Na+—Ca2+ Exchange Currents in Cortical Neurons: Concomitant Forward and Reverse Operation and Effect of Glutamate

Na⁺-Ca²⁺ exchanger-associated membrane currents were studied in cultured murine neocortical neurons, using whole-cell recording combined with intracellular perfusion. A net inward current specifically associated with forward (Na⁺_o-Ca²⁺_i) exchange was evoked at -40 mV by switching external 140 mM Li⁺ to 140 mM Na⁺. The voltage dependence of this current was consistent with that predicted for 3Na⁺:1Ca²⁺ exchange. As expected, the current depended on internal Ca²⁺, and could be blocked by intracellular application of the exchanger inhibitory peptide, XIP. Raising internal Na⁺ from 3 to 20 mM or switching the external solution from 140 mM Li⁺ to 30 mM Na⁺ activated outward currents, consistent with reverse (Na⁺,-Ca²⁺_o) exchange. An external Ca²⁺-sensitive current was also identified as associated with reverse Na⁺-Ca²⁺ exchange based on its internal Na⁺ dependence and sensitivity to XIP. Combined application of external Na⁺ and Ca²⁺ in the absence of internal Na⁺ triggered a 3.3–fold larger inward current than the current activated in the presence of 3 mM internal Na⁺, raising the intriguing possibility that Na⁺-Ca²⁺ exchangers might concurrently operate in both the forward and the reverse direction, perhaps in different subcellular locations. With this idea in mind, we examined the effect of excitotoxic glutamate receptor activation on exchanger operation. After 3–5 min of exposure to 100–200 μM glutamate, the forward exchanger current was significantly increased even when external Na⁺ was reduced to 100 mM, and the external Ca²⁺-activated reverse exchanger current was eliminated.     

Ionic currents in carotid body type I cells isolated from normoxic and chronically hypoxic adult rats

Whole-cell recordings were used to investigate the effects of a 3-week period of hypoxia (10% O₂) on the properties of K⁺ and Ca²⁺ currents in type I cells isolated from adult rat carotid bodies. Chronic hypoxia significantly increased whole-cell membrane capacitance. K⁺ current amplitudes were not affected by this period of hypoxia, but K⁺ current density was significantly reduced in cells from chronically hypoxic rats as compared with normoxically maintained, age-matched controls. K⁺ current density was separated into Ca²⁺-dependent and Ca²⁺-independent components by bath application of 200 μM Cd²⁺, which blocked Ca²⁺ currents and therefore, indirectly, Ca²⁺-dependent K⁺ currents. Ca²⁺-dependent K⁺ current density was not significantly different in control and chronically hypoxic type I cells. Cd²⁺-resistant (Ca²⁺-insensitive) K⁺ current densities were significantly reduced in type I cells from chronically hypoxic rats. Acute hypoxia (Po₂ 15–22 mmHg) caused reversible, selective inhibition of Ca²⁺-dependent K⁺ currents in both groups of cells and Ca²⁺-insensitive K⁺ currents were unaffected by acute hypoxia. Ca²⁺ channel current density was not significantly affected by chronic hypoxia, nor was the degree of Ca²⁺ channel current inhibition caused by nifedipine (5 μM). Acute hypoxia did not affect Ca²⁺ channel currents in either group. Our results indicate that adult rat type I cells undergo a selective suppression of Ca²⁺-insensitive, voltage-gated K⁺ currents in response to chronic hypoxia in vivo. These findings are discussed in relation to the known adaptations of the intact carotid body to chronic hypoxia.     

Regulation of Ca-activated nonselective cationic currents in rat pituitary GH3 cells: involvement in L-type Ca current

