Neuromodulation, development and synaptic plasticity.

Discusses parallels in the mechanisms underlying use-dependent synaptic plasticity during development and long-term potentiation (LTP) and long-term depression (LTD) in neocortical synapses. Neuromodulators, such as norepinephrine, serotonin, and acetylcholine, have also been implicated in regulating both developmental plasticity and LTP/LTD. There are many potential levels of interaction between neuromodulators and plasticity. Ion channels are substrates for modulation in many cell types. The authors discuss examples of modulation of voltage-gated Ca2+ channels and Ca2+-dependent K+ channels and the consequences for neocortical pyramidal cell firing behaviour. At the time when developmental plasticity is most evident in rat cortex, the substrate for modulation is changing as the densities and relative proportions of various ion channels types are altered during ontogeny. The authors review examples of changes in K+ and Ca2+ channels and the consequence for modulation of neuronal activity. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

The role of voltage-gated Ca2+ channels in anoxic injury of spinal cord white matter

Dorsal column axons of the rat spinal cord are partially protected from anoxic injury following blockade of voltage-sensitive Na+ channels and the Na+/--Ca2+ exchanger. To examine the potential contribution of voltage-gated Ca2+ channels to anoxic injury of spinal cord axons, we studied axonal conduction in rat dorsal columns in vitro following a 60-min period of anoxia. Glass microelectrodes were used to record field potentials from the dorsal columns following distal local surface stimulation. Perfusion solutions containing blockers of voltage-gated Ca2+ channels were introduced 60 min prior to onset of anoxia and continued until 10 min after reoxygenation. Pharmacological blocking agents which are relatively selective for L- (verapamil, diltiazem, nifedipine) and N- (omega-conotoxin GVIA) type calcium channels were significantly protective against anoxia-induced loss of conduction, as was non-specific block using divalent cations. Other Ca2+ channel blockers (neomycin and omega-conotoxin MVIIC) that affect multiple Ca2+ channel types were also neuroprotective. Ni2+, which preferentially blocks R-type Ca2+ channels more than T-type channels, was also protective in a dose-dependent manner. These data suggest that the influx of Ca2+, through L-, N- and possibly R-type voltage-gated Ca2+ channels, participates in the pathophysiology of the Ca2+-mediated injury of spinal cord axons that is triggered by anoxia.     

The effects of prepulse-blink reflex trial repetition and prepulse change on blink reflex modification at short and long lead intervals

1. Thin slices of the posterior pituitary can be used as a preparation for the study of biophysical mechanisms underlying neuropeptide secretion. Patch-clamp techniques in this preparation have revealed the properties of ion channels that control the excitability of the nerve terminal membrane and have clarified the relation between Ca2+ and exocytosis. 2. Repetitive electrical activity at high frequencies broadens action potentials to allow more Ca2+ entry and thus enhance exocytosis. Action potential broadening results from the inactivation of a voltage-dependent K+ channel. 3. When repetitive electrical activity is sustained, secretion is depressed. This depression can be attributed in part to action potential failure caused by the opening of a Ca(2+)-activated K+ channel. This channel can be modulated by protein kinases, phosphatases, and G-proteins. 4. The inhibitory neurotransmitter GABA activates a GABAA receptor in the nerve terminal membrane. The gating of the associated Cl- channel depolarizes the membrane slightly to inactivate voltage-gated Na+ channels and block action potential propagation. 5. The response of the nerve terminal GABAA receptor is enhanced by neuroactive steroids and this can potentiate the inhibition of neurosecretion by GABA. The action of neurosteroids at this site could play a role in changes in neuropeptide secretion associated with reproductive transitions. 6. Ca2+ channels in the nerve terminal membrane are inactivated by sustained depolarization and by trains of brief pulses. Ca2+ entry promotes Ca2+ channel inactivation during trains by inhibiting the recovery of Ca2+ channels from inactivation. The inactivation of Ca2+ channels can play a role in defining the optimal frequency and train duration for evoking neuropeptide secretion. 7. Measurements of membrane capacitance in peptidergic nerve terminals have revealed rapid exocytosis and endocytosis evoked by Ca2+ entry through voltage-gated Ca2+ channels. Exocytosis is too rapid to account for the delays in neuropeptide secretion evoked by trains of action potentials. Endocytosis sets in rapidly after exocytosis with a time course comparable to that of the rapid endocytosis observed in nerve terminals at rapid synapses. Our results support the finding in rapid synaptic nerve terminals that endocytosis is inhibited by intracellular Ca2+. Multiple pools of vesicles were revealed, and these pools may reflect different stages in the mobilization and release of neuropeptide.     

