Kinetic analysis of open- and closed-state inactivation transitions in human Kv4.2 A-type potassium channels.

1. We studied the gating kinetics of Kv4.2 channels, the molecular substrate of neuronal somatodendritic A-type currents. For this purpose wild-type and mutant channels were transiently expressed in the human embryonic kidney (HEK) 293 cell line and currents were measured in the whole-cell patch-clamp configuration. 2. Kv4.2 channels inactivated from pre-open closed state(s) with a mean time constant of 959 ms at -50 mV. This closed-state inactivation was not affected by a deletion of the Kv4.2 N-terminus (Delta2-40). 3. Kv4.2 currents at +40 mV inactivated with triple-exponential kinetics. A fast component (tau = 11 ms) accounted for 73 %, an intermediate component (tau = 50 ms) for 23 % and a slow component (tau = 668 ms) for 4 % of the total decay. 4. Both the fast and the intermediate components of inactivation were slowed by a deletion of the Kv4.2 N-terminus (tau = 35 and 111 ms) and accounted for 33 and 56 %, respectively, of the total decay. The slow component was moderately accelerated by the truncation (tau = 346 ms) and accounted for 11 % of the total Kv4.2 current inactivation. 5. Recovery from open-state inactivation and recovery from closed-state inactivation occurred with similar kinetics in a strongly voltage-dependent manner. Neither recovery reaction was affected by the N-terminal truncation. 6. Kv4.2 Delta2-40 channels displayed slowed deactivation kinetics, suggesting that the N-terminal truncation leads to a stabilization of the open state. 7. Simulations with an allosteric model of inactivation, supported by the experimental data, suggested that, in response to membrane depolarization, Kv4.2 channels accumulate in the closed-inactivated state(s), from which they directly recover, bypassing the open state.

Episodic ataxia/myokymia mutations functionally expressed in the Shaker potassium channel.

Episodic ataxia type 1 is a rare, autosomal dominant neurological disorder caused by missense mutations of the Kv1.1 gene from the Shaker K+ channel subfamily. To study the functional effects of the disease-causing mutations in a robust K+ channel background, we introduced seven different episodic ataxia type 1 substitutions into the corresponding, conserved residues of the Shaker K+ channel. K+ channel currents expressed in Xenopus oocytes were studied by electrophysiology. All episodic ataxia type 1 mutations produced functional K+ channels. In a Shaker N-terminal deletion mutant with fast inactivation removed, current amplitudes were significantly reduced in channels harboring an episodic ataxia type 1 mutation. Six of the seven mutations also showed depolarizing shifts (+9 to +36 mV) in the conductance voltage dependence. One mutation (F307I) shifted the midpoint of the conductance-voltage relationship by 23 mV in the hyperpolarizing direction. Episodic ataxia type 1 mutations were also expressed in ShakerH4 with intact N-terminal inactivation. In this construct, current amplitudes for episodic ataxia type 1 mutants were not significantly different from wild-type channels. All mutations altered the voltage range of steady-state inactivation; most changes were coupled to the changes in activation gating. Some episodic ataxia type 1 mutants also caused significant changes in the kinetics of N-type (F307I, E395D) or C-type (F307I, E395D, V478A) inactivation. These results suggest that episodic ataxia type 1 mutations may change K+ channel function by two mechanisms: (i) reduced channel expression and (ii) altered channel gating.

Protein kinase C inhibits Kv1.1 potassium channel function.

The regulation by protein kinase C (PKC) of recombinant voltage-gated potassium (K) channels in frog oocytes was studied. Phorbol 12-myristate 13-acetate (PMA; 500 nM), an activator of PKC, caused persistent and large (up to 90%) inhibition of mouse, rat, and fly Shaker K currents. K current inhibition by PMA was blocked by inhibitors of PKC, and inhibition was not observed in control experiments with PMA analogs that do not activate PKC. However, site-directed substitution of potential PKC phosphorylation sites in the Kv1.1 protein did not prevent current inhibition by PMA. Kv1.1 current inhibition was also not accompanied by changes in macroscopic activation kinetics or in the conductance-voltage relationship. In Western blots, Kv1.1 membrane protein was not significantly reduced by PKC activation. The injection of oocytes with botulinum toxin C3 exoenzyme blocked the PMA inhibition of Kv1. 1 currents. These data are consistent with the hypothesis that PKC-mediated inhibition of Kv1.1 channel function occurs by a novel mechanism that requires a C3 exoenzyme substrate but does not alter channel activation gating or promote internalization of the channel protein.

