Action potentials in primary osteoblasts and in the MG-63 osteoblast-like cell line

Whole-cell patch-clamp analysis revealed a resting membrane potential of −60 mV in primary osteoblasts and in the MG-63 osteoblast-like cells. Depolarization-induced action potentials were characterized by duration of 60 ms, a minimal peak-to-peak distance of 180 ms, a threshold value of −20 mV and a repolarization between the spikes to −45 mV. Expressed channels were characterized by application of voltage pulses between −150 mV and 90 mV in 10 mV steps, from a holding potential of −40 mV. Voltages below −60 mV induced an inward current. Depolarizing voltages above −30 mV evoked two currents: (a) a fast activated and inactivated inward current at voltages between −30 and 30 mV, and (b) a delayed-activated outward current that was induced by voltages above −30 mV. Electrophysiological and pharmacological parameters indicated that hyperpolarization activated strongly rectifying K⁺ (K_ir) channels, whereas depolarization activated tetrodotoxin sensitive voltage gated Na⁺ (Na_v) channels as well as delayed, slowly activated, non-inactivating, and tetraethylammonium sensitive voltage gated K⁺ (K_v) channels. In addition, RT-PCR showed expression of Na_v1.3, Na_v1.4, Na_v1.5, Na_v1.6, Na_v1.7, and K_ir2.1, K_ir2.3, and K_ir2.4 as well as K_v2.1. We conclude that osteoblasts express channels that allow firing of action potentials.     

Purinergic signalling in rat GFSHR-17 granulosa cells: an <Emphasis Type="Italic">in vitro</Emphasis> model of granulosa cells in maturing follicles

Purinergic signalling in rat GFSHR-17 granulosa cells was characterised by Ca²⁺-imaging and perforated patch-clamp. We observed a resting intracellular Ca²⁺-concentration ([Ca²⁺]_i) of 100 nM and a membrane potential of −40 mV. This was consistent with high K⁺− and Cl⁻ permeability and a high intracellular Cl⁻ concentration of 40 mM. Application of ATP for 5–15 s every 3 min induced repeated [Ca²⁺]_i increases and a 30 mV hyperpolarization. The phospholipase C inhibitor U73122 or the IP₃-receptor antagonist 2-aminoethoethyl diphenyl borate suppressed ATP responses. Further biochemical and pharmacological experiments revealed that ATP responses were related to stimulation of P2Y₂ and P2Y₄ receptors and that the [Ca²⁺]_i increase was a prerequisite for hyperpolarization. Inhibitors of Ca²⁺-activated channels or K⁺ channels did not affect the ATP-evoked responses. Conversely, inhibitors of Cl⁻ channels hyperpolarized cells to −70 mV and suppressed further ATP-evoked hyperpolarization. We propose that P2Y₂ and P2Y₄ receptors in granulosa cells modulate Cl⁻ permeability by regulating Ca²⁺-release.     

Three Types of Single Voltage-Dependent Potassium Channels in the Sarcolemma of Frog Skeletal Muscle

Patch-clamp experiments in the sarcolemma of frog skeletal muscle evidenced the presence of three types of voltage-dependent single-channel K⁺ currents. According to their unitary conductance at a membrane voltage of +40 mV, we classified them as 16-, 13-, and 7-pS K⁺ channels. The 16-pS K⁺ channels are active close to a membrane voltage of −80 mV and they do not become inactivated during voltage pulses of 100 ms. Within 10 min after beginning the recording, these channels developed rundown with an exponential time course. The 13-pS K⁺ channels are active near −60 mV; upon a 100-ms depolarization, they exhibited inactivation with an approximate exponential time course. The 7-pS K⁺ channels were recorded at voltages positive to 0 mV. In patches containing all three types of K⁺ channels, the ensemble average currents resemble the kinetic properties of the macroscopic delayed rectifier K⁺ currents recorded in skeletal muscle and other tissues. In conclusion, the biophysical properties of unitary K⁺ currents suggest that these single-channel K⁺ currents may underlie the macroscopic delayed K⁺ currents in frog skeletal muscle fibers. In addition, since the 16- and 13-pS channels were more frequently recorded, both are the main contributors to the delayed K⁺ currents.     

