In vivo functional role of the Drosophila hyperkinetic beta subunit in gating and inactivation of Shaker K+ channels

The physiological roles of the beta, or auxiliary, subunits of voltage-gated ion channels, including Na+, Ca2+, and K+ channels, have not been demonstrated directly in vivo. Drosophila Hyperkinetic (Hk) mutations alter a gene encoding a homolog of the mammalian K+ channel beta subunit, providing a unique opportunity to delineate the in vivo function of auxiliary subunits in K+ channels. We found that the Hk beta subunit modulates a wide range of the Shaker (Sh) K+ current properties, including its amplitude, activation and inactivation, temperature dependence, and drug sensitivity. Characterizations of the existing mutants in identified muscle cells enabled an analysis of potential mechanisms of subunit interactions and their functional consequences. The results are consistent with the idea that via hydrophobic interaction, Hk beta subunits modulate Sh channel conformation in the cytoplasmic pore region. The modulatory effects of the Hk beta subunit appeared to be specific to the Sh alpha subunit because other voltage- and Ca(2+)-activated K+ currents were not affected by Hk mutations. The mutant effects were especially pronounced near the voltage threshold of IA activation, which can disrupt the maintenance of the quiescent state and lead to the striking neuromuscular and behavioral hyperexcitability previously reported.     

Prefrontal cortex involvement in selective letter generation: a functional magnetic resonance imaging study

Leech neurons exposed to salines containing inorganic Ca(2+)-channel blockers generate rhythmic bursts of impulses. According to an earlier model, these blockers unmask persistent Na+ currents that generate plateau-like depolarizations, each triggering a burst of impulses. The resulting increase in intracellular Na+ activates an outward Na+/K+ pump current that contributes to burst termination. We tested this model by examining systematically the effects of six transition metal ions (Co2+, Ni2+, Mn2+, Cd2+, La3+, and Zn2+) on the electrical activity of neurons in isolated leech ganglia. Each ion induced bursting activity, but the amplitude, form, and persistence of bursting differed with the ion used and its concentration relative to Ca2+. All ions tested suppressed chemical synaptic transmission between identified motor neurons, consistent with block of voltage-dependent Ca2+ currents in these cells. In addition, a strong correlation between suppression of synaptic transmission and burst amplitudes was obtained. Finally, burst duration was increased and the rate of repolarization decreased in reduced K+ saline, as expected for pump-dependent repolarization. These results provide further support for the hypothesis that a novel form of oscillatory electrical activity driven by persistent Na+ currents and the Na+/K+ pump occurs in leech ganglia exposed to Ca(2+)-channel blockers.     

Calcium-activated potassium conductances on cultured nontransformed dog pancreatic duct epithelial cells

Pancreatic duct epithelial cells (PDECs) mediate the pancreatic secretion of fluid and electrolytes. Membrane K+ channels on these cells regulate intracellular K+ concentration; in combination with the Na+/H+ antiport and Na+,K+ adenosine triphosphatase (ATPase), they may also mediate serosal H+ secretion, balancing luminal HCO3- secretion. We describe the K+ conductances on well-differentiated and functional nontransformed cultured dog PDECs. Through 86Rb+ efflux studies, we demonstrated Ca(2+)-activated K+ channels that were stimulated by A23187, thapsigargin, and 1-ethyl-2-benzimidazolinone, but not forskolin. These conductances also were localized on the basolateral membrane because 86Rb+ efflux was directed toward the serosal compartment. Of the K+ channel blockers, BaCl2, charybdotoxin, clotrimazole, and quinidine, but not 4-aminopyridine, apamin, tetraethylammonium, or iberiotoxin, inhibited 86Rb+ efflux. This efflux was not inhibited by amiloride, ouabain, and bumetanide, inhibitors of the Na+/H+ antiport, the Na+,K(+)-ATPase pump, and the Na+,K+,2Cl- cotransporter, respectively. When apically permeabilized PDEC monolayers were mounted in Ussing chambers with a luminal-to-serosal K+ gradient, A23187 and 1-ethyl-2-benzimidazolinone stimulated a charybdotoxin-sensitive short-circuit current (Isc) increase. Characterization of K+ channels on these cultured PDECs, along with previous identification of Cl- channels (1), further supports the importance of these cells as models for pancreatic duct secretion.     