AnkyrinG is required for clustering of voltage-gated Na channels at axon initial segments and for normal action potential firing.

Voltage-gated sodium channels (NaCh) are colocalized with isoforms of the membrane-skeletal protein ankyrinG at axon initial segments, nodes of Ranvier, and postsynaptic folds of the mammalian neuromuscular junction. The role of ankyrinG in directing NaCh localization to axon initial segments was evaluated by region-specific knockout of ankyrinG in the mouse cerebellum. Mutant mice exhibited a progressive ataxia beginning around postnatal day P16 and subsequent loss of Purkinje neurons. In mutant mouse cerebella, NaCh were absent from axon initial segments of granule cell neurons, and Purkinje cells showed deficiencies in their ability to initiate action potentials and support rapid, repetitive firing. Neurofascin, a member of the L1CAM family of ankyrin-binding cell adhesion molecules, also exhibited impaired localization to initial segments of Purkinje cell neurons. These results demonstrate that ankyrinG is essential for clustering NaCh and neurofascin at axon initial segments and is required for physiological levels of sodium channel activity.

Inhibition of calcium channels in rat central and peripheral neurons by omega-conotoxin MVIIC.

Inhibition of voltage-dependent calcium channels by omega-conotoxin MVIIC (omega-CTx-MVIIC) was studied in various types of rat neurons. When studied with 5 mM Ba2+ as charge carrier, omega-CTx-MVIIC block of N-type calcium channels in sympathetic neurons was potent, with half-block at 18 nM. Block of N-type channels had a rapid onset (tau approximately 1 sec at 1 microM omega-CTx-MVIIC) and quick reversibility (tau approximately 30 sec). The rate of block was proportional to toxin concentration, consistent with 1:1 binding of toxin to channels, with a rate constant (k on) of approximately 1 X 10(6) M-1. sec-1. Both potency and rate of block were reduced dramatically with increasing concentrations of extracellular Ba2+ omega-CTx-MVIIC also blocked P-type calcium channels in cerebellar Purkinje neurons, but both development and reversal of block were far slower than for N-type channels. The rate of block was proportional to toxin concentration, with k on -1.5 x 10(3) M-1. sec-1 at 5 mM Ba2+. From this value and an unblocking time constant of approximately 200 min, a dissociation constant of approximately 50 nM was estimated. Thus, block of P-type channels is potent but very slow. In hippocampal CA3 pyramidal neurons, omega-CTx-MVIIC blocked approximately 50% of the high-threshold calcium channel current; one component (approximately 20%) was blocked with the rapid kinetics expected for N-type channels, whereas the other component was blocked slowly. The component blocked slowly was reduced but not eliminated by preexposure to 200 nM or 1 microM omega-Aga-IVA.

Visual identification of individual transfected cells for electrophysiology using antibody-coated beads.

Electrophysiological study of transiently transfected cells requires the identification of individual cells that express the protein of interest. We describe a simple, quick and inexpensive method for visually identifying cells that have been co-transfected with an expression plasmid for a lymphocyte surface antigen (CD8-alpha). Transfected cells are incubated briefly with polystyrene microspheres (4.5 microns diameter) that have been precoated with antibody to CD8. Cells expressing CD8 on their surface are decorated with many beads and are thus readily distinguishable from untransfected cells. Beads already coated with antibody are available commercially. The method takes less than five minutes and requires no reagent preparation or special equipment for visualization of the beads.

omega-Conotoxin block of N-type calcium channels in frog and rat sympathetic neurons.