Ionic currents were investigated by a patch clamp technique in a clonal strain of pituitary (GH₃) cells, using the whole cell configuration with Cs⁺ internal solution. Depolarizing pulses positive to 0 mV from a holding potential of −50 mV activated the voltage-dependent L-type Ca²⁺ current (I_Ca,L) and late outward current. Upon repolarization to the holding potential, a slowly decaying inward tail current was also observed. This inward tail current upon repolarization following a depolarizing pulse was found to be enhanced by Bay K 8644, but blocked by nifedipine or tetrandrine. This current was eliminated by Ba²⁺ replacement of external Ca²⁺ as the charge carrier through Ca²⁺ channels, removal of Ca²⁺ from the bath solution, or buffering intracellular Ca²⁺ with EGTA (10 mM). The reversal potential of inward tail current was approximately −25 mV. When intracellular Cl⁻ was changed, the reversal potential of the Ca²⁺-activated currents was not shifted. Thus, this current is elicited by depolarizing pulses that activate I_Ca,L and allow Ca²⁺ influx, and is referred to as Ca²⁺-activated nonselective cationic current (I_CAN). Without including EGTA in the patch pipette, the slowly decaying inward current underlying the long-lasting depolarizing potential after Ca²⁺ spike was also observed with a hybrid current–voltage protocol. Thus, the present studies clearly indicate that Ca²⁺-activated nonselective cationic channels are expressed in GH₃ cells, and can be elicited by the depolarizing stimuli that lead to the activation of I_Ca,L.     

Inositol 1,4,5-trisphosphate activates non-selective cation conductance via intracellular Ca2+ increase in isolated frog taste cells

The effect of intracellular Ca²⁺ increase was analysed in isolated frog taste cells under the whole-cell patch clamp. External application of a Ca²⁺-ionophore, ionomycin (3 μm ) induced the sustained inward current of ?200 ± 17 pA (mean ± SE, n = 23) at – 50 mV in taste cells. The ionomycin-induced response was observed in most of the cells exposed in the drug, but not when 10 mm BAPTA (1,2-bis (o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid) was included in the pipette (eight cells). Steady-state I–V relationships of ionomycin-induced currents were almost linear and reversed at – 8 ± 1 mV (n = 23). The simultaneous removal of Na⁺ and Ca²⁺ from the external solution eliminated the response completely (three cells). Intracellular dialysis with 1 mm Ca²⁺ or 50 μm inositol 1,4,5-trisphosphate (IP₃) in K⁺-internal solution also induced an inward current in the taste cells. The Ca²⁺-induced and IP₃-induced responses were observed in 82% and 36% of the cells dialysed with the drugs, respectively. The Ca²⁺-induced and IP₃-induced currents were inhibited by external Cd²⁺ (1–2 mm ). The reversal potentials of the inward currents were – 15 ± 3 mV (n = 9) in Ca²⁺ dialysis and – 11 ± 3 mV (n = 13) in IP₃ dialysis. The half-maximal Ca²⁺ concentration in the pipette to induce the inward current was ≈ 170 μm . The results suggest that IP₃ can depolarize the taste cell with mediation by intracellular Ca²⁺.     

Modulation of presynaptic Ca2+ currents in frog motor nerve terminals by high pressure

Presynaptic Ca²⁺‐dependent mechanisms have already been implicated in depression of evoked synaptic transmission by high pressure (HP). Therefore, pressure effects on terminal Ca²⁺ currents were studied in Rana pipiens peripheral motor nerves. The terminal currents, evoked by nerve or direct stimulation, were recorded under the nerve perineurial sheath with a loose macropatch clamp technique. The combined use of Na⁺ and K⁺ channel blockers, [Ca²⁺]_o changes, voltage‐dependent Ca²⁺ channel (VDCC) blocker treatments and HP perturbations revealed two components of presynaptic Ca²⁺ currents: an early fast Ca²⁺ current (I_CaF), possibly carried by N‐type (Ca_V2.2) Ca²⁺ channels, and a late slow Ca²⁺ current (I_CaS), possibly mediated by L‐type (Ca_V1) Ca²⁺ channels. HP reduced the amplitude and decreased the maximum (saturation level) of the Ca²⁺ currents, I_CaF being more sensitive to pressure, and may have slightly shifted the voltage dependence. HP also moderately diminished the Na⁺ action current, which contributed to the depression of VDCC currents. Computer‐based modeling was used to verify the interpretation of the currents and investigate the influence of HP on the presynaptic currents. The direct HP reduction of the VDCC currents and the indirect effect of the action potential decrease are probably the major cause of pressure depression of synaptic release.     