Glomerular mesangial cells: electrophysiology and regulation of contraction

Mesangial cells are smooth muscle-like pericytes that abut and surround the filtration capillaries within the glomerulus. Studies of the fine ultrastructure of the glomerulus show that the mesangial cell and the capillary basement membrane form a biomechanical unit capable of regulating filtration surface area as well as intraglomerular blood volume. Structural and functional studies suggest that mesangial cells regulate filtration rate in both a static and dynamic fashion. Mesangial excitability enables a homeostatic intraglomerular stretch reflex that integrates an increase in filtration pressure with a reduction in capillary surface area. In addition, mesangial tone is regulated by diverse vasoactive hormones. Agonists, such as angiotensin II, contract mesangial cells through a signal transduction pathway that releases intracellular stores of Ca2+, which subsequently activate nonselective cation channels and Cl- channels to depolarize the plasma membrane. The change in membrane potential activates voltage-gated Ca2+ channels, allowing Ca2+ cell entry and further activation of depolarizing conductances. Contraction and entry of cell Ca2+ are inhibited only when Ca2+-activated K+ channels (BK(Ca)) are activated and the membrane is hyperpolarized toward the K+ equilibrium potential. The mesangial BK(Ca) is a weak regulator of contraction in unstimulated cells; however, the gain of the feedback is increased by atrial natriuretic peptide, nitric oxide, and the second messenger cGMP, which activates protein kinase G and decreases both the voltage and Ca2+ activation thresholds of BK(Ca) independent of sensitivity. This enables BK(Ca) to more effectively counter membrane depolarization and voltage-gated Ca2+ influx. After hyperpolarizing the membrane, BK(Ca) rapidly inactivates because of dephosphorylation by protein phosphatase 2A. Regulation of ion channels has been linked casually to hyperfiltration during early stages of diabetes mellitus. Determining the signaling pathways controlling the electrophysiology of glomerular mesangial cells is important for understanding how glomerular filtration rate is regulated in health and disease.     

The Müller (glial) cell in normal and diseased retina: a case for single-cell electrophysiology

In the retina of most vertebrates there exists only one type of macroglia, the Müller cell. Müller cells express voltage-gated ion channels, neurotransmitter receptors and various uptake carrier systems. These properties enable the Müller cells to control the activity of retinal neurons by regulating the extracellular concentration of neuroactive substances such as K+, GABA and glutamate. We show here how electrophysiological recordings from enzymatically dissociated mammalian Müller cells can be used to study these mechanisms. Müller cells from various species have Na(+)-dependent GABA uptake carriers, but only cells from primates have additional GABA receptors that activate Cl- channels. Application of glutamate analogues causes enhanced membrane currents recorded from Müller cells in situ but not from isolated cells. We show that mammalian Müller cells have no ionotropic glutamate receptors but respond to increased K+ release from glutamate-stimulated retinal neurons. This response is involved in extracellular K+ clearance and is mediated by voltage-gated (inwardly rectifying) K+ channels which are abundantly expressed by healthy Müller cells. In various cases of human retinal pathology, currents through these channels are strongly reduced or even extinguished. Another type of voltage-gated ion channels, observed in Müller cells from many mammalian species, are Na+ channels. In Müller cells from diseased human retinae, voltage-dependent Na+ currents were significantly increased in comparison to cells from control donors. Thus, the expression of glial ion channels seems to be controlled by neuronal signals. This interaction may be involved in the pathogenesis of retinal gliosis which inevitably accompanies any degeneration of retinal neurons. In particular, Müller cell proliferation may be triggered by mechanisms requiring the activation of Ca(2+)-dependent K+ channels. Ca(2+)-dependent K+ currents are easily elicitable in Müller cells from degenerating retinae and can be blocked by 1 mM TEA (tetraethylammonium). In purified Müller cell cultures, the application of 1 mM TEA greatly reduces the proliferative activity of the cells. These data clearly show that Müller cells are altered in cases of neuronal degeneration and may be crucially involved in pathogenetic mechanisms of the retina.     