Block of N-type Ca channels by omega-conotoxin GVIA (CgTx) was studied in freshly dissociated bullfrog and rat sympathetic neurons. With 2-5 mM Ba as charge carrier, CgTx blocked almost all of the high-threshold Ca channel current recorded in the presence of nimodipine (3 microM) to block L-type Ca channels. Toxin block reversed slowly (time constant approximately 1 hr) in frog cells and even more slowly in rat cells. CgTx block was faster and more potent in rat cells than frog cells. The rate of block was proportional to CgTx concentration, consistent with 1:1 binding of CgTx to channels. When the external Ba concentration was increased, the development of block was slower, consistent with competition between CgTx and Ba for a binding site. The recovery from block was somewhat faster in higher external Ba. Some cells had significant current remaining in saturating concentrations of nimodipine and CgTx, especially with high Ba concentrations in the external solution. The current resistant to nimodipine and CgTx was activated at lower depolarizations than the CgTx-sensitive current and had faster activation and inactivation kinetics, but unlike low-threshold T-type current, the resistant current had rapidly decaying tail currents.

Cysteines in the Shaker K+ channel are not essential for channel activity or zinc modulation.

We investigated whether the cysteine residues in Shaker potassium (K+) channels are essential for activation and inactivation gating or for modulation of activation gating by external zinc (Zn2+). Mutants of the Shaker K+ channel were prepared in which all seven cysteine residues were replaced (C-less). These changes were made in both wild-type Shaker H4 channels and in a deletion mutant (delta 6-46) lacking N-type ("fast") inactivation. Replacement of all cysteines left most functional properties of the K+ currents unaltered. The most noticeable difference between the C-less and wild-type currents was the faster C-type inactivation of the C-less channel which could be attributed largely to the mutation of Cys462. This is consistent with the effects of previously reported mutations of nearby residues in the S6 region. There were also small changes in the activation gating of C-less currents. Modulation by external Zn2+ of the voltage dependence and rate of activation gating is preserved in the C-less channels, indicating that none of the cysteines in the Shaker K+ channel plays an important role in Zn2+ modulation.

Modulation of N-type calcium channels in bullfrog sympathetic neurons by luteinizing hormone-releasing hormone: kinetics and voltage dependence.

Inhibition of Ca channel current by luteinizing hormone-releasing hormone (LHRH) was studied in freshly dissociated bullfrog sympathetic ganglion neurons using whole-cell recording. LHRH inhibited up to 80% of the high-threshold Ca channel current with a half-maximally effective concentration of about 20 nM. LHRH inhibited omega-conotoxin-sensitive but not nimodipine-sensitive current and also did not inhibit Bay K 8644-enhanced currents, suggesting that LHRH inhibits N-type but not L-type channels. Inhibition was faster at higher concentrations of LHRH, reaching a limiting time constant of 2 sec at 0.3-3 mM LHRH. The rate of recovery from block (tau approximately 19 sec) was independent of LHRH concentration. Inhibition of N-type current by LHRH was highly sensitive to the gating state of the channel. Though strongly effective if applied when channels were mostly in the resting state, LHRH had little effect if applied rapidly during a long depolarization that opened the channels. Inhibition could be relieved if channels were activated by short, large test depolarizations or by long, smaller depolarizations. The state-dependent properties of LHRH block could be simulated by a model that assumes that inhibition by LHRH results from activated G-proteins binding to N-type channels and that (1) G-protein binding stabilizes closed gating states and (2) activation of G-protein-bound channels destabilizes the binding of the G-protein to the channel.

Gadolinium block of calcium channels: influence of bicarbonate.

