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The cut-open oocyte Vaseline gap technique is a powerful electrophysiological method for the characterization of ion channels. However, traditional amplifiers for cut-open oocyte Vaseline gap are labor intensive and require significant user expertise. We introduce an innovative, open-source digital amplifier system with high-speed digitization and software-controlled electronics for computer-driven automation. This system compares well to existing commercial systems in terms of conventional specifications of step response (current peak at 25μs and decay of 36μs time constant), current noise (1.0 nA at 3-kHz bandwidth), and dynamic range (96.9 dB). Additionally, it unlocks new methods through close integration of the amplifier and software, including machine-learning techniques for tuning capacitive compensation waveforms, achieving a 100-fold suppression of mean-squared transient current, and impedance measurement methods to identify system components such as membrane capacitance and electrode resistances. For future extensions, the design has unique attributes such as real-time digital signal processing for feedback, multiple input and multiple output, and allows for user customization. By providing open-source access to the circuit board designs, control software, and field-programmable gate array code on GitHub, this approach aims to foster cross-disciplinary collaboration and facilitate instrument customization enabling previously inaccessible electrophysiology experiments., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Two electrode voltage-clamp (TEVC) electrophysiology in Xenopus oocytes is a common approach to studying the physiology and pharmacology of membrane transport proteins. Undergraduates may learn to use TEVC methodology in neuroscience or physiology courses and/or in faculty-mentored research experiences. Challenges with the methodology include the cost of commercially available recording chambers, especially when a lab needs multiple copies, and the additional time and expertise needed to use agar bridges and to stabilize solution flow and minimize noise from solution aspiration. Offering a low-cost and accessible recording chamber that overcomes these challenges would lower the barriers to success for undergraduates while also supporting publication-quality recordings. To address these issues, we developed a recording chamber using stereolithography, a 3D printing process. The physiology (PhISio) recording chamber features two options for solution aspiration that allow for individual preferences, optimizes placement of pre-made agar bridges to achieve laminar flow and reduce the time delays in initiating daily experiments, and minimizes the challenges of changing solution height and aspiration noise during perfusion. We compared the functionality of the PhISio chamber with a commercially available Warner Instruments RC-1Z chamber in electrophysiological recordings of inwardly rectifying potassium channels expressed in Xenopus oocytes. The PhISio chamber produced equivalent results to the RC-1Z chamber with respect to time-dependent solution changes and has several operational advantages for both new and experienced electrophysiologists, providing an affordable and convenient alternative to commercially available TEVC recording chambers., (Copyright © 2023 Faculty for Undergraduate Neuroscience.)

Action potentials (APs) are the functional units of fast electrical signaling in excitable cells. The upstroke and downstroke of an AP is generated by the competing and asynchronous action of Na + - and K + -selective voltage-gated conductances. Although a mixture of voltage-gated channels has been long recognized to contribute to the generation and temporal characteristics of the AP, understanding how each of these proteins function and are regulated during electrical signaling remains the subject of intense research. AP properties vary among different cellular types because of the expression diversity, subcellular location, and modulation of ion channels. These complexities, in addition to the functional coupling of these proteins by membrane potential, make it challenging to understand the roles of different channels in initiating and "temporally shaping" the AP. Here, to address this problem, we focus our efforts on finding conditions that allow reliable AP recordings from Xenopus laevis oocytes coexpressing Na + and K + channels. As a proof of principle, we show how the expression of a variety of K + channel subtypes can modulate excitability in this minimal model system. This approach raises the prospect of studies on the modulation of APs by pharmacological or biological means with a controlled background of Na + and K + channel expression., (© 2018 Corbin-Leftwich et al.)

Mosquitoes use chemical cues to modulate important behaviors such as feeding, mating, and egg laying. The primary chemosensory organs comprising the paired antennae, maxillary palps and labial palps are adorned with porous sensilla that house primary sensory neurons. Dendrites of these neurons provide an interface between the chemical environment and higher order neuronal processing. Diverse proteins located on outer membranes interact with chemicals, ions, and soluble proteins outside the cell and within the lumen of sensilla. Here, we review the repertoire of chemosensory receptors and other membrane proteins involved in transduction and discuss the outlook for their functional characterization. We also provide a brief overview of select ion channels, their role in mammalian taste, and potential involvement in mosquito taste. These chemosensory proteins represent targets for the disruption of harmful biting behavior and disease transmission by mosquito vectors.

