins

Abstract: Ion channels are poised uniquely to initiate, mediate, or regulate such distinct cellular activities as action potential propagation, secretion, and gene transcription. In retrospect, it is not surprising that studies of ion channels have revealed considerable diversities in their primary structures, regulation, and expression. From a functional standpoint, the various mechanisms coopted by cells to regulate channel activity are particularly fascinating. Extracellular ligands, membrane potential, phosphorylation, ions themselves, and diffusible second messengers are all well-established regulators of ion channel activity. Heterotrimeric GTP-binding proteins (G proteins) mediate many of these types of ion channel regulation by stimulating or inhibiting phosphorylation pathways, initiating intracellular cascades leading to elevation of cytosolic Ca2+ or adenosine 3',5'-cyclic monophosphate levels, or by generating various lipid-derived compounds. In some cases, it seems that activated G protein subunits can interact directly with ion channels to elicit regulation. Although there is currently little direct biochemical evidence to support such a mechanism, it is the working hypothesis for the most-studied G protein-regulated ion channels.

Revealing the architecture of a K+ channel pore through mutant cycles with a peptide inhibitor

Abstract: Thermodynamic mutant cycles provide a formalism for studying energetic coupling between amino acids on the interaction surface in a protein-protein complex. This approach was applied to the Shaker potassium channel and to a high-affinity peptide inhibitor (scorpion toxin) that binds to its pore entryway. The assignment of pairwise interactions defined the spatial arrangement of channel amino acids with respect to the known inhibitor structure. A strong constraint was placed on the Shaker channel pore-forming region by requiring its amino-terminal border to be 12 to 15 angstroms from the central axis. This method is directly applicable to sodium, calcium, and other ion channels where inhibitor or modulatory proteins bind with high affinity.

A prokaryotic potassium ion channel with two predicted transmembrane segments from Streptomyces lividans.

Abstract: We report the identification, functional expression, purification, reconstitution and electrophysiological characterization of an up to now unique prokaryotic potassium ion channel (KcsA). It has a rectifying current-voltage relationship and displays subconductance states, the largest of which amounts to A approximately equal to 90 pS. The channel is blocked by Cs- ions and gating requires the presence of Mg2+ ions. The kcsA gene has been identified in the gram-positive soil bacterium Streptomyces lividans. It encodes a predicted 17.6 kDa protein with two potential membrane-spanning helices linked by a central domain which shares a high degree of similarity with the H5 segment conserved among eukaryotic ion channels. Multiple alignments of deduced amino acids suggest that the novel channel has the closest kinship to the S5, H5 and S6 regions of voltage-gated K+ channel families, mainly to the subfamily represented by the Shaker protein from Drosophila melanogaster. Moreover, KcsA is most distantly related to eukaryotic inwardly rectifying channels with two putative predicted transmembrane segments.

Roles of Ion Channels in Initiation of Signal Transduction in Higher Plants.

Abstract: Recently, progress has been made in identifying initial signal reception mechanisms and early events in signaling cascades in higher plants. Ion channels, along with intracellular signaling proteins and second messengers, are critical components mediating early events in higher plant signal transduction. Ion channel-mediated signal transduction in higher plants has notable differences from signaling mechanisms in animal systems. Of the many types of ion channels found in higher plants, there are indications that anion channels, along with Ca2+ channels, play critical and rate-limiting roles in the mediation of early events of signal transduction. We have now begun to obtain the first insights into the modes of regulation, the membrane localization, and in the case of K+ channels, the molecular structure of higher plant ion channels.