Kinetics of Ca2+ release evoked by photolysis of caged InsP3 in rat submandibular cells

Quantitative time-resolved measurements of cytosolic Ca2+ release by photolysis of caged InsP3 have been made in single rat submandibular cells using patch clamp whole-cell recording to measure the Ca2+-activated Cl- and K+ currents. Photolytic release of InsP3 from caged InsP3 at 100 Joules caused transient inward (V(H) = 60 mV) and outward (V(H) = 0 mV) currents, which were nearly symmetric in their time course. The inward current was reduced when pipette Cl- concentration was decreased, and the outward current was suppressed by K+ channel blockers, indicating that they were carried by Cl- and K+, respectively. Intracellular pre-loading of the InsP3 receptor antagonist heparin or the Ca2+ chelator EGTA clearly prevented both inward and outward currents, indicating that activation of Ca2+-dependent Cl- and K+ currents underlies the inward and the outward currents. At low flash intensities, InsP3 caused Ca2+ release which normally activated the K+ and Cl- currents in a mono-transient manner. At higher intensities, however, InsP3 induced an additional delayed outward K+ current (I[K,(delay)]). I[K(delay)] was independent of the initial K+ current, independent of extracellular Ca2+, inhibited by TEA, and gradually prolongated by repeated flashes. The photolytic release of Ca2+ from caged Ca2+ did not mimic the I[K(delay)]. It is suggested that Ca2+ releases from the InsP3-sensitive pools in an InsP3 concentration-dependent manner. Low concentrations of InsP3 induce the transient Ca2+-dependent Cl- and K+ currents, which reflects the local Ca2+ release, whereas high concentrations of InsP3 induce a delayed Ca2+-dependent K+ current, which may reflect the Ca2+ wave propagation.     

Ion channels in the immune system as targets for immunosuppression

The discovery of a diverse and unique subset of ion channels in T lymphocytes has led to a rapidly growing body of knowledge about their functional roles in the immune system. Potent and specific blockers have provided molecular tools to probe channel structure-function relations and to elucidate the involvement of K+, Ca2+, and Cl- channels in T-cell activation and cell volume regulation. Recent advances in analyzing Kv1.3 channel structure-function relationships have defined binding sites for channel blockers, which have now been shown to be effective in suppressing T-cell function in vivo. Ion channels may provide excellent pharmaceutical targets for modulating immune system function.     

Hypotonically induced whole-cell currents in A6 cells: relationship with cell volume and cytoplasmic Ca2+

We investigated changes in whole-cell currents, cell volume, and intracellular calcium concentration ([Ca2+]i) during hypotonic stimulation in whole-cell clamped cultured amphibian renal cells (A6 cells). Upon being exposed to hypotonic solution (80% osmolality), the A6 cells swelled and peaked in the first 5 min, which was followed by a progressive decrease in cell volume termed regulatory volume decrease (RVD). Following the cell swelling, there were large increases in both outward- and inward-currents, which seemed to be carried by K+ efflux and Cl- efflux, respectively. A K+ channel blocker (TEA or quinine) or a Cl- channel blocker (NPPB or SITS) significantly inhibited both currents and RVD, suggesting that the inward- and outward-currents are highly correlated with each other and essential to RVD. Hypotonic stimulation also induced a transient [Ca2+]i increase, of which the time course was essentially similar to that of the currents. When internal and external Ca2+ were deprived to eliminate the Ca2+ transient increase, whole-cell currents and RVD were strongly inhibited. On the other hand, channel blockers TEA and NPPB, which inhibited whole-cell currents and RVD, did not inhibit the [Ca2+]i increase. It is concluded that hypotonic stimulation to A6 cells first induces cell swelling, which is followed by [Ca2+]i increase that leads to the coactivation of K+ and Cl- channels. This coactivation may accelerate K+ and Cl- effluxes, resulting in RVD.     

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.     