All vertebrate inwardly rectifying potassium (Kir) channels are activated by phosphatidylinositol 4,5-bisphosphate (PIP2) (Logothetis, D. E., Petrou, V. I., Zhang, M., Mahajan, R., Meng, X. Y., Adney, S. K., Cui, M., and Baki, L. (2015) Annu. Rev. Physiol. 77, 81-104; Fürst, O., Mondou, B., and D'Avanzo, N. (2014) Front. Physiol. 4, 404-404). Structural components of a PIP2-binding site are conserved in vertebrate Kir channels but not in distantly related animals such as sponges and sea anemones. To expand our understanding of the structure-function relationships of PIP2 regulation of Kir channels, we studied AqKir, which was cloned from the marine sponge Amphimedon queenslandica, an animal that represents the phylogenetically oldest metazoans. A requirement for PIP2 in the maintenance of AqKir activity was examined in intact oocytes by activation of a co-expressed voltage-sensing phosphatase, application of wortmannin (at micromolar concentrations), and activation of a co-expressed muscarinic acetylcholine receptor. All three mechanisms to reduce the availability of PIP2 resulted in inhibition of AqKir current. However, time-dependent rundown of AqKir currents in inside-out patches could not be re-activated by direct application to the inside membrane surface of water-soluble dioctanoyl PIP2, and the current was incompletely re-activated by the more hydrophobic arachidonyl stearyl PIP2. When we introduced mutations to AqKir to restore two positive charges within the vertebrate PIP2-binding site, both forms of PIP2 strongly re-activated the mutant sponge channels in inside-out patches. Molecular dynamics simulations validate the additional hydrogen bonding potential of the sponge channel mutants. Thus, nature's mutations conferred a high affinity activation of vertebrate Kir channels by PIP2, and this is a more recent evolutionary development than the structures that explain ion channel selectivity and inward rectification., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

The gating of Ca²⁺-activated Cl⁻ channels is controlled by a complex interplay among [Ca²⁺](i), membrane potential and permeant anions. Besides Ca²⁺, Ba²⁺ also can activate both TMEM16A and TMEM16B. This study reports the effects of several divalent cations as regulators of TMEM16A channels stably expressed in HEK293T cells. Among the divalent cations that activate TMEM16A, Ca²⁺ is most effective, followed by Sr²⁺ and Ni²⁺, which have similar affinity, while Mg²⁺ is ineffective. Zn²⁺ does not activate TMEM16A but inhibits the Ca²⁺-activated chloride currents. Maximally effective concentrations of Sr²⁺ and Ni²⁺ occluded activation of the TMEM16A current by Ca²⁺, which suggests that Ca²⁺, Sr²⁺ and Ni²⁺ all regulate the channel by the same mechanism.

Polyunsaturated fatty acids such as arachidonic acid (AA) exhibit inhibitory modulation of Kv4 potassium channels. Molecular docking approaches using a Kv4.2 homology model predicted a membrane-embedded binding pocket for AA comprised of the S4-S5 linker on one subunit and several hydrophobic residues within S3, S5 and S6 from an adjacent subunit. The pocket is conserved among Kv4 channels. We tested the hypothesis that modulatory effects of AA on Kv4.2/KChIP channels require access to this site. Targeted mutation of a polar residue (K318) and a nonpolar residue (G314) within the S4-S5 linker as well as a nonpolar residue in S3 (V261) significantly impaired the effects of AA on K (+) currents in Xenopus oocytes. These residues may be important in stabilizing (K318) or regulating access to (V261, G314) the negatively charged carboxylate moiety on the fatty acid. Structural specificity was supported by the lack of disruption of AA effects observed with mutations at residues located near, but not within the predicted binding pocket. Furthermore, we found that the crystal structure of the related Kv1.2/2.1 chimera lacks the structural features present in the proposed AA docking site of Kv4.2 and the Kv1.2/2.1 K (+) currents were unaffected by AA. We simulated the mutagenic substitutions in our Kv4.2 model to demonstrate how specific mutations may disrupt the putative AA binding pocket. We conclude that AA inhibits Kv4 channel currents and facilitates current decay by binding within a hydrophobic pocket in the channel in which K318 within the S4-S5 linker is a critical residue for AA interaction.