ATP-mediated potassium recycling in the cochlear supporting cells

Gap junction-mediated K⁺ recycling in the cochlear supporting cell has been proposed to play a critical role in hearing. However, how potassium ions enter into the supporting cells to recycle K⁺ remains undetermined. In this paper, we report that ATP can mediate K⁺ sinking to recycle K⁺ in the cochlear supporting cells. We found that micromolar or submicromolar levels of ATP could evoke a K⁺-dependent inward current in the cochlear supporting cells. At negative membrane potentials and the resting membrane potential of −80 mV, the amplitude of the ATP-evoked inward current demonstrated a linear relationship to the extracellular concentration of K⁺, increasing as the extracellular concentration of K⁺ increased. The inward current also increased as the concentration of ATP was increased. In the absence of ATP, there was no evoked inward current for extracellular K⁺ challenge in the cochlear supporting cells. The ATP-evoked inward current could be inhibited by ionotropic purinergic (P2X) receptor antagonists. Application of pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS, 50 μM) or pre-incubation with an irreversible P2X7 antagonist oxidized ATP (oATP, 0.1 mM) completely abolished the ATP-evoked inward current at the negative membrane potential. ATP also evoked an inward current at cell depolarization, which could be inhibited by intracellular Cs⁺ and eliminated by positive holding potentials. Our data indicate that ATP can activate P2X receptors to recycle K⁺ in the cochlear supporting cells at the resting membrane potential under normal physiological and pathological conditions. This ATP-mediated K⁺ recycling may play an important role in the maintenance of cochlear ionic homeostasis.     

Evidence for Multidrug Resistance-1 P-Glycoprotein-dependent Regulation of Cellular ATP Permeability

The mechanisms responsible for regulating epithelial ATP permeability and purinergic signaling are not well defined. Based on the observations that members of the ATP-binding cassette (ABC)¹ family of proteins may contribute to ATP release, the purpose of these studies was to assess whether multidrug resistance-1 (MDR1) proteins are involved in ATP release from HTC hepatoma cells. Using a bioluminescence assay to detect extracellular ATP, increases in cell volume increased ATP release ∼3-fold. The MDR1 inhibitors cyclosporine A (10 μm) and verapramil (10 μm) inhibited ATP release by 69% and 62%, respectively (p < 0.001). Similarly, in whole-cell patch-clamp recordings, intracellular dialysis with C219 antibodies to inhibit MDR1 decreased ATP-dependent volume-sensitive Cl⁻ current density from −33.1 ± 12.5 pA/pF to −2.0 ± 0.3 pA/pF (−80 mV, p≤ 0.02). In contrast, overexpression of MDR1 in NIH 3T3 cells increased ATP release rates. Inhibition of ATP release by Gd³⁺ had no effect on transport of the MDR1 substrate rhodamine-123; and alteration of MDR1-substrate selectivity by mutation of G185 to V185 had no effect on ATP release. Since the effects of P-glycoproteins on ATP release can be dissociated from P-glycoprotein substrate transport, MDR1 is not likely to function as an ATP channel, but instead serves as a potent regulator of other cellular ATP transport pathways. Received: 20 November 2000/Revised: 25 May 2001     

TEA-Sensitive Currents Contribute to Membrane Potential of Organ Surface Primo-node Cells in Rats

The primo-vascular (Bonghan) tissue has been identified in most tissues in the body, but its structure and functions are not yet well understood. We characterized electrophysiological properties of the cells of the primo-nodes (PN) on the surface of abdominal organs using a slice patch clamp technique. The most abundant were small round cells (~10 μm) without processes. These PN cells exhibited low resting membrane potential (−36 mV) and did not fire action potentials. On the basis of the current–voltage (I–V) relationships and kinetics of outward currents, the PN cells can be grouped into four types. Among these, type I cells were the majority (69%); they showed strong outward rectification in I–V relations. The outward current was activated rapidly and sustained without decay. Tetraethylammonium (TEA) dose-dependently blocked both outward and inward current (IC₅₀, 4.3 mM at ±60 mV). In current clamp conditions, TEA dose-dependently depolarized the membrane potential (18.5 mV at 30 mM) with increase in input resistance. The tail current following a depolarizing voltage step was reversed at −27 mV, and transient outward current like A-type K⁺ current was not expressed at holding potential of −80 mV. Taken together, the results demonstrate for the first time that the small round PN cells are heterogenous, and that, in type I cells, TEA-sensitive current with limited selectivity to K⁺ contributed to resting membrane potential of these cells.     

