Characterization of voltage-gated ionic channels in cholinergic amacrine cells in the mouse retina

Recent studies have shown that cholinergic amacrine cells possess unique membrane properties. However, voltage-gated ionic channels in cholinergic amacrine cells have not been characterized systematically. In this study, using electrophysiological and immunohistochemical techniques, we examined voltage-gated ionic channels in a transgenic mouse line the cholinergic amacrine cells of which were selectively labeled with green fluorescent protein (GFP). Voltage-gated K(+) currents contained a 4-aminopyridine-sensitive current (A current) and a tetraethylammonium-sensitive current (delayed rectifier K(+) current). Voltage-gated Ca(2+) currents contained a omega-conotoxin GVIA-sensitive component (N-type) and a omega-Aga IVA-sensitive component (P/Q-type). Tetrodotoxin-sensitive Na(+) currents and dihydropyridine-sensitive Ca(2+) currents (L-type) were not observed. Immunoreactivity for the Na channel subunit (Pan Nav), the K channel subunits (the A-current subunits [Kv. 3.3 and Kv 3.4]) and the Ca channel subunits (alpha1(A) [P/Q-type], alpha1(B) [N-type] and alpha1(C) [L-type]) was detected in the membrane fraction of the mouse retina by Western blot analysis. Immunoreactivity for the Kv. 3.3, Kv 3.4, alpha1(A) [P/Q-type], and alpha1(B) [N-type] was colocalized with the GFP signals. Immunoreactivity for alpha1(C) [L-type] was not colocalized with the GFP signals. Immunoreactivity for Pan Nav did not exist on the membrane surface of the GFP-positive cells. Our findings indicate that signal propagation in cholinergic amacrine cells is mediated by a combination of two types of voltage-gated K(+) currents (the A current and the delayed rectifier K(+) current) and two types of voltage-gated Ca(2+) currents (the P/Q-type and the N-type) in the mouse retina.     

Role of L-type Ca2+ channels in transmitter release from mammalian inner hair cells. II. Single-neuron activity

Previously reported changes in the gross sound-evoked cochlear potentials after intracochlear perfusion of nimodipine suggest that dihydropyridine-sensitive Ca2+ channels (L-type) control the sound-evoked release of transmitter from the inner hair cells of the mammalian cochlea. In the present study, we combined recording of the action potentials of single primary auditory afferent neurons with intracochlear perfusion to further investigate the role of voltage-gated Ca2+ channels at this synapse. Spontaneous action potential firing rates were depressed by the L-type channel blocker nimodipine, but were elevated by S(-) BAY K8644, an L-type channel agonist. Sound-evoked responses of single primary afferents were depressed by nimodipine in a manner that was consistent with a block at the inner hair cell-afferent dendrite synapse. Perfusions with solutions containing the N-type channel blocker conotoxin GVIA did not differ in their effects from control artificial perilymph perfusions. The results extend the conclusions of the earlier study by showing that L-type Ca2+ channels are primarily responsible for controlling both spontaneous and sound-evoked transmitter release from inner hair cells. In addition it was found that afferent neurons with widely different spontaneous firing rates were all sensitive to nimodipine and to BAY K8644, suggesting that the multiple synaptic outputs of each inner hair cell are under the control of only one major type of Ca2+ channel.     

BK channel activation by brief depolarizations requires Ca2+ influx through L- and Q-type Ca2+ channels in rat chromaffin cells.