A cDNA encoding a potassium channel of the two-pore domain family (K(2P), KCNK) of leak channels was cloned from the marine sponge Amphimedon queenslandica. Phylogenetic analysis indicated that AquK(2P) cannot be placed into any of the established functional groups of mammalian K(2P) channels. We used the Xenopus oocyte expression system, a two-electrode voltage clamp and inside-out patch clamp electrophysiology to determine the physiological properties of AquK(2P). In whole cells, non-inactivating, voltage-independent, outwardly rectifying K(+) currents were generated by external application of micromolar concentrations of arachidonic acid (AA; EC(50) ∼30 μmol l(-1)), when applied in an alkaline solution (≥pH 8.0). Prior activation of channels facilitated the pH-regulated, AA-dependent activation of AquK(2P) but external pH changes alone did not activate the channels. Unlike certain mammalian fatty-acid-activated K(2P) channels, the sponge K(2P) channel was not activated by temperature and was insensitive to osmotically induced membrane distortion. In inside-out patch recordings, alkalinization of the internal pH (pK(a) 8.18) activated the AquK(2P) channels independently of AA and also facilitated activation by internally applied AA. The gating of the sponge K(2P) channel suggests that voltage-independent outward rectification and sensitivity to pH and AA are ancient and fundamental properties of animal K(2P) channels. In addition, the membrane potential of some poriferan cells may be dynamically regulated by pH and AA.

Kv4/K channel interacting protein (KChIP) potassium channels are a major class of rapidly inactivating K(+) channels in neurons and cardiac muscle. Modulation of Kv4/KChIP channels by polyunsaturated fatty acids (PUFAs) is important in the regulation of cellular excitability and the induction of activity-dependent synaptic plasticity. Using the Xenopus laevis oocyte expression system, we studied the inhibition by PUFAs of the peak outward K(+) current and the accompanying increase in the rate of current inactivation of rKv4.2/rKChIP1b. Inhibitory effects do not depend on KChIP coexpression since Kv4.2 channels lacking an NH(2)-terminal KChIP association region were substantially inhibited by PUFAs and showed strong kinetic modulation. PUFAs accelerated both the fast and slow time constants that describe the kinetics of Kv4/KChIP inactivation. The time course of entry into closed inactivated states was facilitated by PUFAs, but steady-state inactivation and recovery from inactivation were unaltered. PUFA inhibition of Kv4/KChIP current was not use dependent. The concentration-response relationship for arachidonic acid (AA) inhibition of Kv4/KChIP channels mimicked that for activation of TRAAK channels. Internal serum albumin largely prevents the inhibitory effects of externally applied AA, and the membrane-impermeant AA-CoA is inactive when applied externally. Overall, our data suggest that PUFAs inhibit Kv4/KChIP channels by facilitating inactivation from open and closed gating states and that access of the fatty acid to the internal leaflet of the membrane is important. These results improve our understanding of the mechanisms for the inhibitory effects of PUFAs on Kv4/KChIP channel function.

Ion channels establish and regulate membrane potentials in excitable and non-excitable cells. How functional diversification of ion channels contributed to the evolution of nervous systems may be understood by studying organisms at key positions in the evolution of animal multicellularity. We have carried out the first analysis of ion channels cloned from a marine sponge, Amphimedon queenslandica. Phylogenetic comparison of sequences encoding for poriferan inward-rectifier K(+) (Kir) channels suggests that Kir channels from sponges, cnidarians and triploblastic metazoans each arose from a single channel and that duplications arose independently in the different groups. In Xenopus oocytes, AmqKirA and AmqKirB produced K(+) currents with strong inward rectification, as seen in the mammalian Kir2 channels, which are found in excitable cells. The pore properties of AmqKir channels demonstrated strong K(+) selectivity and block by Cs(+) and Ba(2+). We present an original analysis of sponge ion channel physiology and an examination of the phylogenetic relationships of this channel with other cloned Kir channels.