The selectivity of block of voltage-activated barium (Ba2+) currents by lanthanide ions was studied in a rat dorsal root ganglion (DRG) cell line (F11-B9), rat and frog peripheral neurons, and rat cardiac myocytes using the whole-cell patch clamp technique. Gadolinium (Gd3+) produced a dose-dependent and complete inhibition of whole-cell Ba2+ current in all cells studied, including cells expressing identified dihydropyridine-sensitive L-type currents and omega-conotoxin-sensitive N-type currents. Like Gd3+, lutetium (Lu3+) and lanthanum (La3+) blocked all Ba2+ current with little selectivity for different components of the whole-cell current. Gd3+ block of Ba2+ currents was incomplete, however, when sodium bicarbonate (5-22.6 mM) was added to the standard HEPES-buffered external Ba2+ solution. In rat DRG neurons and F11-B9 cells, a fraction of the whole-cell Ba2+ current recorded in the presence of bicarbonate was resistant to block by saturating concentrations of Gd3+ (50-100 microM). The resistant current inactivated more rapidly than the original current giving the appearance that, under these conditions, Gd3+ block is more selective for the slowly inactivating component of the whole-cell current. Bicarbonate modification of Gd3+ block occurred both before and after omega-conotoxin block of N-type currents in rat DRG neurons, suggesting that even in the presence of bicarbonate, Gd3+ block was not selective for N-type currents.

Inhibition by bradykinin of voltage-activated barium current in a rat dorsal root ganglion cell line: role of protein kinase C.

The whole-cell patch-clamp technique was used to record Ba2+ currents through voltage-activated calcium channels in the clonal dorsal root ganglion cell line F11-B9. The pain-producing peptide bradykinin (BK; 100 nM) reduced the sustained Ba2+ current in F11-B9 cells by 30%. In cultures prelabeled with 3H-arachidonic acid and tested under ionic conditions similar to those used for recording Ba2+ currents, BK also induced a concentration-dependent, transient, 2.7-fold accumulation of 3H-diacylglycerol. Both the elevation of 3H-diacylglycerol and the inhibition of Ba2+ current began within 5 sec following BK exposure, and the effective concentration range of BK was similar for the 2 responses. In whole-cell recordings, extracellularly applied 1-oleoyl-2-acetylglycerol (OAG; 0.5-5 microM) mimicked the degree of block and occluded the block of sustained current by BK. Another protein kinase C (PKC) activator, 1,2-dioctanoylglycerol (diC8), blocked 70-100% of sustained current when applied intracellularly or extracellularly at 5 microM, whereas extracellular application of ethylene glycol dioctanoate (5 microM), an analog reported not to stimulate PKC, inhibited only 14% of sustained current. The pseudosubstrate peptide PKC19-36 (2 microM in pipette) and the lipid staurosporine (100 nM in pipette), both inhibitors of PKC, reduced the effects of maximal concentrations of OAG or BK by 55-60%. Dynorphin A applied intracellularly (2 microM) as a control for nonspecific effects of PKC19-36 did not inhibit the block of sustained current by BK. These data are consistent with the hypothesis that BK inhibits whole-cell sustained Ba2+ current in F11-B9 cells via a mechanism that involves activation of PKC.

Expression of sensory neuron antigens by a dorsal root ganglion cell line, F-11.

The F-11 cell line is a fusion product of embryonic rat dorsal root ganglion (DRG) cells with mouse neuroblastoma cell line N18TG-2 (Platika, D., Boulos, M.H., Baizer, L. and Fishman, M.C., Proc. Natl. Acad. Sci. U.S.A., 82 (1985) 3499-3503). F-11 cells were uniformly labelled using a monoclonal antibody (RT-97) to the 200 kDa subunit of neurofilament protein, which labels a subpopulation of adult rat DRG neurons. F-11 cells did not stain for antigenic markers of fibroblasts or Schwann/satellite cells which are also present in DRG. Monoclonal antibodies that recognize cell surface carbohydrates have been shown to label subpopulations of DRG neurons. The stage-specific embryonic antigens SSEA-3 and SSEA-4, and the antigen recognized by B23D8, were expressed by some F-11 cells but not by the neuroblastoma parent of the hybrid cells. SSEA-3 was expressed by about 20% of the F-11 cells, whereas 40-60% expressed SSEA-4 or the antigen recognized by B23D8. The stability of F-11 cell subpopulations for sensory antigen expression was demonstrated by isolating single cells and growing the progeny as clonal lines. In some subclones, nearly 100% of the cells stably expressed SSEA-4 and/or B23D8, or failed to stain with anti-SSEA-4, anti-SSEA-3, or B23D8 over 12 passages. Other subclones were unstable for the expression of these antigens. This study demonstrates the derivation of the F-11 cell line from sensory neurons but also indicates that multiple phenotypes of varying stability are present in this line. This information is important for the use of this line as a model for DRG neurons.