BK channel activation by brief depolarizations requires Ca2+ influx through L- and Q-type Ca2+ channels in rat chromaffin cells. Ca2+- and voltage-dependent BK-type K+ channels contribute to action potential repolarization in rat adrenal chromaffin cells. Here we characterize the Ca2+ currents expressed in these cells and identify the Ca2+ channel subtypes that gate the activation of BK channels during Ca2+ influx. Selective Ca2+ channel antagonists indicate the presence of at least four types of high-voltage-gated Ca2+ channels: L-, N-, P, and Q type. Mean amplitudes of the L-, N-, P-, and Q-type Ca2+ currents were 33, 21, 12, and 24% of the total Ca2+ current, respectively. Five-millisecond Ca2+ influx steps to 0 mV were employed to assay the contribution of Ca2+ influx through these Ca2+ channels to the activation of BK current. Blockade of L-type Ca2+ channels by 5 microM nifedipine or Q-type Ca2+ channels by 2 microM Aga IVA reduced BK current activation by 77 and 42%, respectively. In contrast, blockade of N-type Ca2+ channels by brief applications of 1-2 microM CnTC MVIIC or P-type Ca2+ channels by 50-100 nM Aga IVA reduced BK current activation by only 11 and 12%, respectively. Selective blockade of L- and Q-type Ca2+ channels also eliminated activation of BK current during action potentials, whereas almost no effects were seen by the selective blockade of N- or P-type Ca2+ channels. Finally, the L-type Ca2+ channel agonist Bay K 8644 promoted activation of BK current by brief Ca2+ influx steps by more than twofold. These data show that, despite the presence of at least four types of Ca2+ channels in rat chromaffin cells, BK channel activation in rat chromaffin cells is predominantly coupled to Ca2+ influx through L- and Q-type Ca2+ channels.     

Calcium-dependent inactivation of neuronal calcium channel currents is independent of calcineurin

Dephosphorylation by the Ca2+/calmodulin-dependent phosphatase calcineurin has been suggested as an important mechanism of Ca2+-dependent inactivation of voltage-gated Ca2+ channels. We have tested whether calcineurin plays a role in the inactivation process of two types of high-voltage-activated Ca2+ channels (L and N type) widely expressed in the central nervous system, using the immunosuppressive drug FK506 (tacrolimus), which inhibits calcineurin after binding to intracellular FK506 binding proteins. Inactivation of L- and N-type Ca2+ channels was studied in a rat pituitary tumor cell line (GH3) and chicken dorsal root ganglion neurons, respectively. With the use of antisera directed against the calcineurin subunit B and the 12,000 mol. wt binding protein, we show that both proteins are present in the cytoplasm of GH3 cells and chicken dorsal root ganglion neurons. Ionic currents through voltage-gated Ca2+ channels were investigated in the perforated-patch and whole-cell configurations of the patch-clamp technique. The inactivation of L- as well as N-type Ca2+ currents could be well fitted with a bi-exponential function. Inactivation was largely reduced when Ba2+ substituted for extracellular Ca2+ or when the Ca2+ chelator EGTA was present intracellularly, indicating that both types of Ca2+ currents exhibited Ca2+-dependent inactivation. Extracellular (perforated-patch configuration) or intracellular (whole-cell configuration) application of FK506 to inactivate calcineurin had no effect on the amplitude and time-course of Ca2+ channel current inactivation of either L- or N-type Ca2+ channels. In addition, we found that recovery from inactivation and rundown of N-type Ca2+ channel currents were not affected by FK506. Our results provide direct evidence that the calcium-dependent enzyme calcineurin is not involved in the inactivation process of the two Ca2+ channel types which are important for neuronal functioning, such as gene expression and transmitter release.     

Reconstitution of neurotransmission by determining communication between differentiated PC12 pheochromocytoma and HEL 92.1.7 erythroleukemia cells

Neurotransmitter release was monitored using fura-2-loaded HEL 92.1.7 cells dispersed among differentiated PC12 cells (loaded with another Ca2+ indicator fluo-3) and immobilised using transparent polycarbonate membrane filters with uniform pore size. Depolarisation with K+ caused a rapid rise in Ca2+ concentration in the PC12 cells, followed by a delayed secondary Ca2+ response in simultaneously monitored nearby HEL cells. There was a lag period of about 20 s between the responses of the two cell types. Voltage-gated Ca2+ channels in PC12 cells were inhibited by the P/Q-type (omega-conotoxin MVIIC, omega-agatoxin IVA), N-type (omega-conotoxin GVIA) and L-type channel blockers (nifedipine) as determined using fura-2 or whole-cell patch-clamp recordings. The communication between the cell types on the other hand was sensitive to P/Q- and N-type but not to L-type channel blockers. This suggests that, as in neurons, P/Q- and N-type Ca2+ channels mediate the release of neurotransmitters acting on HEL cells. Theoretically, the procedure employed should be sensitive enough to detect single exocytotic events. Our results demonstrate that a random distribution between effector and target cells is sufficient to allow communication between cells in a manner similar to extrasynaptic transmission.     