Techniques for Membrane Capacitance Measurements

Abstract: Understanding the process whereby cells transduce an external signal to a secretory response (“stimulus—secretion coupling”: Douglas, 1968) has been an important topic of research for many years. The understanding of early events in the cascade in excitable cells, whereby an external signal evokes an electrical response mediated by ion channels, has certainly been revolutionized by the development of the patch-clamp technique. Extensions of the technique, however, have also provided surprising flexibility in reporting events late in the cascade whereby intracellular Ca2+ and other second messengers lead to exocytosis. In 1982, Neher and Marty reported that the patch-clamp technique together with basic impedance analysis could be used to monitor membrane (electrical) capacitance as a single-cell assay of exocytosis and endocytosis. Since exocytosis involves the fusion of secretory granule membrane with the plasma membrane and a corresponding increase in surface area, an increase in membrane capacitance is observed. The excess membrane is reclaimed in the process of endocytosis, which leads to a corresponding decrease in capacitance. Present techniques can detect changes in capacitance on the order of a femtofarad, allowing the fusion of single secretory granules with diameters greater that about 200 nm to be resolved. The temporal resolution possible is on the order of milliseconds (e.g., Breckenridge and Almers, 1987); therefore, capacitance-recording techniques can almost achieve the resolution of synaptic preparations, where the electrical response of a postsynaptic cell serves as a reporter of secretion. Resolution limits of membrane capacitance estimation techniques are discussed in greater detail in Section 5.

Selectivity of Connexin-Specific Gap Junctions Does Not Correlate With Channel Conductance

Abstract: Connexins form a variety of gap junction channels that vary in their developmental and tissue-specific levels of expression, modulation of gating by transjunctional voltage and posttranslational modification, and unitary channel conductance (gamma j). Despite a 10-fold variation in gamma j, whether connexin-specific channels possess distinct ionic and molecular permeabilities is presently unknown. A major assumption of the conventional model for a gap junction channel pore is that gamma j is determined primarily by pore diameter. Hence, molecular size permeability limits should increase and ionic selectivity should decrease with increasing channel gamma j (and pore diameter). Equimolar ion substitution of 120 mmol/L KCl for potassium glutamate was used to determine the unitary conductance ratios for rat connexin40 and connexin43, chicken connexin43 and connexin45, and human connexin37 channels functionally expressed in communication-deficient mouse neuroblastoma (N2A) cells. Comparison of experimental and predicted conductance ratios based on the aqueous mobilities of all ions according to the Goldman-Hodgkin-Katz current equation was used to determine relative anion-to-cation permeability ratios. Direct correlation of junctional conductance with dye transfer of two fluorescein-derivatives (2 mmol/L 6-carboxyfluorescein or 2',7'-dichlorofluorescein) was also performed. Both approaches revealed a range of selectivities and permeabilities for all five different connexins that was independent of channel conductance. These results are not consistent with the conventional simple aqueous pore model of a gap junction channel and suggest a new model for connexin channel conductance and permselectivity based on electrostatic interactions. Divergent conductance and permeability properties are features of other classes of ion channels (eg, Na+ and K+ channels), implying similar mechanisms for selectivity.

Relative densities of synaptic and extrasynaptic GABAA receptors on cerebellar granule cells as determined by a quantitative immunogold method

Abstract: Ion channels gated by the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) are thought to be located in synaptic junctions, but they have also been found throughout the somatodendritic membrane of neurons independent of synapses. To test whether synaptic junctions are enriched in GABAA receptors, and to determine the relative densities of synaptic and extrasynaptic receptors, the alpha 1 and beta 2/3 subunits of the GABAA receptor were localized on cerebellar granule cells using a postembedding immunogold method in cats. Immunoparticle density for the alpha 1 and beta 2/3 subunits was approximately 230 and 180 times more concentrated, respectively, in the synaptic junction made by GABAergic Golgi cell terminals with granule cell dendrites than on the extrasynaptic somatic membrane. Quantification of immunoreactivity revealed one synapse population for the beta 2/3, but appeared to show two populations for the alpha 1 subunit immunoreactivity. The concentration of these subunits on somatic membrane was significantly lower than on the extrasynaptic dendritic membrane. Synaptic junctions with glutamatergic mossy fiber terminals were immunonegative. The results demonstrate that granule cells receiving GABAergic synapses at a restricted location on their distal dendrites exhibit a highly compartmentalized distribution of GABAA receptor in their plasma membrane.

