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.     

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.     

Suppression of glomus cell K conductance by 4-aminopyridine is not related to [Ca]i, dopamine release and chemosensory discharge from carotid body

The hypothesis that suppression of O₂-sensitive K⁺ current is the initial event in hypoxic chemotransduction in the carotid body glomus cells was tested by using 4-aminopyridine (4-AP), a known suppressant of K⁺ current, on intracellular [Ca²⁺]_i, dopamine secretion and chemosensory discharge in cat carotid body (CB). In vitro experiments were performed with superfused–perfused cat CBs, measuring chemosensory discharge, monitoring dopamine release by microsensors without and with 4-AP (0.2, 1.0 and 2.0 mM in CO₂-HCO₃^- buffer) and recording [Ca²⁺]_i by ratio fluorometry in isolated cat and rat glomus cells. 4-AP decreased the chemosensory activities in normoxia but remained the same in hypoxia and in flow interruption. It decreased the tissue dopamine release in normoxia, and showed an additional inhibition with hypoxia. Also, 4-AP did not evoke any rise in [Ca²⁺]_i in glomus cells either during normoxia and hypoxia, although hypoxia stimulated it. Thus, the lack of stimulatory effect on chemosensory discharge, inhibition of dopamine release and unaltered [Ca²⁺]_i by 4-AP are not consistent with the implied meaning of the suppressant effect on K⁺ current of glomus cells.     

Kinetic and pharmacological properties of Ca currents in postganglionic sympathetic neurones projecting to muscular and cutaneous effectors

Voltage-gated Ca²⁺ channels are expressed in neurones and greatly influence neuronal activity by activating Ca²⁺-dependent K⁺ channels. The whole cell patch-clamp technique was used to compare the kinetic and pharmacological properties of voltage-dependent Ca²⁺ currents in two groups of sympathetic neurones identified by the fluorescent tracer Fast Blue: putative muscular sympathetic neurones (MSN) and putative cutaneous sympathetic neurones (CSN). The tracer was injected into the muscular part of the diaphragm (to mark MSN) and into the skin of the ear (to mark CSN). The capacitance of MSN (23.0 pF) was larger than the capacitance of CSN (12.6 pF). The maximum current in MSN (1.3 nA) was also larger than in CSN (0.93 nA). However, the current density was larger in CSN (77.3 pA/pF) than in MSN (57.7 pA/pF) and the current activation rate was faster in CSN (0.27 nA/ms) than in MSN (0.19 nA/ms). V_1/2 and slope factors of activation and inactivation were not significantly different for MSN and CSN. The majority of Ca²⁺ current was available for activation in both categories of neurones at resting membrane potential. Ca²⁺ currents in MSN and CSN were blocked by nifedipine (7.0 and 3.6%, respectively), ω-Agatoxin-IVA (23.0 and 25.6%, respectively) and ω-conotoxin-GVIA (67.0 and 65.1%, respectively). We found that CSN are twice as small, have higher Ca²⁺ current density and their Ca²⁺ activation rate is faster in comparison to MSN. Such properties may lead to faster rise of Ca²⁺ concentration in the cytoplasm of the CSN comparing to MSN and more effectively dampen their activity due to more effective activation of Ca²⁺-dependent K⁺ current. Both kinds of neurones express high proportion of N and P/Q Ca²⁺ current.     

Ca channel currents in type I carotid body cells of normoxic and chronically hypoxic neonatal rats

Whole-cell patch-clamp recordings were used to study voltage-gated Ca²⁺ channel currents in type I carotid body cells of young rats born and reared in normoxia or in a chronically hypoxic (CH) environment (10% O₂). Currents activated at potentials of −40 mV and more positive, and typically peaked at 0 mV in both groups of cells. Steady-state inactivation curves were similar in the two populations. Ca²⁺ currents were significantly larger in CH type I cells, but this was accounted for by the increased size of CH cells: current density was similar in both cell types. Nifedipine (5 μM) always partially inhibited currents and Bay K 8644 (2–5 μM) always enhanced currents, indicating the presence of L-type channels. In a small number of cells from each group, the N-type channel blocker ω-conotoxin GVIA caused partial, irreversible inhibition, but in most cells was without discernible effect. These results indicate that type I cells possess L-type Ca²⁺ channels, that N-type are expressed in some cells and that non-L, non-N-type channels are also present. Furthermore, chronic hypoxia does not appear to cause specific adaptive changes in the properties of Ca²⁺ channels in type I cells.     