Endothelium-smooth muscle interactions in blood vessels

1. Blood vessel tone is determined both by smooth muscle and endothelial functions. In coronary arteries taken from rat (Fisher-Lewis) cardiac transplanted hearts, the inducible form of NOS (iNOS) in smooth muscle is more active, while acetylcholine-induced nitric oxide production in the endothelium is greatly diminished. This causes a greatly reduced myogenic constriction, in pressurized septal arteries taken from immunologically challenged transplanted hearts. 2. The sarcoplasmic reticulum (SR) of smooth muscle and the endoplasmic reticulum (ER) of endothelial cells sequester Ca2+ from the cytoplasm. This reduces the intracellular concentration of free Ca2+, which is necessary for the activation of cellular processes. The release of Ca2+ from internal stores occurs through ryanodine and IP3 recoptors located on the SR membrane. 3. The superficial SR/ER also interacts with ion exchangers and pumps in the plasma membrane. This allows for the superficial SR/ER to function in Ca2+ extrusion; for example, inhibition of the SR/ER Ca(2+)-ATPase (SERCA) partially inhibits the rate of loss Ca2+ from the cell. Recent data suggest that the SR Ca(2+)-ATPase and the Na(+)-Ca2+ exchanger of smooth muscle cells function in series; that is, Ca2+ uptake by the SR followed by release towards the exchanger to mediate extrusion. This interaction between the SERCA of the superficial SR and ion exchangers and pumps creates intracellular Ca2+ gradients. 4. The SERCA of the superficial, peripherally distributed SR/ER also serves to regulate Ca2+ entry from the extracellular space. This occurs in part by inhibition of the superficial buffer barrier function of the SR as well as by depletion of stimulated Ca2+ entry. 5. Ca2+ entry is also regulated in endothelial and smooth muscle cells by the membrane potential. Membrane hyperpolarization increases the driving force for Ca2+ entry into endothelial cells, which lack voltage-gated Ca2+ channels, and reduces open state probability of voltage-gated Ca2+ channels in vascular smooth muscle cells. The two cell types have electrical contact and interact in a dynamic manner to regulate blood vessel diameter.     

Voltage-controlled gating in a large conductance Ca2+-sensitive K+channel (hslo)

Large conductance calcium- and voltage-sensitive K+ (MaxiK) channels share properties of voltage- and ligand-gated ion channels. In voltage-gated channels, membrane depolarization promotes the displacement of charged residues contained in the voltage sensor (S4 region) inducing gating currents and pore opening. In MaxiK channels, both voltage and micromolar internal Ca2+ favor pore opening. We demonstrate the presence of voltage sensor rearrangements with voltage (gating currents) whose movement and associated pore opening is triggered by voltage and facilitated by micromolar internal Ca2+ concentration. In contrast to other voltage-gated channels, in MaxiK channels there is charge movement at potentials where the pore is open and the total charge per channel is 4-5 elementary charges.     