Protein kinase C is involved in neurokinin receptor modulation of N- and L-type Ca2+ channels in DRG neurons of the adult rat

Whole cell patch-clamp techniques were used to examine neurokinin receptor modulation of Ca2+ channels in small to medium size dorsal root ganglia neurons (<40 pF) that express mainly N- and L-type Ca2+ currents. Low concentrations of substance P enhanced Ca2+ currents (5-40%, <0.2 microM), while higher concentrations applied cumulatively reversed these enhancements (5-28% reductions, >0.5 microM). This apparent inhibition by high concentrations of substance P was blocked by the administration of the NK3 antagonist SB 235,375 (0.2 microM). The NK1 agonist, [Sar9,Met11]-substance P (0.05 to 1.0 microM) did not alter Ca2+ currents; whereas the NK2 agonist, [betaAla8]-neurokinin A (4-10), enhanced Ca2+ currents (5-36% increase, 0.05-0.5 microM). The enhancement was reversed by the NK2 antagonist MEN 10,376 (0.2 microM) but unaffected by the NK3 antagonist SB 235,375 (0.2 microM). The NK3 agonist [MePhe7]-neurokinin B (0.5-1.0 microM) inhibited Ca2+ currents (6-24% decrease). This inhibition was not prevented by the NK2 antagonist MEN 10,376 (0.2 microM) but was blocked by the NK3 antagonist SB 235,375 (0.2 microM). Both the enhancement and inhibition of Ca2+ currents by neurokinin agonists were reversed by the protein kinase C inhibitor bisindolylmaleimide I HCl (0.2-0.5 microM). Following inhibition of Ca2+ channels by [MePhe7]-neurokinin the facilitatory effect of BayK 8644 (5 microM) was increased and the inhibitory effect of the N-type Ca2+ channel blocker w -conotoxin GVIA (1 microM) was diminished, suggesting that the NK3 agonist inhibits N-type Ca2+ channels. Similarly, block of all but N-type Ca2+ channels, revealed that [betaAla8]-neurokinin A (4-10) enhanced the currents while [MePhe7]-neurokinin B inhibited the currents. Inhibition of all but L-type Ca2+ channels, revealed that [betaAla8]-neurokinin A (4-10) enhanced the currents while [MePhe7]-neurokinin B had no effect. Activation of protein kinase C with low concentrations of phorbol-12,13-dibutyrate enhanced Ca2+ currents, but high concentrations inhibited N- and L-type Ca2+ currents. In summary, these data suggest that in adult rat dorsal root ganglia neurons, NK2 receptors enhance both L- and N-type Ca2+ channels and NK3 receptors inhibit N-type Ca2+ channels and that these effects are mediated by protein kinase C phosphorylation of Ca2+ channels.     

Autoradiography of L-type and N-type calcium channels in aged rat hippocampus, entorhinal cortex, and neocortex

The present study examined brains from 6, 17, and 32 month old male (F344x BN)F1 rats to determine whether there was any age-related change in the distribution or density of L-type and N-type Ca2+ channels in hippocampus, entorhinal cortex, and neocortex, areas commonly involved in the generation of epileptic seizures. The L-type channel antagonist PN200-110 and the N-type channel antagonist omega-conotoxin GVIA were used to determine specific binding densities and the autoradiographic distribution of ligand binding was quantified by computer-assisted densitometry. One-way ANOVA noted a significant variance in the mean value of binding density between different age groups only in neocortex laminae IV-VI for [(3)H]PN200-110 binding (P < 0.05). Post-hoc testing indicated that the mean value of the 17 month old group was significantly less than those of the 6 and 32 month old groups (P < 0.05). These results indicate no overall age-related change in the number of L-type and N-type Ca2+ channels in brain areas frequently involved in seizure activity and suggest that age-related changes in brain Ca2+ physiology may be associated with changes in voltage-gated Ca2+ channel function rather than channel number.     