Ca2+ permeation in cyclic nucleotide-gated channels

Cyclic nucleotide-gated (CNG) channels conduct Na+, K+ and Ca2+ currents under the control of cGMP and cAMP. Activation of CNG channels leads to depolarization of the membrane voltage and to a concomitant increase of the cytosolic Ca2+ concentration. Several polypeptides were identified that constitute principal and modulatory subunits of CNG channels in both neurons and non-excitable cells, co-assembling to form a variety of heteromeric proteins with distinct biophysical properties. Since the contribution of each channel type to Ca2+ signaling depends on its specific Ca2+ conductance, it is necessary to analyze Ca2+ permeation for each individual channel type. We have analyzed Ca2+ permeation in all principal subunits of vertebrates and for a principal subunit from Drosophila melanogaster. We measured the fractional Ca2+ current over the physiological range of Ca2+ concentrations and found that Ca2+ permeation is determined by subunit composition and modulated by membrane voltage and extracellular pH. Ca2+ permeation is controlled by the Ca2+-binding affinity of the intrapore cation-binding site, which varies profoundly between members of the CNG channel family, and gives rise to a surprising diversity in the ability to generate Ca2+ signals.     

The differentiation of DSM-III-R psychotic depression in later life from nonpsychotic depression: comparisons of brain changes measured by multispectral analysis of magnetic resonance brain images, neuropsychological findings, and clinical features

P2U/2Y-receptors elicit multiple signaling in Madin-Darby canine kidney (MDCK) cells, including a transient increase of [Ca2+]i, activation of phospholipases C (PLC) and A2 (PLA2), protein kinase C (PKC) and mitogen-activated protein kinase (MAPK). This study examines the involvement of these signaling pathways in the inhibition of Na+,K+,Cl- cotransport in MDCK cells by ATP. The level of ATP-induced inhibition of this carrier ( approximately 50% of control values) was insensitive to cholera and pertussis toxins, to the PKC inhibitor calphostin C, to the cyclic nucleotide-dependent protein kinase inhibitors, H-89 and H-8 as well as to the inhibitor of serine-threonine type 1 and 2A phosphoprotein phosphatases okadaic acid. ATP led to a transient increase of [Ca2+]i that was abolished by a chelator of Ca2+i, BAPTA. However, neither BAPTA nor the Ca2+ ionophore A231287, or an inhibitor of endoplasmic reticulum Ca2+-pump, thapsigargin, modified ATP-induced inhibition of Na+,K+, Cl- cotransport. An inhibitor of PLC, U73122, and an inhibitor of MAPK kinase (MEK), PD98059, blocked ATP-induced inositol-1,4, 5-triphosphate production and MAPK phosphorylation, respectively. However, these compounds did not modify the effect of ATP on Na+,K+, Cl- cotransport activity. Inhibitors of PLA2 (AACOCF3), cycloxygenase (indomethacin) and lypoxygenase (NDGA) as well as exogenous arachidonic acid also did not affect ATP-induced inhibition of Na+,K+,Cl- cotransport. Inhibition of the carrier by ATP persisted in the presence of inhibitors of epithelial Na+ channels (amiloride), Cl- channels (NPPB) and Na+/H+ exchanger (EIPA) and was insensitive to cell volume modulation in anisosmotic media and to depletion of cells with monovalent ions, thus ruling out the role of other ion transporters in purinoceptor-induced inhibition of Na+,K+,Cl- cotransport. Our data demonstrate that none of the known purinoceptor-stimulated signaling pathways mediate ATP-induced inhibition of Na+,K+,Cl- cotransport and suggest the presence of a novel P2-receptor-coupled signaling mechanism.     

Role of natriuretic peptides in ion transport mechanisms

Natriuretic peptides (NP) act as ligands on the guanylyl cyclase family of receptors. The NP binding site on these receptors is extracellular and the guanylyl cyclase and protein kinase domains are intracellular. The guanylyl cyclase receptor catalyzes the synthesis of the second messenger molecule, cGMP, which activates protein kinase. This in turn is involved in the phosphorylation of various ion transport proteins. Ion transport proteins, which are modulated by NP and are thought to underlie the natriuretic and diuretic actions of NP, include: (a) calcium-activated K+ channels; (b) ATP-sensitive K+ channels; (c) inwardly-rectifying K+ channels; (d) outwardly-rectifying K+ channels; (e) L-type Ca2+ channels; (f) Cl- channels including cystic fibrosis transmembrane conductance regulator Cl- channels; (g) Na+- K+ 2Cl- co-transporter; (h) Na+- K+ ATPase; (i) Na+ channels; (j) stretch-activated channels; and (k) water channels. It appears that NP modulate the kinetics, rather than the conductance, of ion channels. Some of these channels, like the Ca2+, ATP-sensitive K+ and stretch-activated channels, are also involved in NP secretion. In addition, the structural properties of the NP, e.g., ovCNP-22 and ovCNP-39, appear to confer on them the ability to form ion channels. These CNP-formed ion channels can modify the trans-membrane signal transduction and second messenger systems underlying NP-induced pathological effects.     