Arachidonic acid (AA) was found to inhibit the function of whole-cell voltage-gated (VG) calcium currents nearly 16 years ago. There are now numerous examples demonstrating that AA and other polyunsaturated fatty acids (PUFAs) modulate the function of VG ion channels, primarily in neurons and muscle cells. We will review and extract some common features about the modulation by PUFAs of VG calcium, sodium, and potassium channels and discuss the impact of this modulation on the excitability of neurons and cardiac myocytes. We will describe the fatty acid nature of the membrane, how fatty acids become available to function as modulators of VG channels, and the physiologic importance of this type of modulation. We will review the evidence for molecular mechanisms and assess our current understanding of the structural basis for modulation. With guidance from research on the structure of fatty acid binding proteins, the role of lipids in gating mechanosensitive (MS) channels, and the impact of membrane lipid composition on membrane-embedded proteins, we will highlight some avenues for future investigations.

The amyloid precursor protein (APP) undergoes sequential cleavages to generate various polypeptides, including the amyloid-beta protein (Abeta), which forms amyloid plaques in Alzheimer's disease (AD), secreted APPalpha (sAPPalpha) which enhances memory, and the APP intracellular domain (AICD), which has been implicated in the regulation of gene transcription and calcium signaling. The beta-site APP cleaving enzyme 1 (BACE1) cleaves APP in an activity-dependent manner to form Abeta, AICD, and secreted APPbeta. Because this neural activity was shown to diminish synaptic transmission in vitro [Kamenetz F, Tomita T, Hsieh H, Seabrook G, Borchelt D, Iwatsubo T, Sisodia S, Malinow R (2003) Neuron 37:925-937], the prevailing notion has been that this pathway diminishes synaptic function. Here we investigated the role of this pathway in vivo. We studied transgenic mice overproducing APP that do not develop AD pathology or memory deficits but instead exhibit enhanced spatial memory. We showed enhanced synaptic plasticity in the hippocampus that depends on prior synaptic activity. We found that the enhanced memory and synaptic plasticity are abolished by the ablation of one or both copies of the BACE1 gene, leading to a significant decrease in AICD but not of any other APP cleavage products. In contrast to the previously described negative effect of BACE1-mediated cleavage of APP on synaptic function in vitro, our in vivo work indicates that BACE1-mediated cleavage of APP can facilitate learning, memory, and synaptic plasticity.

The CREB transcription factor mediates neuronal plasticity in many systems, but the relationship between CREB levels and CRE-mediated transcription in individual neurons in vivo is unclear. In FVB/N nontransgenic mice, we observed that Purkinje cells showed low basal levels of Ser(133)-phosphorylated CREB protein yet displayed strong CRE-directed transcription. Transgenic mice overexpressing CREB in Purkinje cells and dentate gyrus granule cells showed a decreased CRE-lacZ signal in the same cells, indicating repression of ATF/CREB family function. Dentate region long-term potentiation was not altered by these changes in CREB expression. CREB transgenic mice demonstrated an inability to perform the rotarod task, without signs of overt ataxia. Our results demonstrate that the level of phosphorylated CREB protein is not a reliable indicator of CRE-mediated function. Furthermore, we conclude that CRE-mediated transcription may be linked to only a subset of cerebellum-mediated motor behaviors and may not be universally required for long-lasting synaptic potentiation.

Kv4/K channel-interacting protein (KChIP) potassium channels are a major class of rapidly inactivating K channels in brain and heart. Considering the importance of alternative splicing to the quantitative features of KChIP gating modulation, a previously uncharacterized splice form of KChIP1 was functionally characterized. The KChIP1b splice variant differs from the previously characterized KChIP1a splice form by the inclusion of a novel amino-terminal region that is encoded by an alternative exon that is conserved in mouse, rat, and human genes. The expression of KChIP1b mRNA was high in brain but undetectable in heart or liver by RT-PCR. In cerebellar tissue, KChIP1b and KChIP1a transcripts were expressed at nearly equal levels. Coexpression of KChIP1b or KChIP1a with Kv4.2 channels in oocytes slowed K current decay and destabilized open-inactivated channel gating. Like other KChIP subunits, KChIP1b increased Kv4.2 current amplitude and KChIP1b also shifted Kv4.2 conductance-voltage curves by -10 mV. The development of Kv4.2 channel inactivation accessed from closed gating states was faster with KChIP1b coexpression. Deletion of the novel amino-terminal region in KChIP1b selectively altered the subunit's modulation of Kv4.2 closed inactivation gating. The role of the KChIP1b NH2-terminal region was further confirmed by direct comparison of the properties of the NH2-terminal deletion mutant and the KChIP1a subunit, which is encoded by a transcript that lacks the novel exon. The features of KChIP1b modulation of Kv4 channels are likely to be conserved in mammals and demonstrate a role for the KChIP1 NH2-terminal region in the regulation of closed inactivation gating.