Pharmacology of N-type Ca2+ channels distributed in cardiovascular system (Review)

Irregular functions in Ca2+ channels are intimately involved in many aspects of cardiovascular diseases. We can obtain a wide variety of L-type Ca2+ channel antagonists to treat hypertension and angina pectoris. Dihydropyridines (DHPs) have, first of all, been extensively developed due to their high selectivity for L-type Ca2+ channel and safety in pharmacological aspects. In contrast, many lines of evidence suggest that clinical efficacy of those DHPs are limited and undesirable effects are sometimes observed because of the specific distribution of L-type Ca2+ channels. As well as the L-type, peripherally distributed N-type Ca2+ channel plays a key role in cardiovascular regulation through autonomic nervous system. Recently, we developed a unique DHP derivative, cilnidipine (FRC8653) which has a dual antagonistic action on both L-type and N-type Ca2+ channels. Our recent studies with this DHP have made it clear that the N-type Ca2+ channel is also a new therapeutic target in cardiovascular diseases. We review the recent advances in pharmacology of the N-type Ca2+ channel and therapeutic implications of their antagonists.     

K(+)-evoked dopamine release depends on a cytosolic Ca2+ pool regulated by N-type Ca2+ channels.

Membrane depolarization evoked by 25-40 mM K+ elicited an immediate increase of somatic and neuritic [Ca2+]i in cultured dopaminergic neurons as measured by digital fluorescence microscope imaging. The rise of neuritic [Ca2+]i was inhibited by N-type but not L-type Ca2+ channel blockers, while the rise of somatic [Ca2+]i was prevented by both L- and N-type Ca2+ channel blockers. Similarly, depolarization-induced [3H]dopamine release was selectively attenuated by N-type Ca2+ channel blockers. The present results suggest that [3H]dopamine release from mesencephalic neuronal cell cultures relates to a Ca(2+)-dependent mechanism regulated by N-type channels located in the vicinity of the exocytotic sites within neuritic processes.     

Functional roles of ion channels in lymphocytes.

The application of patch-clamp and video-imaging techniques has enabled responses of lymphocytes to be examined at the level of individual cells. Eight distinct types of ion channel activity have been revealed in T lymphocytes. A variety of external stimuli shifts the pattern of channel activity from the resting state, which is dominated by voltage-gated K+ channels. Channel regulation is achieved both by acute modulation and by altered expression in the membrane. During mitogen stimulation, Ca2+ channels and Ca(2+)-activated K+ channels become active and appear to underlie Ca2+ oscillations. These acute changes are followed by increased expression of voltage-gated K+ channels. In response to osmotic challenge in hypotonic media, cell swelling initiates activation of Cl- channels, which may, in turn, indirectly activate K+ channels and trigger a regulatory decrease in cell volume.     

Hypotonic swelling increases L-type calcium current in smooth muscle cells of the human stomach

The purpose of the present study was to characterize the Ca2+ channels in smooth muscle cells from human stomach and to examine the effects of osmotic swelling on the channel activity. Ca2+ channel current with either Ca2+ or Ba2+ as charge carrier was recorded from freshly isolated smooth muscle cells using the conventional whole-cell patch clamp technique. The degree of cell swelling as a result of hypotonic challenge was monitored using a video image analysis system. The changes in intracellular Ca2+ concentration ([Ca2+]i) were measured by microfluorimetry. The pharmacological and voltage activation profile suggests a typical dihydropyridine-sensitive L-type Ca2+ current. Cell swelling, induced by hypotonic challenge, enhanced the amplitude of currents through L-type Ca2+ channels without significant effects on steady-state voltage dependency. After treatment with the L-type Ca2+ channel agonist Bay K 8644 (0.1-2 microM), no further significant increase in calcium channel current or corresponding [Ca2+]i transients were provoked by the swelling. The above results demonstrated that the presence of L-type Ca2+ current in smooth muscle cells of the human stomach and the augmentation of the current are closely associated with the volume increase resulting from hypotonic swelling.     