Concentration-dependent modulations of potassium and calcium currents of rat osteoblastic cells by arachidonic acid

We show that the voltage-gated K+ and Ca2+ currents of rat osteoblastic cells are strongly modulated by arachidonic acid (AA), and that these modulations are very sensitive to the AA concentration. At 2 or 3 microM, AA reduces the amplitude and accelerates the inactivation of the K+ current activated by depolarization; at higher concentrations (> or = 5 microM), AA still blocks this K+ current, but also induces a very large noninactivating K+ current. At 2 or 3 microM, AA enhances the T-type Ca2+ current, close to its threshold of activation, whereas at 10 microM, it blocks that current. AA (1-10 microM) also blocks the dihydropyridine-sensitive L-type Ca2+ current. Thus, the effect of AA on Ca2+ entry through voltage-gated Ca2+ channels can change qualitatively with the AA concentration: at 2 or 3 microM, AA will favor Ca2+ entry through T channels, both by lowering the voltage-gated K+ conductance and by increasing the T current, whereas at 10 microM, AA will prevent Ca2+ entry through voltage-gated Ca2+ channels, both by inducing a K+ conductance and by blocking Ca2+ channels.     

Rat group I metabotropic glutamate receptors inhibit neuronal Ca2+ channels via multiple signal transduction pathways in HEK 293 cells

We have shown previously that metabotropic glutamate receptors with group I-like pharmacology couple to N-type and P/Q-type calcium channels in acutely isolated cortical neurons using G proteins most likely belonging to the Gi/Go subclass. To better understand the potential mechanisms forming the basis for group I mGluR modulation of voltage-gated calcium channels in the CNS, we have examined the ability of specific mGluRs to couple to neuronal N-type (alpha1B-1/alpha2delta/beta1b) and P/Q-type (alpha1A-2/alpha2delta/beta1b) voltage-gated calcium channels in an HEK 293 heterologous expression system. Using the whole cell patch-clamp technique where intracellular calcium is buffered to low levels, we have shown that group I receptors inhibit both N-type and P/Q-type calcium channels in a voltage-dependent fashion. Similar to our observations in cortical neurons, this voltage-dependent inhibition is mediated almost entirely by N-ethylmaleimide (NEM)-sensitive heterotrimeric G proteins, strongly suggesting that these receptors can use Gi/Go-like G proteins to couple to N-type and P/Q-type calcium channels. However, inconsistent with the apparent NEM sensitivity of group I modulation of calcium channels, modulation of N-type channels in group I mGluR-expressing cells was only partially sensitive to pertussis toxin (PTX), indicating the potential involvement of both PTX-sensitive and -resistant G proteins. The PTX-resistant modulation was voltage dependent and entirely resistant to NEM and cholera toxin. A time course of treatment with PTX revealed that this toxin caused group I receptors to slowly shift from using a primarily NEM-sensitive G protein to using a NEM-resistant form. The PTX-induced switch from NEM-sensitive to -resistant modulation was also dependent on protein synthesis, indicating some reliance on active cellular processes. In addition to these voltage-dependent pathways, perforated patch recordings on group I mGluR-expressing cells indicate that another slowly developing, calcium-dependent form of modulation for N-type channels may be seen when intracellular calcium is not highly buffered. We conclude that group I mGluRs can modulate neuronal Ca2+ channels using a variety of signal transduction pathways and propose that the relative contributions of different pathways may exemplify the diversity of responses mediated by these receptors in the CNS.     

A new role for IP3 receptors: Ca2+ release during nuclear vesicle fusion

During nuclear assembly, vesicles derived from the mitotic disassembly of the nuclear membranes reform the nuclear envelope. The vesicles first bind to chromosomes, specifically recognize other nuclear vesicles and then fuse to enclose the chromosomes. The proteins that mediate these events are largely unknown. Using reconstituted extracts of Xenopus eggs, we found that nuclear vesicle fusion required elevated (microM) concentrations of free Ca2+ [Sullivan KMC. Busa WB. Wilson KL. (1993) Cell, 73, 1411-1422]. Our data suggest that Ca2+ is released from the vesicle lumen by the activation of IP3 receptors (ligand-gated Ca2+ channels). We propose that the role of IP3 receptors during nuclear assembly may be analogous to that of voltage-gated Ca2+ channels during regulated secretion: to provide a microdomain of high cytosolic Ca2+ that triggers fusion. In this article, we will briefly describe current ideas about nuclear assembly and disassembly, and summarize the evidence that IP3 receptors are required for nuclear vesicle fusion. We will discuss parallels between our results and the role of voltage-gated Ca2+ channels, and Ca2+, in regulated exocytosis. Finally, we will address the question of how IP3 receptors are activated during nuclear vesicle fusion: is there a signal that stimulates IP3 production, or is the channel activated directly?     