The Jellyfish Green Fluorescent Protein: A New Tool for Studying Ion Channel Expression and Function Neurotechnique

Abstract: John Marshall,* Raymond Molloy,t Guy W. J. Moss,* James R. Howe,* and Thomas E. Hughestt *Deparment of Pharmacology tSection of Neurobiology SDepartment of Ophthalmology and Visual Science Yale University School of Medicine New Haven, Connecticut 06520 Summary Two methods are described for using the jellyfish green fluorescent protein (GFP) as a reporter gene for ion channel expression. GFP fluorescence can be used to identify the transfected cells, and to estimate the relative levels of ion channel expression, in cotrans- fection experiments. A GFP-NMDAR1 chimera can be constructed that produces a functional, fluorescent receptor subunit. These methods should facilitate studies of ion channel expression, localization, and processing. Introduction A problem in transfecting cloned ion channels into mam- malian cells is identifying the cells that express the chan- nel and/or localizing the channel within a cell. A variety of strategies have been employed to overcome this problem, including generating stable cell lines and using antibodies, fluorescent enzyme substrates, or the luciferase gene. Al- though powerful in certain applications, each of these ap- proaches has limitations. It has been shown recently that expression of the green fluorescent protein (GFP) from the jellyfish Aequorea victo- ria (Prasher et al., 1992) in Escherichia coil or Caenorhab- ditis elegans produces a protein that is strongly fluores- cent, and that this fluorescence does not appear to depend upon exogenous substrates or coenzymes (Chalfie et al., 1994). Subsequently, GFP was used to construct chimeric proteins that were functional and fluorescent in Drosophila melanogaster (Wang and Hazelrigg, 1994). Here we de- scribe expression of GFP in human embryonic kidney cells (HEK 293; Graham et al., 1977). It produces a robust sig- nal, making it possible to identify cells for patch-clamp studies or to follow chimeric receptor proteins. Results Cotransfection of GFP with Cloned Receptors Identifies the Transfected Cells GFP expressed under the control of a cytomegalovirus (CMV) promoter is readily detected in HEK 293 cells 2 days after the transfection (Figures 1A-1C). When GFP is expressed alone, or in pairwise combination with an ion channel, the signal produced is a bright green fluores- cence when viewed with conventional epifluorescence op- tics for fluorescein isothiocyanate. This signal, which is resistant to photobleaching, makes it possible to combine both bright-field and epifluorescence illumination so that the transfected and untransfected cells are visible. The green fluorescence is apparent in both living cells and those fixed with paraformaldehyde. Although adjacent HEK 293 cells are known to form gap junctions, there was no evidence that GFP spread to surrounding cells. The expression of GFP does not appear to affect the cells ad- versely, and strong signals can be found in living cells at least 5 days after the transfection. Whole-cell recordings from cotransfected, fluorescent cells reveal that they invariably express the introduced ion channels (n = 42). Such expression is absent from cells that do not exhibit detectable fluorescence. In addition, there is cell to cell variability in the intensity of the GFP- associated signal (Figure 1C), and there is a correlation between the intensity of the fluorescence and the level of expression of functional ion channels. Figures 2A and 2B show whole-cell recordings from two cells cotransfected with cDNAs encoding GFP and the fully edited (R) version of glutamate receptor 6 (GluR6; Egebjerg et al., 1991). The recording shown in (A) was made from an intensely fluorescent cell, whereas the recording in (B) was obtained from a nearby cell that exhibited a much weaker fluores- cent signal. Both cells were voltage clamped at -80 mV, and inward currents were evoked by the application of 10 I~M kainate. The current recorded from the intensely fluorescent cell is about 40 times larger than the corre- sponding current in the weakly fluorescent cell. A similar correlation between the intensity of the fluorescent signal and functional channel expression was observed for cells that coexpressed GFP and a Ca2+-activated K + channel that was cloned from bovine aorta (bSIo; Moss et al., 1995). Single-channel currents through Ca2÷-activated K ÷ chan- nels in membrane patches isolated from intensely or weakly fluorescent cells from the same transfection are shown in Figures 2C and 2D, respectively. The single- channel activity recorded at +20 mV is 3-4 times higher in the patch from the intensely fluorescent cell. It was con- sistently true that patches from intensely fluorescent cells showed substantially higher levels of channel activity than patches isolated from weakly fluorescent cells. GFP did not appear to alter the macroscopic properties of currents through GluR6(R) channels, or the properties of recombinant Ca2÷-activated K ÷ channels studied in patches from GFP-identified cells. These latter channels were very similar in their properties to the corresponding native channels expressed in smooth muscle cells from which the cDNA clone was isolated. We therefore found no indication that GFP alters the properties of ion channels with which it is coexpressed. A NMDAR1-GFP Chimeric Protein Makes It Possible to Follow Functional NMDA Receptor Proteins in Living Cells When GFP is fused to the 3' end of the N-methyi-D- aspartate (NMDA) receptor subunit NMDAR1, a strongly