Intracellular Action of Spermine on Neuronal Ca2+ and K+ Currents

Intra-and extracellular effects of the polyamine spermine on electrical activity and membrane currents of identified neurons in the abdominal ganglion of Aplysia californica were studied under current-and voltage-clamp conditions. Lonophoretic injection of spermine reduced the amplitude of action potentials and altered their time course as well as spontaneous discharge activity. Investigation of membrane currents showed that intracellular spermine suppressed the total outward current but increased the inward rectifier current. After separation of ion currents it was found that the voltage-activated, delayed K⁺ outward current and the Ca²⁺ inward current were reduced by intracellular spermine in a dose- and voltage-dependent manner. The block of the K⁺ current can be described by a voltage-dependent reaction, where one spermine molecule binds to one channel. The binding constant K_b, at zero voltage, and the effective valency, zδ, had values of 176/M and 0.41 for cell R-15, 223/M and 0.64 for cell L-11, and 137/M and 0.42 for cell L-3. Apparently, more than one spermine cation is needed to block one Ca²⁺ channel, since the coefficient n, which absorbs the molecularity and cooperativity of the reaction, had non-integral values between 1.34 and 2.22. The binding constant K_b and the effective valency zδ had values of 265/M and 0.64 for cell R-15, 821M and 0.56 for cell L-4, and 263/M and 0.51 for cell L-6. Intracellular spermine also blocked the Ca²⁺-activated K⁺ current induced by ionophoretic Ca²⁺-injections, but increased the current at prolonged times after spermine injection. Extracellular spermine had no effect on electrical activity or on membrane currents. The results indicate that intracellular spermine affects the electrical discharge activity of neurons by acting as a blocker and/or modulator at voltage-dependent membrane conductances.     

Ionic currents on type-I cells of the rabbit carotid body measured by voltage-clamp experiments and the effect of hypoxia

Type-I cells (from rabbit embryos) in primary culture were studied in voltage-clamp experiments using the whole cell arrangement of the patch-clamp technique. With a pipette solution containing 130 mM K⁺ and 3 mM Mg-ATP, large outward currents were obtained positive to a threshold of about −30 mV by clamping cells from −50 mV to different test pulses (−80 to 50 mV). Negative to −30 mV, the slope conductance was low (outward rectification). The outward currents were blocked by external Cs⁺ (5 mM) and partially blocked by TEA (5 mM) and Co²⁺ (1 mM). The initial part of the outward currents during depolarizing voltage pulses exhibited a transient Ca²⁺ inward component partially superimposed to a Ca²⁺-dependent outward current. Inward currents were further characterized by replacing K⁺ with Cs⁺ in the intra- and extracellular solution in order to minimize the outward component and by using 1.8 mM Ca²⁺ or 10.8 mM Ba²⁺ as charge carrier. Slow-inactivating inward currents were recorded at test potentials ranging from −50 to 40 mV (holding potential −80 mV). The maximal amplitude, measured at 10 mV in the U-shaped I–V curve, amounted to 247 ± 103pA(n = 3). This inward current was insensitive to 3 μM TTX, but blocked by 1 mM Co²⁺ and partially reduced by 10 μM D600 and 3 μM PN 200-110. In contrast to outward currents, the inward currents exhibited a ‘run-down’ within about 10 min. Lowering the pO₂ from the control of 150 Torr (air-gassed medium) to 28 Torr had no apparent effect on inward currents, but depressed reversibly outward currents by 28%. In conclusion, it is suggested that type-I cells possess voltage-activated K⁺ and Ca²⁺ channels which might be essential for chemoreception in the carotid body.     