Use of an albumin-binding domain for the selective immobilisation of recombinant capture antibody fragments on ELISA plates

Human embryonic kidney cells (HEK 293) are widely used as an expression system in studies of ion channels. However, their endogenous ionic currents remain largely unidentified. To characterize these currents, we performed patch clamp experiments on this expression system. In whole-cell voltage clamp mode, the HEK 293 cells showed mainly outward currents using physiological concentrations of Na+ and K+ and symmetric concentrations of Cl- (150 mM) across the plasma membranes. K+ currents contributed to a small portion of these outward currents, since a shift of the reversal potentials of only approximately 20 mV was seen with a change of extracellular K+ concentration from 3 to 150 mM. In contrast, the reversal potential shifted approximately 25 mV when extracellular Cl- was reduced to 50 mM, indicating that most of the outward currents are carried by Cl-. In inside-out patches, several distinct Cl- currents were identified. They were: (1) 350 pS Cl- current, which was voltage-activated and had a moderate outward rectification; (2) 240 pS Cl- current with a weak outward rectification; and (3) 55 pS Cl- current, which was voltage-activated, sensitive to DIDS, and showed a strong outward rectification. Activation of these Cl- currents did not require an elevation of free Ca2+ level in the cytosol. Besides these three currents, we observed two other Cl- currents with much smaller conductances (25 and 16 pS, respectively). Two different K+ currents were seen in the HEK 293 cells, with one of them (125 pS) showing inward rectification and the other (70 pS) outward rectification. Moreover, a 50 pS cation channel was recorded in these cells. The presence of a variety of ion channels in the HEK 293 cells suggests that a great precaution needs to be taken when this expression system is used in studies of several similar ion channels.     

Control of ionic currents in guard cell vacuoles by cytosolic and luminal calcium

Activity of vacuolar ion channels can be regulated by the cytosolic free Ca2+ concentration ([Ca2+]cyt). Using the whole-vacuole mode of patch-clamp with Vicia faba guard cell vacuoles, three distinct cation currents were apparent that were differentially regulated by [Ca2+]cyt. At 'zero' to 100 nM [Ca2+]cyt, instantaneous currents typical of Fast Vacuolar (FV) channels were activated. A 10 fold KCl gradient directed out of the vacuole increased FV currents (up to fivefold) at negative potentials compared with the currents in symmetrical KCl. At [Ca2+]cyt higher than 100 nM, instantaneous currents became smaller and voltage-independent (non-rectifying) and were typical of Vacuolar K(+)-selective (VK) channels. These currents were less sensitive to a KCl gradient than were the FV currents, being stimulated less than twofold at negative potentials. Reversal potentials measured in the presence of a KCl gradient indicated a high K+ permeability of both FV and VK currents. At [Ca2+]cyt higher than 600 nM time-dependent currents elicited by positive potentials were typical of Slow Vacuolar (SV) channel activation. When the Ca2+ mole fraction in the cytosolic or luminal solution was varied the reversal potential of SV currents (determined by tail current analysis) passed through maximum or minimum values. The resultant calculated apparent permeability ratios varied with ionic conditions but indicated high Ca2+ and K+ permeabilities. If a Cl- permeability was assumed then the apparent PCa was lower. However, substitution of Cl- by the larger (impermeant) anion gluconate had no effect on the reversal potential of SV tail currents in the presence of Ca2+ and a K+ gradient, demonstrating that the assumption of Cl- permeability of the SV channel is invalid. Single-channel SV currents also decreased with increasing cytosolic Ca2+ mole fraction. These data indicate that the SV channel is highly cation selective, shows characteristics typical of a multi-ion pore and derives ion selectivity by Ca2+ binding. The SV channel currents could also be Mg(2+)-activated and were demonstrated to be Mg(2+)-permeable in the absence of Ca2+. The apparent permeability ratio (PMg:PK) also varied under different ionic conditions. The results indicate not only that FV, VK and SV channels are all present in a single cell type, but also that each is differentially regulated by [Ca2+]cyt. The respective roles of these channels in vacuolar ion release are discussed, and possible conditions are presented in which these channels could be activated by disparate signalling pathways during stomatal closure.     