Localization of Ca2+ channel subtypes on rat spinal motor neurons, interneurons, and nerve terminals

Ca2+ channels in distinct subcellular compartments of neurons mediate voltage-dependent Ca2+ influx, which integrates synaptic responses, regulates gene expression, and initiates synaptic transmission. Antibodies that specifically recognize the alpha1 subunits of class A, B, C, D, and E Ca2+ channels have been used to investigate the localization of these voltage-gated ion channels on spinal motor neurons, interneurons, and nerve terminals of the adult rat. Class A P/Q-type Ca2+ channels were present mainly in a punctate pattern in nerve terminals located along the cell bodies and dendrites of motor neurons. Both smooth and punctate staining patterns were observed over the surface of the cell bodies and dendrites with antibodies to class B N-type Ca2+ channels, indicating the presence of these channels in the cell surface membrane and in nerve terminals. Class C and D L-type and class E R-type Ca2+ channels were distributed mainly over the cell soma and proximal dendrites. Class A P/Q-type Ca2+ channels were present predominantly in the presynaptic terminals of motor neurons at the neuromuscular junction. Occasional nerve terminals innervating skeletal muscles from the hindlimb were labeled with antibodies against class B N-type Ca2+ channels. Staining of the dorsal laminae of the rat spinal cord revealed a complementary distribution of class A and class B Ca2+ channels in nerve terminals in the deeper versus the superficial laminae. Many of the nerve terminals immunoreactive for class B N-type Ca2+ channels also contained substance P, an important neuropeptide in pain pathways, suggesting that N-type Ca2+ channels are predominant at synapses that carry nociceptive information into the spinal cord.     

Ca2+ currents in compensated hypertrophy and heart failure

Transmembrane voltage-gated Ca2+ channels play a central role in the development and control of heart contractility which is modulated by the concentration of free cytosolic calcium ions (Ca2+). Ca2+ channels are closed at the normal membrane resting potential of cardiac cells. During the fast upstroke of the action potential (AP), they are gated into an open state by membrane depolarisation and thereby transduce the electrical signal into a chemical signal. In addition to its contribution to the AP plateau, Ca2+ influx through L-type Ca2+ channels induces a release of Ca2+ ions from the sarcoplasmic reticulum (SR) which initiates contraction. Because of their central role in excitation-contraction (E-C) coupling, L-type Ca2+ channels are a key target to regulate inotropy [1]. The role of T-type Ca2+ channels is more obscure. In addition to a putative part in the rhythmic activity of the heart, they may be implicated at early stages of development and during pathology of contractile tissues [2]. Despite therapeutic advances improving exercise tolerance and survival, congestive heart failure (HF) remains a major problem in cardiovascular medicine. It is a highly lethal disease; half of the mortality being related to ventricular failure whereas sudden death of the other patients is unexpected [3]. Although HF has diverse aetiologies, common abnormalities include hypertrophy, contractile dysfunction and alteration of electrophysiological properties contributing to low cardiac output and sudden death. A significant prolongation of the AP duration with delayed repolarisation has been observed both during compensated hypertrophy (CH) and in end-stage HF caused by dilated cardiomyopathy (Fig. 1A) [4-8]. This lengthening can result from either an increase in inward currents or a decrease in outward currents or both. A reduction of K+ currents has been demonstrated [6,9]. Prolonged Na+/Ca2+ exchange current may also be involved [9]. In contrast, there is a large variability in the results concerning Ca2+ currents (ICa). The purpose of this paper is to review results obtained in various animal models of CH and HF with special emphasis on recent studies in human cells. We focus on: (i) the pathophysiological role of T-type Ca2+ channels, present in some animal models of hypertrophy; (ii) the density and properties of L-type Ca2+ channels and alteration of major physiological regulations of these channels by heart rate and beta-adrenergic receptor stimulation; and (iii) recent advances in the molecular biology of the L-type Ca2+ channel and future directions.     