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.     

Arachidonic acid inhibits both K and Ca currents in isolated type I cells of the rat carotid body

Whole-cell patch-clamp recordings were used to investigate the effects of arachidonic acid (AA) on K⁺ and Ca²⁺ channels in isolated rat type I carotid body cells. AA (2–20 μM) produced a concentration-dependent inhibition of both K⁺ currents and Ca²⁺ channel currents. The effects of AA on K⁺ currents were unaffected by indomethacin (5 μM), phenidone (5 μM) or 1-aminobenzotriazole (3 mM), suggesting that AA did not exert its effects via cyclo-oxygenase, lipoxygenase or cytochrome P-450 (cP-450) metabolism. Our results suggest that AA directly and non-selectively inhibits ionic currents in rat type I carotid body cells.     

Cat carotid body chemosensory discharge (in vitro) is insensitive to charybdotoxin

Charybdotoxin (ChTX), a venom protein, suppresses Ca²⁺-activated K⁺ (K_Ca⁺) currents in the glomus cell of neonatal rat carotid body. If it works similarly for cat carotid body chemoreceptors, charybdotoxin is expected to stimulate the chemosensory discharge during normoxia, and particularly hypoxia and hypercapnia. We studied the effects of charybdotoxin (20–40 nM) in vitro (perfused/superfused) on the cat carotid chemosensory discharge, and simultaneously tissue PO₂ (PtiO₂), as a measure of positive control. ChTX (20 nM) only increased PtiO₂ and decreased carotid chemosensory discharge during hypoxia, indicating vasodilation. We conclude that K_Ca⁺ channels do not appear to play a significant role in chemotransduction in the cat carotid body.     

Selective block of Ca2+-dependent K+ current in crayfish neuromuscular system and chromaffin cells by sea anemone Bunodosoma cangicum venom

The effects of the nematocyst venom of the sea anemone Bunodosoma cangicum on depolarization-activated currents were studied in opener crayfish muscle fibers and in cultured bovine chromaffin cells. The venom selectively and reversibly blocked the Ca²⁺ -dependent K⁺ current (I_K(Ca)) present in crayfish muscle in a dose-dependent manner without affecting voltage-gated Ca²⁺ or K⁺ currents. Furthermore, the venom also reduced I_K(Ca) in chromaffin cells, without modifying voltage-gated Na⁺, Ca²⁺, or K⁺ currents. Synaptic transmission in crayfish muscle was also affected by the venom. Repetitive excitatory and inhibitory postsynaptic currents (each associated with a presynaptic action potential) were evoked by each nerve stimulus, suggesting that presynaptic I_K(Ca) may control the electrical activity of excitatory and inhibitory presynaptic fibers. We conclude that B. cangicum venom includes a toxin that selectively and reversibly blocks Ca²⁺ -dependent K⁺ currents in crayfish muscle and in bovine chromaffin cells, and modifies excitatory and inhibitory synaptic transmission, probably abolishing a similar conductance at the presynaptic fibers. © 1995 Wiley-Liss, Inc.     

Involvement of the glutamate transporter and the sodium–calcium exchanger in the hypoxia-induced increase in intracellular Ca in rat hippocampal slices