Expression of recombinant calcium channels support secretion in a mouse pheochromocytoma cell line

We have characterized a recently established mouse pheochromocytoma cell line (MPC 9/3L) as a useful model for studying neurotransmitter release and neuroendocrine secretion. MPC 9/3L cells express many of the proteins involved in Ca2+-dependent neurotransmitter release but do not express functional endogenous Ca2+-influx pathways. When transfected with recombinant N-type Ca2+ channel subunits alpha1B,beta2a,alpha2delta (Cav2.2), the cells expressed robust Ca2+ currents that were blocked by omega-conotoxin GVIA. Activation of N-type Ca2+ currents caused rapid increases in membrane capacitance of the MPC 9/3L cells, indicating that the Ca2+ influx was linked to exocytosis of vesicles similar to that reported in chromaffin or PC12 cells. Synaptic protein interaction (synprint) sites, like those found on N-type Ca2+ channels, are thought to link voltage-dependent Ca2+ channels to SNARE proteins involved in synaptic transmission. Interestingly, MPC 9/3L cells transfected with either LC-type (alpha1C, beta2a, alpha2delta, Cav1.2) or T-type (alpha1G, beta2a, alpha2delta, Cav3.1) Ca2+ channel subunits, which do not express synprint sites, expressed appropriate Ca2+ currents that supported rapid exocytosis. Thus MPC 9/3L cells provide a unique model for the study of exocytosis in cells expressing specific Ca2+ channels of defined subunit composition without complicating contributions from endogenous channels. This model may help to distinguish the roles that different Ca2+ channels play in Ca2+-dependent secretion.     

Reciprocal synaptic interactions between rod bipolar cells and amacrine cells in the rat retina.

Reciprocal synaptic transmission between rod bipolar cells and presumed A17 amacrine cells was studied by whole cell voltage-clamp recording of rod bipolar cells in a rat retinal slice preparation. Depolarization of a rod bipolar cell evoked two identifiable types of Ca2+ current, a T-type current that activated at about -70 mV and a current with L-type pharmacology that activated at about -50 mV. Depolarization to greater than or equal to -50 mV also evoked an increase in the frequency of postsynaptic currents (PSCs). The PSCs reversed at approximately ECl (the chloride equilibrium potential), followed changes in ECl, and were blocked by gamma-aminobutyric acidA (GABAA) and GABAC receptor antagonists and thus were identified as GABAergic inhibitory PSCs (IPSCs). Bipolar cells with cut axons displayed the T-type current but lacked an L-type current and depolarization-evoked IPSCs. Thus L-type Ca2+ channels are placed strategically at the axon terminals to mediate transmitter release from rod bipolar cells. The IPSCs were blocked by the non-N-methyl-D-aspartate (non-NMDA) receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione, indicating that non-NMDA receptors mediate the feed-forward bipolar-to-amacrine excitation. The NMDA receptor antagonist 3-((RS)-2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid had no consistent effect on the depolarization-evoked IPSCs, indicating that activation of NMDA receptors is not essential for the feedforward excitation. Tetrodotoxin (a blocker of voltage-gated Na+ channels) reversibly suppressed the reciprocal response in some cells but not in others, indicating that graded potentials are sufficient for transmitter release from A17 amacrine cells, but suggesting that voltage-gated Na+ channels, under some conditions, can contribute to transmitter release.     

Ethanol blocks cytosolic Ca2+ responses triggered by activation of GABA(A) receptor/Cl- channels in cultured proliferating rat neuroepithelial cells.