Metabotropic glutamate receptors: electrophysiological properties and role in plasticity

Electrophysiological research on mGluRs is now very extensive, and it is clear that activation of mGluRs results in a large number of diverse cellular actions. Studies of mGluRs and on ionic channels has clearly demonstrated that mGluR activation has a widespread and potent inhibitory action on both voltage-gated Ca2+ channels and K+ channels. Inhibition of N-type Ca2+ channels, and inhibition of Ca(++)-dependent K+ current, IAHP, and IM being particularly prominent. Potentiation of activation of both Ca2+ and K+ channels has also been observed, although less prominently than inhibition, but mGluR-mediated activation of non-selective cationic channels is widespread. In a small number of studies, generation of an mGluR-mediated slow excitatory postsynaptic potential has been demonstrated as a consequence of the effect of mGluR activation on ion channels, such as activation of a non-selective cationic channels. Although certain mGluR-modulation of channels is a consequence of direct G-protein-linked action, for example, inhibition of Ca2+ channels, many other effects occur as a result of activation of intracellular messenger pathways, but at present, little progress has been made on the identification of the messengers. The field of study of the involvement of mGluRs in synaptic plasticity is very large. Evidence for the involvement of mGluRs in one form of LTD induction in the cerebellum and hippocampus is now particularly impressive. However, the role of mGluRs in LTP induction continues to be a source of dispute, and resolution of the question of the exact involvement of mGluRs in the induction of LTP will have to await the production of more selective ligands and of selective gene knockouts.     

Calcium signalling in glial cells

Glial cells respond to a variety of external stimuli such as neurotransmitters, hormones or even mechanical stress by generating complex changes in the cytoplasmic Ca2+ concentration. This Ca2+ signal is controlled by an interplay of different mechanisms including plasmalemmal and intracellular Ca2+ channels, Ca2+ transporters and cytoplasmic Ca2+ buffers. In astrocytes, the Ca2+ signal can travel as waves within the syncytium spreading via gap junctions which might be regarded as a possible means for interglial communication. Ca2+ signalling is also an important medium for neurone-glia interaction: neuronal activity can trigger Ca2+ signals in glial cells and, in turn, there is evidence that glial Ca2+ signals can elicit responses in neurones. While glial cells are not equipped with the proper channels to generate action potentials, Ca2+ signalling could be the instrument by which these cells integrate and propagate signals in the CNS.     

Ca2+ channels, ryanodine receptors and Ca(2+)-activated K+ channels: a functional unit for regulating arterial tone

Local calcium transients ('Ca2+ sparks') are thought to be elementary Ca2+ signals in heart, skeletal and smooth muscle cells. Ca2+ sparks result from the opening of a single, or the coordinated opening of many, tightly clustered ryanodine receptor (RyR) channels in the sarcoplasmic reticulum (SR). In arterial smooth muscle, Ca2+ sparks appear to be involved in opposing the tonic contraction of the blood vessel. Intravascular pressure causes a graded membrane potential depolarization to approximately -40 mV, an elevation of arterial wall [Ca2+]i and contraction ('myogenic tone') of arteries. Ca2+ sparks activate calcium-sensitive K+ (KCa) channels in the sarcolemmal membrane to cause membrane hyperpolarization, which opposes the pressure induced depolarization. Thus, inhibition of Ca2+ sparks by ryanodine, or of KCa channels by iberiotoxin, leads to membrane depolarization, activation of L-type voltage-gated Ca2+ channels, and vasoconstriction. Conversely, activation of Ca2+ sparks can lead to vasodilation through activation of KCa channels. Our recent work is aimed at studying the properties and roles of Ca2+ sparks in the regulation of arterial smooth muscle function. The modulation of Ca2+ spark frequency and amplitude by membrane potential, cyclic nucleotides and protein kinase C will be explored. The role of local Ca2+ entry through voltage-dependent Ca2+ channels in the regulation of Ca2+ spark properties will also be examined. Finally, using functional evidence from cardiac myocytes, and histological evidence from smooth muscle, we shall explore whether Ca2+ channels, RyR channels, and KCa channels function as a coupled unit, through Ca2+ and voltage, to regulate arterial smooth muscle membrane potential and vascular tone.     