Metabolism of juvenile hormone during adult development of Dermacentor variabilis (Acari: Ixodidae).

Juvenile hormone (JH)-I and -III were used as model substrates to study the in vitro metabolism of JH in the hemolymph and body homogenates of the American dog tick, Dermacentor variabilis (Say). Ester hydrolysis was the principal pathway of JH metabolism in hemolymph and homogenates. JH also was converted into JH-diol primarily by body homogenates, indicating the presence of JH epoxide hydrolase activity. JH epoxide hydrolase activity, alpha-naphthyl acetate esterase activity, and protein concentration per milligram wet weight were significantly lower (t test, alpha = 0.05) in homogenates of partially fed, virgin and replete, mated females of D. variabilis compared with unfed, virgin females. The decline in these factors was probably because of the influx of water into the tissues caused by the blood meal. In addition, the epoxide hydrolase and alpha-naphthyl acetate esterase activity per milligram tissue protein decreased significantly during this time. Mating of fed females rather than feeding alone caused a significant decline in the tissue JH esterase activity per milligram wet weight but not per milligram protein. The JH esterase activity per milligram protein was significantly higher in partially fed, virgin and replete, mated females compared with unfed females, indicating that feeding may actually increase JH esterase activity on a protein basis. JH-III was metabolized 1.4 times faster than JH-I by the hemolymph of partially fed, virgin females. The inhibitors O,O-diisopropyl phosphorofluoridate and octylthio-1,1,1-trifluoropropan-2-one at 10(-4) M inhibited JH and alpha-naphthyl acetate esterase activity in hemolymph and body homogenate.

Multiple components of both transient and sustained barium currents in a rat dorsal root ganglion cell line.

1. Currents through voltage-activated Ca2+ channels in rat dorsal root ganglion (DRG) x mouse neuroblastoma hybrid (F-11) cells were studied using the whole-cell patch clamp technique with 30 mM-Ba2+ as charge carrier. Two components of the inward Ba2+ current were distinguished on the basis of voltage dependence and time course. Each component could be further subdivided based on pharmacology. 2. A transient inward current activated at test potentials positive to -40 mV, peaked within 20 ms and then decayed during a 200 ms depolarization. The peak amplitude of the transient current occurred between -10 and +10 mV. With a 300 ms conditioning pulse, half-inactivation of the transient current occurred at -30 mV. A sustained inward current activated at test potentials positive to -30 mV and reached a maximum at +20 to +30 mV. The sustained current showed little voltage-dependent inactivation over 200 ms. The amplitudes of both the transient and sustained currents were increased by perfusing with Ba2+ instead of Ca2+. 3. Most F-11 cells had both the transient and sustained Ba2+ currents although the relative amount of the two currents varied with culture conditions. The transient current was more prominent in cells fed with a 'growth' medium (15-20% serum) whereas the sustained current was increased in cells fed with a 'differentiation' medium (1% serum plus growth factors). F-11 cells can be used to study transient current in relative isolation from sustained Ca2+ current under certain culture conditions. The neuroblastoma parent of the F-11 cell line, N18TG-2 cells, exhibited little or no voltage-dependent Ba2+ current. 4. Brief application of omega-conotoxin fraction GVIA (10 microM) produced a long-lasting block of 81% of the sustained current and 27% of the transient current. 5. The transient and sustained Ba2+ currents in F-11 cells were reversibly blocked by brief exposure to Cd2+ or Ni2+. Block of the sustained current was evident with 100 nM-Cd2+ whereas the threshold concentration for Ni2+ block was 1 microM. Cd2+ and Ni2+ were equipotent blockers of the transient current. Dose-response curves for Cd2+ and Ni2+ block of both sustained and transient currents had shallow slopes suggesting that the block was more complex than a simple bimolecular interaction between blocker and one blocking site. Dose-response curves were fitted by a model that included two binding sites for each divalent blocker.(ABSTRACT TRUNCATED AT 400 WORDS)