Effect of Growth Hormone-Releasing Peptide-2 (GHRP-2) and GH-Releasing Hormone (GHRH) on the cAMP Levels and GH Release from Cultured Acromegalic Tumours

There is a difference between the sheep and rat somatotrophs in the response to growth hormone-releasing peptide-2 (GHRP-2), which raises the question of what the response may be in human somatotrophs. In the present study, cells were obtained from seven human acromegalic tumours and the effects of GHRP-2 were studied. Cells were dissociated and kept in primary culture for 1–3 weeks before experimentation. Application of GHRP-2 for 30 min induced a significant increase in GH secretion from the cultured cells from all seven tumours whereas human GH-releasing hormone (hGHRH) at a dose of 10 nM induced a significant GH release in only four of seven tumours. The intracellular levels of cAMP in all seven tumours were significantly increased by both 10 nM GHRP-2 and GHRH, but the response to GHRH was significantly higher than the response to GHRP-2. The adenylyl cyclase inhibitor, MDL 12330A, blocked the effect of GHRH and GHRP-2 on intracellular cAMP levels, whereas the Ca²⁺ channel blocker Co²⁺ (0.5 mM) did not attenuate the cAMP response. For the tumours in which GH secretion was increased by GHRH and GHRP-2, the cAMP antagonist Rp-cAMP blocked the GH response to GHRH but not to GHRP-2. When a protein kinase A (PKA) inhibitor (H₈₉) was applied, GHRH stimulated GH release was blocked, but cAMP accumulation was not affected. The response to GHRP-2 was not altered by H₈₉. Calphostin C [a protein kinase C (PKC) inhibitor] reduced the effect of GHRP-2 on the secretion of GH but did not affect the response to GHRH. Both GHRH and GHRP-2 increased the intracellular Ca²⁺ concentration in a concentration-dependent manner. We conclude that (1) GHRH increases GH secretion from human GH tumours via the cAMP pathway whereas GHRP-2 increases GH secretion mainly via the PKC pathway; (2) GHRH increases cAMP (without GH release) in a subset of tumours whereas GHRP-2 increases cAMP levels (slightly) and GH secretion in all tumours; and (3) GHRP-2 and GHRH do not act on the same receptor on human somatotrophs derived from acromegalic tumours.     

Nicotinic and muscarinic acetylcholine responses in differentiated PC12 cells

Nicotinic and muscarinic acetylcholine (ACh) responses were investigated in PC12 cells using the conventional whole-cell and nystatin perforated patch techniques. With the nystatin perforated patch, ACh induced three kinds of ionic currents: a rapid transient inward current, a subsequent transient outward current and a long-lasting slow inward current, whereas only a transient inward current was recorded by conventional whole-cell patch. The transient rapid inward current was mimicked by nicotine, but not by muscarine. On the contrary, the transient outward current and the long-lasting slow inward current were mimicked by muscarine but not by nicotine. Both nicotinic and muscarinic antagonists inhibited the transient inward current and the subsequent outward current in a concentration-dependent manner. The current-voltage relationship for the nicotine-induced transient current showed an inward rectification and the reversal potential was close to the Na⁺ equilibrium potential. The ACh-, muscarine-, CCh- and oxotremorine-M induced outward currents increased in a sigmoidal fashion with an increase in the concentration. Neither McN-A-343, an M1 agonist, nor oxotremorine, an M2 agonist, mimicked the muscarinic response. The reversal potential of the muscarinic response was close to the K⁺ equilibrium potential. The muscarinic response was not affected by pre-treatment with pertussis toxin but was enhanced by pre-treatment with Li⁺. In the cells perfused with Ca²⁺-free external solution, only the first application of ACh induced the muscarinic response. Calmodulin antagonists reversibly blocked the muscarinic response in a concentration-dependent manner. Neither protein kinase C inhibitor (H-7), protein kinase A inhibitor (H-8), nor Ca-calmodulin dependent kinase II inhibitor (KN-62) affected the muscarinic response. It was concluded that the ACh-induced rapid inward current was passing through non-selectivecation channels coupled with nicotinic ACh receptors. On the other hand, the muscarinic response is mediated by the activation of M3 receptors coupled to IAP-insensitive G-protein which stimulates the phosphatidylinositol pathway through phospholipase C. Consequently, Ca²⁺ was released by the increase in IP₃. Finally, Ca²⁺-calmodulin binding may lead to opening of the K⁺ channels.     