Measurement of intracellular free zinc in living cortical neurons: routes of entry

We used the ratioable fluorescent dye mag-fura-5 to measure intracellular free Zn2+ ([Zn2+]i) in cultured neocortical neurons exposed to neurotoxic concentrations of Zn2+ in concert with depolarization or glutamate receptor activation and identified four routes of Zn2+ entry. Neurons exposed to extracellular Zn2+ plus high K+ responded with a peak cell body signal corresponding to a [Zn2+]i of 35-45 nM. This increase in [Zn2+]i was attenuated by concurrent addition of Gd3+, verapamil, omega-conotoxin GVIA, or nimodipine, consistent with Zn2+ entry through voltage-gated Ca2+channels. Furthermore, under conditions favoring reverse operation of the Na+-Ca2+ exchanger, Zn2+ application induced a slow increase in [Zn2+]i and outward whole-cell current sensitive to benzamil-amiloride. Thus, a second route of Zn2+ entry into neurons may be via transporter-mediated exchange with intracellular Na+. Both NMDA and kainate also induced rapid increases in neuronal [Zn2+]i. The NMDA-induced increase was only partly sensitive to Gd3+ or to removal of extracellular Na+, consistent with a third route of entry directly through NMDA receptor-gated channels. The kainate-induced increase was highly sensitive to Gd3+ or Na+ removal in most neurons but insensitive in a minority subpopulation ("cobalt-positive cells"), suggesting that a fourth route of neuronal Zn2+ entry is through the Ca2+-permeable channels gated by certain subtypes of AMPA or kainate receptors.     

Unique regulatory properties of the type 2a Ca2+ channel beta subunit caused by palmitoylation

Beta subunits of voltage-gated Ca2+ channels are encoded in four genes and display additional molecular diversity because of alternative splicing. At the functional level, all forms are very similar except for beta2a, which differs in that it does not support prepulse facilitation of alpha1C Ca2+ channels, inhibits voltage-induced inactivation of neuronal alpha1E Ca2+ channels, and is more effective in blocking inhibition of alpha1E channels by G protein-coupled receptors. We show that the distinguishing properties of beta2a, rather than interaction with a distinct site of alpha1, are because of the recently described palmitoylation of cysteines in positions three and four, which also occurs in the Xenopus oocyte. Essentially, all of the distinguishing features of beta2a were lost in a mutant that could not be palmitoylated [beta2a(Cys3,4Ser)]. Because protein palmitoylation is a dynamic process, these findings point to the possibility that regulation of palmitoylation may contribute to activity-dependent neuronal and synaptic plasticity. Evidence is presented that there may exist as many as three beta2 splice variants differing only in their N-termini.     