GABA(A) receptor/Cl- channels and voltage-gated Ca2+ channels are believed to be important sites of ethanol action in the CNS. Acute exposure of ethanol potentiates GABA(A) receptor/Cl- channel activity and inhibits voltage-gated Ca2+ channels in a number of preparations, mostly post-mitotic neurons. The effects of ethanol on these channels in primary cultures of undifferentiated neural precursor cells remain unknown. To address this issue, we examined the effects of ethanol on GABA(A) agonist-activated elevation of cytosolic Ca2+ in an in vitro model of the cortical neuroepithelium derived from rat basic fibroblast growth factor-expanded neural precursor cells. We found a potent inhibition of GABA(A)-activated elevation of cytosolic Ca2+ by ethanol in actively proliferating cells. Since we had recently demonstrated that GABA(A) receptor activation depolarizes these cells and elevates their cytosolic Ca2+, we tested whether the effects of ethanol involved both GABA(A) receptors and voltage-gated Ca2+ channels. Both extracellular K+- and muscimol-induced cytosolic Ca2+ elevations were abolished by nitrendipine, indicating that both depolarizing stimuli triggered Ca2+ influx through L-type voltage-gated Ca2+ channels. Exposure of proliferating cells to different concentrations of ethanol revealed that the drug was more potent in blocking muscimol-induced compared to K+-evoked cytosolic Ca2+ elevations.These results raise the possibility that ethanol blocks GABAergic stimulation of cytosolic Ca2+ levels in proliferating precursors primarily by interacting with GABA(A) receptor/Cl- channels and secondarily with voltage-gated Ca2+ channels.     

Computer simulations of N-methyl-D-aspartate receptor-induced membrane properties in a neuron model.

1. To evaluate the role of N-methyl-D-aspartate (NMDA) receptors in simulations of the lamprey spinal locomotor network, we developed a computer-simulated electrical model of a neuron that contains NMDA channels in addition to voltage-gated Na+, K+, and Ca2+ channels and Ca(2+)-activated K+ channels [K(Ca) channels]. 2. The voltage dependence of the Mg2+ block of the Na(+)-K+ current flow through the NMDA channel was modeled according to a scheme of open-channel block. To account for the regulation of K(Ca) channels by NMDA and membrane voltage, we modeled two separate Ca2+ pools that had different voltage dependencies and dynamics. 3. Pacemaker-like membrane potential oscillations could be elicited in the model neuron, which resembled those observed experimentally in the presence of bath-applied NMDA and tetrodotoxin. The effect of changing different channel parameters were tested to determine under which conditions such membrane potential oscillations could occur. 4. The oscillation amplitude was determined by the potential levels at which the NMDA channels and voltage-dependent K+ channels, respectively, were activated. The oscillation frequency and the relative durations of the de- and hyperpolarized phases of the oscillations were determined by the balance between the depolarizing (NMDA channels) and hyperpolarizing [K(Ca) channels] currents. 5. Simulated alterations of the Mg2+ concentration and the K+ conductance as well as injection of constant current caused changes of the oscillations corresponding to those observed experimentally. The de- and hyperpolarizing phases could be reset by brief current pulses. 6. We conclude that the present model can account for the effects of bath-applied NMDA on spinal neurons. This permits an incorporation of NMDA-receptor-mediated properties in simulation models of the lamprey locomotor network.     

Effects of activin A on ionic channels in human FSH-secreting tumour cells.

1. Effects of activin A on ionic channels were examined in human FSH-secreting tumour cells using electrophysiological techniques. 2. Under voltage clamp with the conventional whole-cell clamp technique, the voltage-gated Na+ channel, the T- and L-type Ca2+ channels, the delayed K+ channel and the A-channel were observed. 3. With the nystatin-perforated whole-cell clamp technique, the same voltage-gated channels were recorded. Activin A (10(-7) M) increased the amplitude of the L-type Ca2+ current, whereas it decreased the amplitude of the delayed K+ current. 4. Under current clamp with the perforated whole-cell clamp technique, more than 80% of the cells exhibited spontaneous action potentials. Application of 10(-7) M activin A depolarized the membrane with a conductance increase and augmented action potential frequency. The reversal potential of the activin A-induced current was -20 to 0 mV. The activin A-induced current was abolished in a Na(+)-free extracellular solution, indicating that the membrane depolarization caused by activin A was due to the conductance increase to Na+ ions through non-selective cation channels.     