Hippocampal slices prepared from adult rats were loaded with fura-2 and the intracellular free Ca²⁺ concentration ([Ca²⁺]_i) in the CA1 pyramidal cell layer was measured. Hypoxia (oxygen–glucose deprivation) elicited a gradual increase in [Ca²⁺]_i in normal Krebs solution. At high extracellular sodium concentrations ([Na⁺]_o), the hypoxia-induced response was attenuated. In contrast, hypoxia in low [Na⁺]_o elicited a significantly enhanced response. This exaggerated response to hypoxia at a low [Na⁺]_o was reversed by pre-incubation of the slice at a low [Na⁺]_o prior to the hypoxic insult. The attenuation of the response to hypoxia by high [Na⁺]_o was no longer observed in the presence of antagonist to glutamate transporter. However, antagonist to Na⁺–Ca²⁺ exchanger only slightly influenced the effects of high [Na⁺]_o. These observations suggest that disturbance of the transmembrane gradient of Na⁺ concentrations is an important factor in hypoxia-induced neuronal damage and corroborates the participation of the glutamate transporter in hypoxia-induced neuronal injury. In addition, the excess release of glutamate during hypoxia is due to a reversal of Na⁺-dependent glutamate transporter rather than an exocytotic process.     

Two types of potassium channels in the PC12 cell line

Activation of K⁺ channels in the PC12 cell line was studied by comparing⁸⁶Rb⁺ efflux under depolarizing and non-depolarizing conditions. Evidence for both Ca²⁺-dependent and voltage-dependent K⁺ channels was obtained by studying depolarization-induced⁸⁶Rb⁺ efflux in solutions of varying Ca²⁺ concentration and in the presence of K⁺ and Ca²⁺ channel blocking agents.     

The voltage‐dependent conductances of rat neocortical layer I neurons

Whole cell patch-clamp techniques were used to study voltage-dependent sodium (Na⁺), calcium (Ca²⁺), and potassium (K⁺) conductances in acutely isolated neurons from cortical layer I of adult rats. Layer I cells were identified by means of γ-aminobutyric acid (GABA) immunocytochemistry. Positive stainings for the Ca²⁺-binding protein calretinin in a subset of cells, indicated the presence of Cajal–Retzius (C-R) cells. All investigated cells displayed a rather homogeneous profile of voltage-dependent membrane currents. A fast Na⁺ current activated at about −45 mV, was half-maximal steady-state inactivated at −66.6 mV, and recovery from inactivation followed a two-exponential process (τ₁ = 8.4 ms and τ₂ = 858.8 ms). Na⁺ currents declined rapidly with two voltage-dependent time constants, reaching baseline current after some tens of milliseconds. In a subset of cells (< 50%) a constant current level of < 65 pA remained at the end of a 90 ms step. A transient outward current (I_fast) activated ≈–40 mV, declined rapidly with a voltage-insensitive time constant (τ≈ 350 ms) and was relatively insensitive to tetraethylammonium (TEA, 20 mm ). I_fast was separated into two components based on their sensitivity to 4-aminopyridine (4-AP): one was blocked by low concentrations (40 μm ) and a second by high concentrations (6 mm ). After elimination of I_fast by a conditioning prepulse (50 ms to −50 mV), a slow K⁺ current (I_KV) could be studied in isolation. I_KV was only moderately affected by 4-AP (6 mm ), while TEA (20 mm ) blocked most (> 80%) of the current. I_KV activated at about −40 mV, declined monoexponentially in a voltage-dependent manner (τ≈ 850 ms at −30 mV), and revealed an incomplete steady-state inactivation. In addition to I_fast and I_KV, indications of a Ca²⁺-dependent outward current component were found. When Na⁺ currents, I_fast, and I_KV were blocked by tetrodotoxin (TTX, 1 μm ), 4-AP (6 mm ) and TEA (20 mm ) an inward current carried by Ca²⁺ was found. Ca²⁺ currents activated at depolarized potentials at about −30 mV, were completely blocked by 50 μm cadmium (Cd²⁺), were sensitive to verapamil (≈ 40% block by 10 μm ), and were not affected by nickel (50 μm ). During current clamp recordings, isolated layer I neurons displayed fast spiking behaviour with short action potentials (≈ 2 ms, measured at half maximal amplitude) of relative small amplitude (≈ 83 mV, measured from the action potential threshold).     

Novel inward rectifier blocked by Cd in crayfish muscle

The characteristics of a voltage- and time-dependent inward rectifying current were examined with voltage clamp techniques in crayfish muscle. The inward current, carried by K⁺, was activated by hyperpolarization. Although this inward current increased with the extracellular K⁺ concentration ([K⁺]_o), the voltage-dependence of the underlying conductance was independent of [K⁺]_o. The current was unaffected by Cs⁺ and Ba²⁺, but was blocked by low concentrations of Cd²⁺. Therefore, this inward rectifier is different than previously described ones.     