Pituitary adenylate cyclase-activating polypeptide causes rapid Ca2+ release from intracellular stores and long lasting Ca2+ influx mediated by Na+ influx-dependent membrane depolarization in bovine adrenal chromaffin cells

Pituitary adenylate cyclase-activating polypeptide (PACAP) has been reported to increase intracellular Ca2+ concentrations ([Ca2+]i) and catecholamine release in adrenal chromaffin cells. We measured [Ca2+]i with fura-2 and recorded ion currents and membrane potentials with the whole cell configuration of the patch-clamp technique to elucidate the mechanism of PACAP-induced [Ca2+]i increase in bovine adrenal chromaffin cells. PACAP caused [Ca2+]i to increase due to Ca2+ release and Ca2+ influx, and this was accompanied by membrane depolarization and inward currents. The Ca2+ release was suppressed by ryanodine, an inhibitor of caffeine-sensitive Ca2+ stores, but was unaffected by cinnarizine, an inhibitor of inositol trisphosphate-induced Ca2+ release. Ca2+ influx and inward currents were both inhibited by replacement of extracellular Na+, and Ca2+ influx was inhibited by nicardipine, an L-type Ca2+ channel blocker, or by staurosporine, a protein kinase C (PKC) inhibitor, but was unaffected by a combination of omega- conotoxin-GVIA, omega-agatoxin-IVA, and omega-conotoxin- MVIIC, blockers of N-, P-, and Q-type Ca2+ channels. Moreover, 1-oleoyl-2-acetyl-sn-glycerol, a PKC activator, induced inward currents and Ca2+ influx. These results indicate that PACAP causes both Ca2+ release, mainly from caffeine-sensitive Ca2+ stores, and Ca2+ influx via L-type Ca2+ channels activated by membrane depolarization that depends on PKC-mediated Na+ influx.     

A novel small conductance Ca2+-activated K+ channel blocker from Oxyuranus scutellatus taipan venom. Re-evaluation of taicatoxin as a selective Ca2+ channel probe

Taicatoxin, isolated from the venom of the Australian taipan snake Oxyuranus scutellatus, has been previously regarded as a specific blocker of high threshold Ca2+ channels in heart. Here we show that taicatoxin (in contrast to a range of other Ca2+ channel blockers) interacts with apamin-sensitive, small conductance, Ca2+-activated potassium channels on both chromaffin cells and in the brain. Taicatoxin displays high affinity recognition of 125I-apamin acceptor-binding sites, present on rat synaptosomal membranes (Ki = 1.45 +/- 0.22 nM) and also specifically blocks affinity-labeling of a 33-kDa 125I-apamin-binding polypeptide on rat brain membranes. Taicatoxin (50 nM) completely blocks apamin-sensitive after-hyperpolarizing slow tail K+ currents generated in rat chromaffin cells (mean block 97 +/- 3%, n = 12) while only partially reducing total voltage-dependent Ca2+ currents (mean block 12 +/- 4%, n = 6). In view of these findings, the use of taicatoxin as a specific ligand for Ca2+ channels should now be reconsidered.     

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.     

Intracellular calcium, currents, and stimulus-response coupling in endothelial cells

Vascular endothelium appears to be a unique organ. It not only responds to numerous hormonal and chemical signals but also senses changes in physical parameters such as shear stress, producing mediators that modulate the responses of numerous cells, including vascular smooth muscle, platelets, and leukocytes. In many cases, the initial response of endothelial cells to these diverse signals involves elevation of cytosolic Ca2+ and activation of Ca(2+)-dependent enzymes, including nitric oxide synthase and phospholipase A2. Both the release of Ca2+ from intracellular stores, most likely the endoplasmic reticulum, and the influx of Ca2+ from the extracellular space contribute to the [Ca2+]i increase. The most important trigger for Ca2+ release is inositol 1,4,5-trisphosphate, which is generated by the action of phospholipase C, a plasmalemmal enzyme activated in many cases by the receptor-G protein cascade. Ca2+ influx appears to be related to the activity of receptor-G protein-enzyme complex and to the degree of fullness of the endoplasmic reticulum but does not involve voltage-gated Ca2+ channels. The magnitude of the Ca2+ influx depends on the electrochemical gradient, which is modulated by the membrane potential, Vm. Under basal conditions, Vm is dominated by a large inward rectifier K+ current. Some stimuli, e.g., acetylcholine, have been shown to hyperpolarize Vm, thus increasing the electrochemical gradient for Ca2+, which appears to be modulated by activation of Ca(2+)-dependent K+ and Cl- currents. However, the lack of potent and specific blockers for many of the described or postulated channels (e.g., nonselective cation channel, Ca(2+)-activated Cl- channel) makes an estimation of their effect on endothelial cell function rather difficult. Possible future directions of research and clinical implications are discussed.     