Pore Loops: An Emerging Theme in Ion Channel Structure Minireview

Abstract: Roderick MacKinnon Department of Neurobiology Harvard Medical School Boston, Massachusetts 02115 Recent studies using mutagenesis to alter ion channel function have shown that the selectivity filter in many ion channels is formed by pore loops, relatively short polypep- tide segments that extend into an aqueous pore from one side of the membrane. Why do ion channels use pore loops to form their selective ion-binding sites? The purpose of this perspective is to address this question in the context of other well-characterized proteins. To begin, Figure 1 summarizes the ion channels that are thought to contain pore loops. The voltage-gated K ÷, Na ÷, and Ca 2+ channels and cation channels gated by in- tracellular cyclic nucleotides contain pore loops formed by the linker connecting the fifth and sixth membrane- spanning regions of each subunit or domain (Figure 1A) (MacKinnon and Miller, 1989; Noda et al., 1989; Hartmann et al., 1991 ; Pusch et al., 1991 ; Terlau et al., 1991; Yellen et al., 1991; Yool and Schwarz, 1991; Backx et al., 1992; Satin et al., 1992; Goulding et al., 1993; Kim et al., 1993; Mikala et al., 1993; Root and MacKinnon, 1993; Yang et al., 1993; Eismann et al., 1994). In the case of homotetra- meric K ÷ channels, four identical loops, one from each subunit, extend into the pore. The same is probably true for the cyclic nucleotide-gated channels, but their subunit stoichiometry has not been established. In voltage-gated Na ÷ and Ca 2÷ channels, each of the four homologous do- mains contributes a loop to the ion conduction pore: loop residues determine whether the channel conducts Na ÷ or Ca 2÷ (Heinemann et al., 1992). Pore loops are not limited to those ion channels with membrane topologies described in Figure 1A. Inward recti- fier K ÷ channels (Ho et al., 1993; Kubo et al., 1993a, 1993b) and ATP-gated cation channels (Brake et al., 1994; Valera et al., 1994) have not been studied as extensively, but they probably also have a pore loop (homologous to that in voltage-gated K ÷ channels) in the setting of a much simpler overall transmembrane topology (Figure 1 B). Very recently, studies from several laboratories have com- pletely revised our view of the transmembrane topology of glutamate receptor ion channels, and in so doing have added to the growing list of ion channels thought to contain pore loops (Figure 1C) (Hollmann et al., 1994; Stern-Bach et al., 1994; Wo and Oswald, 1994, 1995a, 1995b; Bennet and Dingeldine, 1995). In the new model for glutamate receptor ion channels, a region known to be important for ion conduction (and previously thought to be a membrane- spanning segment) very nicely fits the description of a pore loop. In the absence of direct structural information, what do we know about the structure of any pore loop? Mutagene- sis studies on a

Actions of anesthetics on ligand-gated ion channels: role of receptor subunit composition