Extracellular ATP-induced currents in astrocytes: Involvement of a cation channel

Whole-cell currents were measured with the perforated patch clamp technique in cultured rat astrocytes to analyze the underlying ionic mechanism for a P₂-purinoceptor-mediated depolarization. ATP (100 μM) induced an inward current with a mean amplitude of 130 pA and an EC₅₀ of 17 μM. The response desensitized during a 1 min application. Replacement of extracellular Na⁺ with NMDG or K⁺ abolished the ATP-evoked inward current. Replacement of Na⁺ with choline, however, resulted in an ATP-evoked response of one-third the amplitude in normal solution. This is indicative of a cation rather than Na⁺ channel. However, due to difficulties in voltage-clamping these gap junction-coupled cells at voltages different from the membrane resting potential, the current reversal potential could not be determined. Measurements with K⁺-sensitive microelectrodes showed that 100 μM ATP lowered the intracellular K⁺ concentration. Replacement of extracellular Ca²⁺ or Cl^? did not alter the ATP-induced inward currents. Fura-2 imaging experiments revealed a transient rise of the intracellular Ca²⁺ concentration during ATP application. Removal of extracellular Ca²⁺ did not influence the peak response; it did, however, shorten the time course. These results and previous observations that the permeability changes are caused by a P_2× receptor are indicative of an ATP-sensitive cation conductance. In addition, cytoplasmic Ca²⁺ is increased by mobilization from intracellular stores, and by additional influx across the cell membrane. Extracellular ATP released by neurons could evoke K⁺ release from astrocytes as well as be a mediator for cation changes that signal cell activation processes when released by damaged cells. © 1994 Wiley-Liss, Inc.     

K-current modulated by PO2 in type I cells in rat carotid body is not a chemosensor

According to the membrane channel hypothesis of carotid body O₂ chemoreception, hypoxia suppresses K⁺ currents leading to cell depolarization, [Ca²⁺]_i rise, neurosecretion, increased neural discharge from the carotid body. We show here that tetraethylammonium (TEA) plus 4-aminopyridine (4-AP) which suppressed the Ca²⁺ sensitive and other K⁺ currents in rat carotid body type I cells, with and without low [Ca²⁺]_o plus high [Mg²⁺]_o, did not essentially influence low

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effects on [Ca²⁺]_i and chemosensory discharge. Thus, hypoxia may suppress the K⁺ currents in glomus cells but K⁺ current suppression of itself does not lead to chemosensory excitation. Therefore, the hypothesis that K⁺–O₂ current is linked to events in chemoreception is not substantiated. K⁺–O₂ current is an epiphemenon which is not directly linked with O₂ chemoreception.     

Three Native Somatostatin Isoforms Differentially Affect Membrane Voltage‐Sensitive Ion Currents in Goldfish Somatotrophs

Message encoding for three isoforms of somatostatin (SS) peptides, SS‐14, goldfish brain (gb)SS‐28 and [Pro²]SS‐14, are expressed in goldfish hypothalamus and pituitary tissues. All three native goldfish SSs are active in reducing basal and stimulated growth hormone (GH) responses in cultured goldfish pituitary cells, although with different potencies and efficacies. In the present study, we examined the effects of these three endogenous SSs on electrophysiological properties of goldfish somatotrophs and their physiological relevance. Voltage‐sensitive K⁺, Ca²⁺ and Na⁺ channels in identified goldfish somatotrophs in primary culture were isolated using whole‐cell, amphotericin B‐perforated patch‐clamp techniques. None of the three SSs affected Na⁺ currents but all three SSs increased maximal K⁺ current magnitude, with SS‐14 being the most effective. [Pro²]SS14 did not affect Ba²⁺ currents through voltage‐sensitive Ca²⁺ channels but SS14 decreased the magnitude of early and late Ba²⁺ currents, whereas gbSS‐28 reduced that of the late Ba²⁺ current. Under current‐clamp conditions, SS14 and gbSS28 attenuated evoked action potential magnitudes by 34% and 18%, respectively, although [Pro²]SS14 had no effects. However, all three SSs decreased basal intracellular Ca²⁺ levels ([Ca²⁺]_i) and suppressed basal GH release. These data suggest that, although the ability of SS‐14 and gbSS‐28 to decrease basal [Ca²⁺]_i and GH release can be explained, at least in part, by their attenuating effects on cell excitability and current flow through voltage‐sensitive Ca²⁺channels, [Pro²]SS14‐induced reduction in GH responses and [Ca²⁺]_i cannot be explained by changes in Ca²⁺ channel properties.     