Calcium electrogenesis in neocortical pyramidal neurons in vivo

Much of what is known about Ca2+ electrogenesis in neocortical cells has been derived from in vitro studies. Since Ca2+ currents are controlled by various modulators, comparing these findings to in vivo data is essential. Here, we analysed tetrodotoxin (TTX)-resistant, presumably Ca2+-mediated potentials in intracellularly recorded neocortical neurons in vivo. TTX was applied locally to block Na+ channels. Its effectiveness was demonstrated by the elimination of fast spikes and orthodromic responses. In response to depolarizing current pulses bringing the membrane potential beyond approximately -33 mV, 71% of neurons generated high-threshold Ca2+ spikes averaging 17 mV. This is in contrast with in vitro findings, where high-threshold spikes could only be elicited following the blockade of K+ conductances. Consistent with this, neurons dialysed with K+ channel blockers in vivo generated high-threshold spikes that had a lower threshold (approximately -40 mV) and, with intracellular Cs+, a larger amplitude, indicating the presence of K+ currents opposing the activation of Ca2+ channels. Only 15% of cortical cells displayed low-threshold Ca2+ spikes. To compare high-threshold Ca2+ spikes evoked by synaptic stimuli or current injection, another group of cortical neurons was dialysed with QX-314 and Cs+, in the absence of extracellular TTX. Synaptic stimuli applied on a background of membrane depolarization elicited presumed Ca2+ spikes whose amplitude varied in a stepwise fashion. Thus, although there are numerous similarities between in vivo and in vitro data, some significant differences were found, which suggest that the high-voltage activated Ca2+ currents and/or the K+ conductances that oppose them are subjected to different modulatory influences in vivo than in vitro.     

Pathways of Rb+ influx and their relation to intracellular [Na+] in the perfused rat heart. A 87Rb and 23Na NMR study

The aims of this study were to characterize the routes of influx of the K+ congener, Rb+, into cardiac cells in the perfused rat heart and to evaluate their links to the intracellular Na+ concentration ([Na+]i) using 87Rb and 23Na nuclear magnetic resonance (NMR) spectroscopy. The rate constant for Rb+ equilibration in the extracellular space was 8.5 times higher than that for the intracellular space. The sensitivity of the rate of Rb+ accumulation in the intracellular space of the perfused rat heart to the inhibitors of the K+ and Na+ transport systems has been analyzed. The Rb+ influx rates were measured in both beating and arrested hearts: both procaine (5 mmol/L) and lidocaine (1 mmol/L) halved the Rb+ influx rate. In procaine-arrested hearts, the Na+,K(+)-ATPase inhibitor ouabain (0.6 mmol/L) decreased Rb+ influx by 76 +/- 24% relative to that observed in untreated but arrested hearts. Rb+ uptake was insensitive to the K+ channel blocker 4-aminopyridine (1 mmol/L). The inhibitor of Na+/K+/2 Cl- cotransport bumetanide (30 mumol/L) decreased Rb+ uptake only slightly (by 9 +/- 8%). Rb+ uptake was dependent on [Na+]i: it increased by 58 +/- 34% when [Na+]i was increased with the Na+ ionophore monensin (1 mumol/L) and decreased by 48 +/- 9% when [Na+]i was decreased by the Na+ channel blockers procaine and lidocaine. Dimethylamiloride (15 to 20 mumol/L), an inhibitor of the Na+/H+ exchanger, slightly reduced [Na+]i and Rb+ entry into the cardiomyocytes (by 15 +/- 5%). 31P NMR spectroscopy was used to monitor the energetic state and intracellular pH (pHi) in a parallel series of hearts. Treatment of the hearts with lidocaine, 4-aminopyridine, dimethylamiloride, or bumetanide for 15 to 20 minutes at the same concentrations as used for the Rb+ and Na+ experiments did not markedly affect the levels of the phosphate metabolites or pHi. These data show that under normal physiological conditions, Rb+ influx occurs mainly through Na+,K(+)-ATPase; the contribution of the Na+/K+/2 Cl- cotransporter and K+ channels to Rb+ influx is small. The correlation between Rb+ influx and [Na+bdi during infusion of drugs that affect [Na+]i indicates that, in rat hearts at 37 degrees C, Rb+ influx can serve as a measure of Na+ influx. We estimate that, at normothermia, at least 50% of the Na+ entry into beating cardiac cells is provided by the Na+ channels, with only minor contributions (< 15%) from the Na+/K+/2 Cl- cotransporter and the Na+/H+ exchanger.     