Hippocampal synaptic plasticity was studied in transgenic mice over-expressing human alpha-synuclein containing the A30P Parkinson's disease mutation. Medial perforant path-dentate granule cell synapses showed enhanced paired-pulse depression (PPD) for short interpulse intervals (< 200 ms), without differences in basal transmission. Extracellular calcium reduction failed to rescue the enhanced PPD. Paired-pulse facilitation in the CA1 region was normal in slices from transgenic mice, but enhanced synaptic depression was revealed upon repetitive stimulation of the Schaffer collaterals. Long-term potentiation in the CA1 field was not impaired in slices from transgenic mice. These results suggest that mutant alpha-synuclein accumulation impairs short-term changes in synaptic strength when neurotransmitter availability is limited due to enhanced release probability or repetitive synaptic activity.

A number of peptide toxins from venoms of spiders and cone snails are high affinity ligands for voltage-gated calcium channels and are useful tools for studying calcium channel function and structure. Using whole-cell recordings from rat sympathetic ganglion and cerebellar Purkinje neurons, we studied toxins that target neuronal N-type (Ca(V)2.2) and P-type (Ca(V)2.1) calcium channels. We asked whether different toxins targeting the same channels bind to the same or different sites on the channel. Five toxins (omega-conotoxin-GVIA, omega-conotoxin MVIIC, omega-agatoxin-IIIA, omega-grammotoxin-SIA, and omega-agatoxin-IVA) were applied in pairwise combinations to either N- or P-type channels. Differences in the characteristics of inhibition, including voltage dependence, reversal kinetics, and fractional inhibition of current, were used to detect additive or mutually occlusive effects of toxins. Results suggest at least two distinct toxin binding sites on the N-type channel and three on the P-type channel. On N-type channels, results are consistent with blockade of the channel pore by omega-CgTx-GVIA, omega-Aga-IIIA, and omega-CTx-MVIIC, whereas grammotoxin likely binds to a separate region coupled to channel gating. omega-Aga-IIIA produces partial channel block by decreasing single-channel conductance. On P-type channels, omega-CTx-MVIIC and omega-Aga-IIIA both likely bind near the mouth of the pore. omega-Aga-IVA and grammotoxin each bind to distinct regions associated with channel gating that do not overlap with the binding region of pore blockers. For both N- and P-type channels, omega-CTx-MVIIC binding produces complete channel block, but is prevented by previous partial channel block by omega-Aga-IIIA, suggesting that omega-CTx-MVIIC binds closer to the external mouth of the pore than does omega-Aga-IIIA.

The opening of voltage-gated sodium, potassium, and calcium ion channels has a steep relationship with voltage. In response to changes in the transmembrane voltage, structural movements of an ion channel that precede channel opening generate a capacitative gating current. The net gating charge displacement due to membrane depolarization is an index of the voltage sensitivity of the ion channel activation process. Understanding the molecular basis of voltage-dependent gating of ion channels requires the measurement and computation of the gating charge, Q. We derive a simple and accurate semianalytic approach to computing the voltage dependence of transient gating charge movement (Q-V relationship) of discrete Markov state models of ion channels using matrix methods. This approach allows rapid computation of Q-V curves for finite and infinite length step depolarizations and is consistent with experimentally measured transient gating charge. This computational approach was applied to Shaker potassium channel gating, including the impact of inactivating particles on potassium channel gating currents.

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.

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.

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.

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.

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.

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.

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.

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)

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.

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.

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.

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.