Electrophysiological and morphological properties of light and dark cells isolated from mudpuppy taste buds

Isolated Necturus taste receptor cells were studied by giga-seal whole-cell recording and electron microscopy to correlate electrophysiological properties with taste cell structural features. Dark (type I) cells were identified by the presence of dense granular packets in the supranuclear and apical regions of the cytoplasm. In response to a series of depolarizing voltage commands from a holding potential of ?80 mV, these cells exhibited a transient, TTX-sensitive inward Na⁺ current, a sustained outward K⁺ current, and a slowly inactivating inward Ca⁺⁺ current. Light (type II) cells were identified by a lack of granular packets and by an abundance of smooth endoplasmic reticulum distributed throughout the cell. In addition, isolated light cells had clear vesicular inclusions in the cytoplasm and blebs on the plasma membrane. Light cells were divided into two functional populations based upon electrophysiological criteria: cells with inward and outward currents, and cells with outward currents only. Light cells with inward and outward currents had voltage-activated Na⁺, K⁺, and Ca⁺⁺ currents with properties similar to those of dark cells. In contrast, the second group of light cells had only voltageactivated outward K⁺ currents in response to depolarizing voltage commands. These data suggest that dark cells and light cells with inward and outward currents are capable of generating action potentials and releasing neurotransmitters onto gustatory afferent neurons in response to taste stimulation. In contrast, light cells with outward currents only likely serve a different function in the taste bud. © 1994 Wiley-Liss, Inc.     

Voltage-gated calcium currents in whole-cell patch-clamped bullfrog dorsal root ganglion cells: effects of cell size and intracellular solutions

Acutely dissociated bullfrog dorsal root ganglion (DRG) cells could be divided into two classes by measurement of cell capacitance. A bimodal distribution of cell capacitance was found and a value of 75 pF was used to divide frog DRG cells into ‘small’ and ‘large’ types. Two distinct voltage-activated Ca²⁺ currents were evoked in both classes of cells: a rapidly inactivating, low-voltage-activated current and a slowly-inactivating, high-voltage-activated current. When the recording pipette contained CsCl, greater peak inward current values and densities were seen in large cells compared to small cells. No significant differences were observed in the distribution of low-and high-voltage-activated currents in small and large cells. Replacement of pipette solutions containing CsCl with solutions containing equimolar concentrations of Cs glutamate,l-arginine Cl, orN-methyl-d-glucamine significantly increased both the reversal potential and the maximum amplitude of the Ca²⁺ currents in both small and large DRG cells. These increases indicate that internal substitutions with organic ions suppresses outward currents more effectively than does CsCl. In contrast to findings with CsCl, when organic ions were used in the pipette solution a significantly higher proportion of low-threshold Ca²⁺ channels was observed in small cells compared to large cells. These observations indicate that when organic solutions were used internally, significant differences in the proportion of low-threshold to high-threshold Ca²⁺ channels were observed in small and large cells. The composition of the internal solution is a critical variable when determining the type and amount of inward Ca²⁺ current in different types of neurons.     

The contribution of voltage-gated Ca currents to K channel activation during ovine adrenal chromaffin cell development

Prior to the development of adrenal innervation, the adrenal medulla is capable of responding to low blood oxygen directly. However, this response is lost once adrenal innervation is established. Previous work by our group has outlined mechanisms involved in this direct hypoxic response and the means by which innervation causes the loss of the direct hypoxic response in the ovine adrenal. The current study further investigates mechanisms which may underlie the developmental loss of the direct hypoxic response by concentrating on two aspects of cell function which regulate catecholamine secretion: the contribution of different types of Ca²⁺ channels to the total Ca²⁺ current and the contribution of each Ca²⁺ channel type to K⁺ channel activation. We identified that Ca²⁺ current size at −40 to −10 mV is increased in amplitude in fetal chromaffin cells. This is not due to the increased prevalence or size of T-type Ca²⁺ currents present at these voltages. The relative contribution of L-, N- or P/Q-type Ca²⁺ channels to total Ca²⁺ current and to activation of the K⁺ current is unchanged during chromaffin cell development, however K⁺ current density increases with age. Our results indicate that there is a developmental shift in relative expression of T-type, but not L-, N- or P/Q-type, Ca²⁺ channels in ovine chromaffin cells. The increased K⁺ current density in adult cells may result in an altered response to an equal stimulus, while larger Ca²⁺ current at negative voltages in fetal cells may facilitate Ca²⁺ entry and catecholamine secretion in response to small depolarisations such as those induced by hypoxia.     