L-glutamate binding site on N18-RE-105 neuroblastoma hybrid cells is not coupled to an ion channel.

We studied the properties of the N18-RE-105 neuronal cell line to determine if its glutamate binding site represents a neurotransmitter receptor. In immunocytochemical experiments, these cells stained strongly for neurofilament, but not for glial fibrillary acidic protein. In whole-cell patch clamp experiments, cells exhibited voltage-dependent Na+, Ca2+, and K+ currents characteristic of neurons. However, perfusion with L-glutamate or other excitatory amino acids did not evoke the inward current expected of a receptor/channel complex. In binding studies, the maximum accumulation of L-[3H]glutamate by washed membrane vesicles at 37 degrees C was 69 pmol/mg protein, and half-maximal accumulation occurred at 0.64 microM. This accumulation was blocked completely by quisqualate, partially by DL-2-amino-4-phosphonobutyric acid and L-cystine, but not at all by 1 mM kainate or N-methylaspartate. L-[3H]Glutamate accumulation was stimulated by Cl-, but reduced by Na+, 0.01% digitonin, or hyperosmotic (400 mM glucose) assay medium. The release of L-[3H]glutamate from vesicles was much faster in the presence of 100 microM unlabelled glutamate than 100 microM unlabelled quisqualate or DL-2-amino-4-phosphonobutyric acid. Thus, although N18-RE-105 cells possess many neuronal properties, the results obtained are not those expected from reversible binding of L-glutamate to a receptor/channel complex, but are consistent with a Cl- -stimulated sequestration or exchange process.

Amino acid receptors and uptake systems in the mammalian central nervous system.

The inhibitory and excitatory amino acid neurotransmitter receptors in the mammalian central nervous system mediate functionally opposite synaptic responses yet appear to share certain structural features. Recent conceptual advances in this field have relied heavily on information obtained by single channel analyses, by the expression of receptors in oocytes, and by autoradiographic studies of receptor distribution among brain receptors. This article reviews the pharmacology, cellular physiology, and regional distribution of these receptors and discusses their role in several well-characterized neurological disease states. Also reviewed are the recent advances made in purifying (in some instances cloning) the receptors and uptake sites involved in synaptic transmission in the brain. Throughout, the emphasis is on synthesis and concept rather than on methodological detail.

Ultrastructural localization of acetylcholinesterase in the synganglion of the tick, Dermacentor variabilis (Say).

Light- and electron-microscopic enzyme cytochemistry was used to localize acetylcholinesterase (AChE) activity in the synganglion (brain) of the tick Dermacentor variabilis. High AChE activity was observed throughout the neuropil as well as adjacent to most neuronal perikarya. Intracellular activity was not observed by light microscopy. By electron microscopy, reaction product was localized at the plasma membrane of glia and neurons. Enzyme activity was not associated with the olfactory globuli neurons. In other types of neurons, small amounts of reaction product were observed in the Golgi apparatus and nuclear envelope. Large neurosecretory neurons contained activity that appeared to be associated with deep invaginations of the plasma membrane as well as intracellular membranes. AChE activity was also associated with processes of both neurons and glia. In most peripheral nerves AChE activity was associated with virtually all axons. Clearly then, AChE is associated with glia and non-cholinergic neurons as well as with presumed cholinergic neurons. The widespread localization and large amounts of AChE in the tick brain exceeds that reported for other invertebrates and vertebrates. As has been suggested for other animals, AChE in the tick brain may have functions in addition to its known role in cholinergic neurotransmission.