Abstract: Molecular cloning of cDNAs coding for ligand-gated ion channel subunits makes it possible to study the pharmacology of recombinant receptors with defined subunit compositions. Many laboratories have used these techniques recently to study actions of agents that produce general anesthesia. We review the effects of volatile and intravenous anesthetics on recombinant GABAA, glycine, AMPA, kainate, NMDA, and 5HT3 receptors. Evidence for and against specific ligand-gated ion channel subunits as targets responsible for anesthesia or the side effects of anesthetic agents is discussed for each type of receptor. Subunit specific actions of some of the agents suggest that construction and testing of certain chimeric receptor subunits may be useful for defining the amino acid sequences responsible for anesthetic actions.

Regulation of ion channels by ABC transporters that secrete ATP.

Abstract: When epithelial cells of patients with cystic fibrosis (CF) were found to have a low permeability to chloride (1), there was much excited anticipation that the molecule responsible would be rapidly identified, resulting in a deeper understanding of the pathogenesis and even treatment of the disease. This well-founded optimism was due to the success of the patch clamp in analyzing the details of ion channel regulation in many cells, including secretory epithelia, the site of the CF defect. Indeed, the apical membrane of CF epithelia was found to contain a C1channel that, unlike those in normal epithelia, could not be opened by cyclic AMP-dependent protein kinase (PKA) (2) (it had been known that sweat secretion induced by cyclic AMP was defective in CF). The single-channel conductance of the misregulated C1channel

The role of conserved leucines in the M2 domain of the acetylcholine receptor in channel gating.

Abstract: A highly conserved leucine is found in the middle of the porelining (M2) domain of the members of the ligand-gated ion channel family. Two very different roles have been proposed for this leucine. In one model, this residue swings into the lumen of the channel during desensitization to form the nonconducting desensitized state, whereas in the other model, the leucines from each subunit interact with each other to form a constriction in the channel that constitutes the actual gate of the channel. We examined the role of this leucine in the muscle-type acetylcholine receptor by replacing it with the polar amino acid threonine. Replacement of the leucine in any one subunit slows desensitization and shifts the dose-response relationship toward lower concentrations. Replacement of leucines in additional subunits leads to progressively larger shifts in the dose-response curves. The shift depends only on the number of leucines replaced, not on which particular subunits contain the mutation; in other words, the mutations act independently. At the single-channel level, the mutation greatly increases the channel mean open time. We conclude that the role of the conserved leucine is to set the mean open time of the channel through interactions with other regions of the receptor rather than to serve as the gate per se of the ion channel.

Structural conservation of ion conduction pathways in K channels and glutamate receptors

Abstract: Single channel recordings demonstrate that ion channels switch stochastically between an open and a closed pore conformation. In search of a structural explanation for this universal open/close behavior, we have uncovered a striking degree of amino acid homology across the pore-forming regions of voltage-gated K channels and glutamate receptors. This suggested that the pores of these otherwise unrelated classes of channels could be structurally conserved. Strong experimental evidence supports a hairpin structure for the pore-forming region of K channels. Consequently, we hypothesized the existence of a similar structure for the pore of glutamate receptors. In ligand-gated channels, the pore is formed by M2, the second of four putative transmembrane segments. A hairpin structure for M2 would affect the subsequent membrane topology, inverting the proposed orientation of the next segments, M3. We have tested this idea for the NR1 subunit of the N-methyl-D-aspartate receptor. Mutations that affected the glycosylation pattern of the NR1 subunit localize both extremes of the M3-M4 linker to the extracellular space. Whole cell currents and apparent agonist affinities were not affected by these mutations. Therefore it can be assumed that they represent the native transmembrane topology. The extracellular assignment of the M3-M4 linker challenged the current topology model by inverting M3. Taken together, the amino acid homology and the new topology suggest that the pore-forming M2 segment of glutamate receptors does not transverse the membrane but, rather, forms a hairpin structure, similar to that found in K channels.