Rectification Properties and Ca2+ Permeability of Glutamate Receptor Channels in Hippocampal Cells

Excitatory amino acids exert a depolarizing action on central nervous system cells through an increase in cationic conductances. Non-NMDA receptors have been considered to be selectively permeable to Na⁺ and K⁺, while Ca²⁺ influx has been thought to occur through the NMDA receptor subtype. Recently, however, the expression of cloned non-NMDA receptor subunits has shown that α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors are permeable to Ca²⁺ whenever the receptor lacks a particular subunit (edited GluR-B). The behaviour of recombinant glutamate receptor channels predicts that Ca²⁺ would only permeate through receptors that show strong inward rectification and vice versa, i.e. AMPA receptors with linear current-voltage relationships would be impermeable to Ca²⁺ . Using the whole-cell configuration of the patch-clamp technique, we have studied the Ca²⁺ permeability and the rectifying properties of AMPA receptors, when activated by kainate, in hippocampal neurons kept in culture or acutely dissociated from differentiated hippocampus. Cells were classified according to whether they showed outward rectifying (type I), inward rectifying (type II) or almost linear (type III) current-voltage relationships for kainate-activated responses. AMPA receptors of type I cells (52.2%) were mostly Ca²⁺-impermeable (P_caIP_cs= 0.1) while type II cells (6.5%) expressed Ca²⁺-permeable receptors (P_caIP_cs=0.9).Type III cells (41.3%) showed responses with low but not negligible Ca²⁺ permeability (P_caIP_cs= 0.18). The degree of Ca²⁺ permeability and inward rectification were well correlated in cultured cells, i.e. more inward rectification corresponded to higher Ca²⁺ permeability. In acutely dissociated neurons, the restricted activation of the receptors located either in dendritic or somatic membranes revealed that inward rectifying (i.e. Ca²⁺-permeable) AMPA receptors are preferentially located in the dendritic shaft (i.e. synaptic field). Our results indicate that oligomeric AMPA receptors of different subunit composition are coexpressed in dissimilar proportions in different cells, which would explain the incomplete inward rectification and graded Ca²⁺ permeability. In addition, Ca²⁺-permeable AMPA receptors may exhibit non-homogeneous subcellular distribution.     

Developmental and activity-dependent changes in K currents in satellite glial cells in mouse superior cervical ganglion

Voltage-gated K⁺ currents were recorded from freshly dissociated satellite glial cells wrapping around ganglion cells in mouse superior cervical ganglion (SCG) by whole-cell recordings of patch clamp techniques. Both inward and outward K⁺ currents during membrane hyperpolarization and depolarization were observed in these glial cells. The current-voltage relation of these K⁺ currents became almost linear in cells obtained more than 4 weeks after birth. The magnitude of the density of inward K⁺ currents, which were elicited during membrane hyperpolarization and were eliminated by external barium, progressively increased during the first month after birth. This developmental increase in the magnitude of inward K⁺ current density was not affected by decentralization of SCG done by transection of cervical sympathetic trunk (CST) 5 days after birth. In adult mice, the magnitude of the inward K⁺ current density decreased after chronic conduction blockade of CST by local application of tetrodotoxin. On the other hand, the magnitude of the inward K⁺ current density increased after daily intraperitoneal injection of reserpine and this increase was abolished by pre-treatment of decentralization of SCG. These results suggested that preganglionic innervation was not prerequisite for developmental increase in the inward K⁺ currents and preganglionic neuronal activity upregulates the inward K⁺ currents in adult mice. Neuronal regulation of glial K⁺ channel expression would assist in K+ clearance from periganglionic space to maintain neuronal activity.