Docosahexaenoic acid block of neuronal voltage-gated K+ channels: subunit selective antagonism by zinc

The omega-3 polyunsaturated fatty acid docosahexaenoic acid is highly enriched in neuronal membranes, and several studies suggest that DHA is critical for neuronal development. We have investigated the effects of exogenously applied DHA on voltage-gated K+ channels using patch-clamp techniques. DHA produced a concentration-dependent inhibition of the sustained outward current in isolated neocortical neurons. This blocking action was examined in more detail with two cloned neuronal K+ channels (Kv1.2 and Kv3.1a) expressed in mammalian fibroblasts. DHA produced a potent inhibition of depolarization-activated K+ currents from cells expressing these channels (Kd values, 1.8 +/- 0.1 muM and 690 +/- 60 nM, for Kv1.2 and Kv3.1a, respectively, at +40 mV). The DHA block of both channel types was rapidly reversed (approximately 2 sec) by bovine serum albumin, which binds the fatty acid. Micromolar concentrations of extracellular Zn2+ non-competitively antagonized DHA inhibition of Kv1.2 channels, whereas there was little effect on DHA block of Kv3.1a channels. Experiments with membrane patches from Kv1.2 transfected cells demonstrated that the DHA block occurred from the outside, suggesting that the fatty acid interacts directly with an external domain of the ion channel. DHA may serve as a local messenger molecule that selectively modulates the activity of certain voltage-gated K+ channels in a Zn2(+)-dependent fashion.     

Thiopental and methohexital depress Ca2+ entry into and glutamate release from cultured neurons

BACKGROUND: Although barbiturates activate alpha-aminobutyric acid type A receptors as part of their hypnotic effect, these drugs also inhibit voltage-gated calcium channels. The authors determined if barbiturates could decrease neuronal intracellular Ca2+ transients and the resulting glutamate release. METHODS: Neonatal rat cerebellar granule neurons were isolated and cultured on coverslips and studied at 37 degrees C. Spectrofluorometric assays were used during identical conditions to monitor intracellular Ca2+ with the Ca2+ -sensitive fluorophore fura-2 and glutamate release by a glutamate dehydrogenase-coupled assay, which produced the reduced form of nicotinamide-adenine dinucleotide phosphate in proportion to the amount of glutamate released. Neurons were depolarized by a rapid increase in external [K+] from 5 to 55 mM. Control responses were compared with those in the presence of 10, 30, and 100 microM thiopental; 3, 10, and 30 microM methohexital; decreased external [Ca2+]; or voltage-gated calcium channel blockers. RESULTS: Thiopental and methohexital depressed the intracellular Ca2+ transient peak and plateau in a dose-dependent manner, as did decreased Ca2+. The intermediate dose of either drug caused approximately 50% decrease in peak intracellular Ca2+ and 60% decrease in glutamate release. In the presence of specific L- and/or N-type voltage-gated calcium channel blockade by nicardipine or omega-conotoxin-GVIA, respectively, 30 microM thiopental further decreased the intracellular Ca2+ transient. Thiopental caused a dose-dependent decrease in glutamate release, which was proportional to the decreased peak intracellular Ca2+. CONCLUSIONS: Thiopental and methohexital depress the depolarization-induced increase in intracellular Ca2+ and the accompanying glutamate release, actions which can contribute to the anesthetic and neuronal protective effects of these drugs.     

Potassium channels in epithelial cells

The broad array of K- channels in epithelial cells includes voltage-dependent (mainly outward) and Ca(2+)-activated channels, and K+ channels modulated by adenosine triphosphate (ATP). Voltage-dependent K+ channels mediate Na+/K+ absorption and secretion; typically, they are found in the basolateral membrane and exhibit burst activity. Ca(2+)-activated K+ (Ca2+/K+) channels regulate activity by decreasing Ca2+ influx via voltage-gated Ca2+ channels. Ca2+/K+ channels exhibit conductances of 4-300 pS, and have a low open probability (< 10(-7)) at the level of the resting membrane potential. ATP-sensitive channels have been observed mainly in insulin-secreting pancreatic beta-cells and in the urinary tract, where the open state is rapidly closed by ATP. The channels are voltage-dependent, exhibit burst activity, and, in the case of the urinary-tract cells, are Ca2+ dependent. Chemical compounds that selectively open or block K+ channels have been exploited to characterize channels found in different cells, but no opener or blocker has been found that specifically affects only one type of K+ channel. Specialized model systems and recombinant techniques have led to a general understanding of the structure of K+ channels, but many important details remain to be determined.