Neuronal synchronization and ionic mechanisms for propagation of excitation in the functional network of immortalized GT1-7 neurons: optical imaging with a voltage-sensitive dye

Immortalized gonadotropin releasing hormone (GnRH) neurons (GT1 cell line) in culture release GnRH in a pulsatile manner, suggesting that GT1 cells form a functional neuronal network. Optical imaging techniques and a voltage-sensitive fluorescent dye (RH795) were used to study the mechanism of neuronal synchronization and intercellular communication in cultured GT1-7 cells (one of the subclones of the GT1 cell line). The majority (79%) of GT1-7 cells in contact with one another revealed synchronized fluctuations in spontaneous neuronal activity. When a cell in contact with other cells was electrically stimulated, the evoked excitation was propagated to neighbouring cells. The ionic mechanisms involved in the propagation of electrical signals between interconnected GT1-7 cells were investigated using various blockers of Na+, Ca2+ and K+ channels. The propagation of stimulus-evoked excitation was prevented by the voltage-dependent Na+ channel blocker tetrodotoxin. It was also prevented by the voltage-dependent Ca2+ channel blockers, Ni+ (nonselective), nimodipine (L-type) and flunarizine (T-type > L-type), but not apparently affected by omega-agatoxin IVA (P- and Q-type) and omega-conotoxin MVIIA (N-type). The propagation was not influenced by the K+ channel blockers, quinine, tetraethylammonium and Ba2+, but in some cases, it was enhanced by 4-aminopyridine (4-AP) and prevented by apamin. These results suggest that voltage-dependent Na+ channels and L- and T-type Ca2+ channels are involved in the propagation of electrical signals in the GT1-7 neuronal network. Ionic mechanisms, through 4-AP- or apamin-sensitive K+ channels, also seem to be involved in the regulation of signal propagation. These mechanisms may underlie the functioning of the neuronal network formed by immortalized GnRH neurons.     

Analysis of leak current properties in the lobster stretch receptor neurone

Experiments were performed to characterize the so-called leak current of the slowly adapting stretch receptor neurone of the European lobster with respect to its ionic basis, its kinetics and its pharmacology. Estimates of the leak current were obtained by subtraction of a Na-K pump current and of an unspecific impalement current from a non-dynamic ('instantaneous') current, recorded in a voltage range from approximately -120 to approximately -30 mV, after blockage of spike-generating currents and a hyperpolarization-activated inwardly rectifying current (Q-current). The leak current, estimated in this way, was seen to reverse direction at the cell's K+ equilibrium voltage, thus indicating that it is carried by K+ passing through channels which, also, proved to be permeable to Rb+ and NH4+, but not permeable to Na+ or Cl- to any significant extent. Kinetically, the leak current was found to be characterized by being enhanced by increases in extracellular K+ and by being subject to outward rectification, most distinctly at elevated extracellular [K+]. In quantitative terms, these kinetic properties could be accounted for by a mathematical model comprising (1) a one-site two-barrier Eyring formulation describing ion permeation through membrane channels and (2) an ordinary dose-response relationship describing the channel-opening effect of K+ at an extracellular regulatory site. Pharmacologically, the leak current proved to be distinguished by being reversibly blockable, in a non-voltage dependent manner, by CO2+ (Kd = 0.9 mM, Hill coefficient 1.1) and procaine, but not by Ba2+, Gd3+, bupivacaine (a local anesthetic), or other K+ channel blockers such as TEA, 4-AP and Cs+. It is concluded that, in native unimpaled cells, the K+ carried leak current (1) is setting the resting voltage together with the (mainly) Na(+)-carried Q-current and the Na-K pump current, (2) is determining the cell's firing threshold, together with the spike generating currents, and (3) is also stabilizing the cell's membrane excitability in conditions of varying extracellular [K+], by virtue of its K+ sensitivity.