Effect of charybdotoxin and leiurotoxon I on potassium currents in bullfrog sympathetic ganglion and hippocampal neurons

The effects of charybdotoxin and leiurotoxin I were examined on several classes of K⁺ currents in bullfrog sympathetic ganglion and hippocampal CA1 pyramidal neurons. Highly purified preparations of charybdotoxin selectively blocked a large voltage- and Ca²⁺-dependent K⁺ current (I_c) responsible for action potential repolarization (IC₅₀ = 6 nM) while leiurotoxin I selectively blocked a small Ca²⁺-dependent K⁺ conductance (I_AHP) responsible for the slow afterhyperpolarization following an action potential (IC₅₀ = 7.5 nM) in bullfrog sympathetic ganglion neurons. Neither of the toxins had a significant effects on other K⁺ currents (M-current [I_M], A-current [I_A] and the delayed rectifier [I_KD] present in these cells. Leiurotoxin I at a concentration of 20 nM had no detectable effect on currents in hippocampal CA1 pyramidal neurons. This lack of effect on I_AHP in central neurons suggests that the channels underlying slow AHPs in those neurons are pharmacologically distinct from analogous channels in peripheral neurons.     

Effects of Na gradient on the intracellular Ca oscillation in the sympathetic ganglion cell: Na---Ca exchange in the neurone cell soma?

(+)-Tubocurarine ((+)-Tc:10–100 μM) reduced the duration of the afterhyperpolarization, which was induced by the activation of Ca²⁺-dependent K⁺-conductance (G_K,Ca) following an action potential in the bullfrog sympathetic ganglion cell, but did not affect the maximum rates of rise and fall of Na⁺- and Ca²⁺-dependent action potentials. The amplitudes of slow rhythmic membrane hyperpolarizations produced by rhythmic rises in the G_K,Ca were also decreased by (+)-Tc without a change in their intervals. Thus, (+)-Tc appears to block the Ca²⁺-dependent K⁺-channel of the bullfrog sympathetic ganglion cell.     

Endogenous voltage-gated potassium channels in human embryonic kidney (HEK293) cells

Endogenous voltage-gated potassium currents were investigated in human embryonic kidney (HEK293) and Chinese hamster ovary (CHO) cells using whole-cell voltage clamp recording. Depolarizing voltage steps from −70 mV triggered an outwardly rectified current in nontransfected HEK293 cells. This current had an amplitude of 296 pA at +40 mV and a current density of 19.2 pA/pF. The outward current was eliminated by replacing internal K⁺ with Cs⁺ and suppressed by the K⁺ channel blockers tetraethylammonium and 4-aminopyridine. Raising external K⁺ attenuated the outward current and shifted the reversal potential towards positive potentials as predicted by the Nernst equation. The current had a fast activation phase but inactivated slowly. These features implicate delayed rectifier (I_K)-like channels as mediators of the observed current, which was comparable in size to I_K currents in many other cells. A small native inward rectifier current but no transient outward current I_A, the M current I_M, or Ca²⁺-dependent K⁺ currents were detected in HEK293 cells. In contrast to these findings in HEK293 cells, little or no I_K-like current was detected in CHO cells. The difference in endogenous voltage-activated currents in HEK293 and CHO cells suggest that CHO cell lines are a preferred system for exogenous K⁺ channel expression. J. Neurosci. Res. 52:612–617, 1998. © 1998 Wiley-Liss, Inc.     