Microscopic heterogeneity in unitary N-type calcium currents in rat sympathetic neurons.

1. Single N-type calcium (Ca2+) channels in rat superior cervical ganglion neurons display complex patterns of activity in both inactivating and non-inactivating gating modes. Unitary currents were elicited by holding the patch at -90 mV and stepping to +30 mV for 740 ms. Barium (110 mM) was used as the charge carrier. The dihydropyridine agonist (+)-202-791 was included in the bath to ensure that single channel recordings showed no L-type Ca2+ channel mode 2 activity. Using this protocol, we characterized three additional patterns of N-type Ca2+ channel activity named: (1) LLP for large unitary current amplitude (i = -0.92 pA) and low open probability (Po = 0.26); (2) SLP for small unitary current amplitude (i = -0.77 pA) and low open probability (Po = 0.25); and (3) SHP for its small unitary current (i = -0.77 pA) and higher open probability (Po = 0.39). 2. Transitions among these patterns of activity occur more slowly than transitions between closed and open states, resulting in significant clustering of like sweeps. Thus, the complicated gating of single N-type Ca2+ channels can be dissected into multiple, independent modes, each with the same reproducible pattern of activity. 3. This heterogeneous activity is not unique to sympathetic neurons, for inactivating (4), non-inactivating (4), SLP (4) and SHP (3 patches) gating modes were also observed in cell-attached patch recordings (n = 4) of single N-type Ca2+ channels in differentiated phaeochromocytoma (PC12) cells. 4. The 1568 sweeps from four single N-type Ca2+ channel recordings that used the same voltage protocol were categorized by mode to determine the frequency of occurrence of each. Of the 54% of sweeps that showed activity, 42% were inactivating and 58% were non-inactivating. The contribution by each mode to the sustained current was estimated using the equation: I = NPoi, where N is the frequency of occurrence of each mode and Po and i are the mean values of open probability and unitary current amplitude respectively. The LLP mode contributed 18%, the SLP mode 16%, and the SHP mode 66% of the sustained whole cell N-type Ba2+ current. 5. The variability in the incidence among these modes in other cell types may resolve some of the controversy surrounding the characterization of N- and L-type whole cell Ca2+ current components in peripheral neurons. In addition, the number of different modes provides a source of plasticity that may be a target of modulation by neurotransmitters and cellular signals.     

Modulation of Ca(2+) signaling by K(+) channels in a hypothalamic neuronal cell line (GT1-1)

The pulsatile release of gonadotropin releasing hormone (GnRH) is driven by the intrinsic activity of GnRH neurons, which is characterized by bursts of action potentials correlated with oscillatory increases in intracellular Ca(2+). The role of K(+) channels in this spontaneous activity was studied by examining the effects of commonly used K(+) channel blockers on K(+) currents, spontaneous action currents, and spontaneous Ca(2+) signaling. Whole-cell recordings of voltage-gated outward K(+) currents in GT1-1 neurons revealed at least two different components of the current. These included a rapidly activating transient component and a more slowly activating, sustained component. The transient component could be eliminated by a depolarizing prepulse or by bath application of 1.5 mM 4-aminopyridine (4-AP). The sustained component was partially blocked by 2 mM tetraethylammonium (TEA). GT1-1 cells also express inwardly rectifying K(+) currents (I(K(IR))) that were activated by hyperpolarization in the presence of elevated extracellular K(+). These currents were blocked by 100 microM Ba(2+) and unaffected by 2 mM TEA or 1.5 mM 4-AP. TEA and Ba(2+) had distinct effects on the pattern of action current bursts and the resulting Ca(2+) oscillations. TEA increased action current burst duration and increased the amplitude of Ca(2+) oscillations. Ba(2+) caused an increase in the frequency of action current bursts and Ca(2+) oscillations. These results indicate that specific subtypes of K(+) channels in GT1-1 cells can have distinct roles in the amplitude modulation or frequency modulation of Ca(2+) signaling. K(+) current modulation of electrical activity and Ca(2+) signaling may be important in the generation of the patterns of cellular activity responsible for the pulsatile release of GnRH.