The ouabain-induced [Ca]i increase in leech Retzius neurones is mediated by voltage-dependent Ca channels

In leech Retzius neurones the inhibition of the Na⁺–K⁺ pump by ouabain causes an increase in the cytosolic free calcium concentration ([Ca²⁺]_i). To elucidate the mechanism of this increase we investigated the changes in [Ca²⁺]_i (measured by Fura-2) and in membrane potential that were induced by inhibiting the Na⁺–K⁺ pump in bathing solutions of different ionic composition. The results show that Na⁺–K⁺ pump inhibition induced a [Ca²⁺]_i increase only if the cells depolarized sufficiently in the presence of extracellular Ca²⁺. Specifically, the relationship between [Ca²⁺]_i and the membrane potential upon Na⁺–K⁺ pump inhibition closely matched the corresponding relationship upon activation of the voltage-dependent Ca²⁺ channels by raising the extracellular K⁺ concentration. It is concluded that the [Ca²⁺]_i increase caused by inhibiting the Na⁺–K⁺ pump in leech Retzius neurones is exclusively due to Ca²⁺ influx through voltage-dependent Ca²⁺ channels.     

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.     

The mechanism of the actions of oxaliplatin on ion currents and action potentials in differentiated NG108-15 neuronal cells

Oxaliplatin (OXAL) is a platinum-based chemotherapeutic agent which is effective against advanced or metastatic gastrointestinal cancer. However, the mechanisms responsible for the development of the neuropathy induced by this agent remain unclear. In this study, we attempted to evaluate the possible effects of OXAL on ion currents and action potentials (APs) in NG108-15 cells differentiated with dibutyryl cyclic-AMP. Application of OXAL decreased the peak amplitude of voltage-gated Na⁺ current (I_Na) with no change in the overall current–voltage relations of the currents. This agent also produced a concentration-dependent slowing of I_Na inactivation. A further application of ranolazine reversed OXAL-induced slowing of I_Na inactivation. Unlike ranolazine or riluzole, OXAL had no effect on persistent I_Na elicited by long ramp pulses. OXAL (100 μM) also had little or no effect on the peak amplitude of L-type Ca²⁺ currents in NG108-15 cells, while it suppressed delayed-rectifier K⁺ current. In current-clamp recordings, OXAL alone reduced the amplitude of APs; however, it did not alter the duration of APs. However, after application of tefluthrin, OXAL did increase the duration of APs. Moreover, OXAL decreased the peak amplitude of I_Na with a concomitant reduction of current inactivation in HEK293T cells expressing SCN5A. The effects of OXAL on ion currents presented here may contribute to its neurotoxic actions in vivo.     

Protective effect of MgSO4 infusion on NMDA receptor binding characteristics during cerebral cortical hypoxia in the newborn piglet

This study tests the hypothesis that magnesium, a selective non-competitive antagonist of the NMDA receptor, will attenuate hypoxia-induced alteration in NMDA receptors and preserve MK-801 binding characteristics during cerebral hypoxia in vivo. Anesthetized, ventilated and instrumented newborn piglets were divided into three groups: normoxic controls were compared to untreated hypoxic and Mg²⁺-treated hypoxic piglets. Cerebral hypoxia was induced by lowering the FiO₂ to 5–7% and confirmed biochemically by a decrease in the levels of phosphocreatine (82% lower than control). The Mg²⁺-treated group received MgSO₄ 600 mg/kg over 30 min followed by 300 mg/kg administered during 60 min of hypoxia. Plasma Mg²⁺ concentrations increased from1.6 ± 0.1mg/dl to17.7 ± 3.3mg/dl.³H-MK-801 binding was used as an index of NMDA receptor modification. TheB_max in control, hypoxic and Mg²⁺-treated hypoxic piglets was1.09 ± 0.17, 0.70 ± 0.25and0.96 ± 0.14pmoles/mg protein, respectively. TheK_d for the same groups were10.02 ± 2.04, 4.88 ± 1.43and8.71 ± 2.23nM, respectively. TheB_max andK_d in the hypoxic group were significantly lower compared to the control and Mg²⁺-treated hypoxic groups, indicating a preservation of NMDA receptor number and affinity for MK-801 during hypoxia with Mg²⁺. The activity of Na⁺, K⁺ ATPase, a marker of neuronal membrane function, was lower in the hypoxic group compared to the control and Mg²⁺-treated hypoxic groups. These findings show that MgSO₄ prevents the hypoxia-induced modification of the NMDA receptor and attenuates neuronal membrane dysfunction. We suggest that the administration of Mg²⁺ prior to and during hypoxia may be neuroprotective in vivo, possibly by reducing the NMDA receptor-mediated influx of calcium.