Basolateral K+ Conductance Establishes Driving Force for Cation Absorption by Outer Sulcus Epithelial Cells

Outer sulcus epithelial cells were recently found to actively reabsorb cations from the cochlear luminal fluid, endolymph, via nonselective cation channels in the apical membrane. Here we determined the transport properties of the basolateral membrane with the whole-cell patch clamp technique; the apical membrane contributed insignificantly to the recordings. Outer sulcus epithelial cells exhibited both outward and inward currents and had a resting membrane potential of −90.4 ± 0.7 mV (n= 78), close to the Nernst potential for K⁺ (−95 mV). The reversal potential depolarized by 54 mV for a tenfold increase in extracellular K⁺ concentration with a K⁺/Na⁺ permeability ratio of 36. The most frequently observed K⁺ current was voltage independent over a broad range of membrane potentials. The current was reduced by extracellular barium (10⁻⁵ to 10⁻³ m), amiloride (0.5 mm), quinine (1 mm), lidocaine (5 mm) and ouabain (1 mm). On the other hand, TEA (20 mm), charybdotoxin (100 nm), apamin (100 nm), glibenclamide (10 μm), 4-aminopyridine (1 mm) and gadolinium (1 mm) had no significant effect. These data suggest that the large K⁺ conductance, in concert with the Na⁺,K⁺-ATPase, of the basolateral membrane of outer sulcus cells provides the driving force for cation entry across the apical membrane, thereby energizing vectorial cation absorption by this epithelium and contributing to the homeostasis of endolymph.     

Fluoxetine Inhibits K+ Transport Pathways (K+ Efflux, Na+-K+-2Cl− Cotransport, and Na+ Pump) Underlying Volume Regulation in Corneal Endothelial Cells

We have studied regulatory volume responses of cultured bovine corneal endothelial cells (CBCEC) using light scattering. We assessed the contributions of fluoxetine (Prozac) and bumetanide-sensitive membrane ion transport pathways to such responses by determining K⁺ efflux and influx. Cells swollen by a 20% hypo-osmotic solution underwent a regulatory volume decrease (RVD) response, which after 6 min restored relative cell volume by 98%. Fluoxetine inhibited RVD recovery; 20 μm by 26%, and 50 μm totally. Fluoxetine had a triphasic effect on K⁺ efflux; from 20 to 100 μm it inhibited efflux 2-fold, whereas at higher concentrations the efflux first increased to 1.5-fold above the control value, and then decreased again. Cells shrunk by a 20% hyperosmotic solution underwent a regulatory volume increase (RVI) which also after 6 min restored the cell volume by 99%. Fluoxetine inhibited RVI; 20 μm by 25%, and 50 μm completely. Bumetanide (1 μm) inhibited RVI by 43%. In a Cl⁻-free medium, fluoxetine (50–500 μm) progressively inhibited bumetanide-insensitive K⁺ influx. The inhibitions of RVI and K⁺ influx induced by fluoxetine 20 to 50 μm were similar to those induced by 1 μm bumetanide and by Cl⁻-free medium. A computer simulation suggests that fluoxetine can interact with the selectivity filter of K⁺ channels. The data suggest that CBCEC can mediate RVD and RVI in part through increases in K⁺ efflux and Na-K-2Cl cotransport (NKCC) activity. Interestingly, the data also suggest that fluoxetine at 20 to 50 μm inhibits NKCC, and at 100–1000 μm inhibits the Na⁺ pump. One possible explanation for these findings is that fluoxetine could interact with K⁺-selective sites in K⁺ channels, the NKC cotransporter and the Na⁺ pump.     

Hyperpolarization-activated Ca2+-permeable Channels in the Plasma Membrane of Tomato Cells

The hyperpolarization of the electrical plasma membrane potential difference has been identified as an early response of plant cells to various signals including fungal elicitors. The hyperpolarization-activated influx of Ca²⁺ into tomato cells was examined by the application of conventional patch clamp techniques. In both whole cell and single-channel recordings, clamped membrane voltages more negative than −120 mV resulted in time- and voltage-dependent current activation. Single-channel currents saturated with increasing activities of Ca²⁺ and Ba²⁺ from 3 to 26 mm and the single channel conductance increased from 4 pS to 11 pS in the presence of 20 mm Ca²⁺ or Ba²⁺, respectively. These channels were 20–25 and 10–13 times more permeable to Ca²⁺ than to K⁺ and to Cl⁻, respectively. Channel currents were strongly inhibited by 10 μm lanthanum and 50% inhibited by 100 μm nifedipine. This evidence suggests that hyperpolarization-activated Ca²⁺-permeable channels provide a mechanism for the influx of Ca²⁺ into tomato cells. Received: 13 February 1996/Revised: 12 August 1996     

Block of a Ca<Superscript>2+</Superscript>-activated Potassium Channel by Cocaine

The primary target for cocaine is believed to be monoamine transporters because of cocaine’s high-affinity binding that prevents re-uptake of released neurotransmitter. However, direct interaction with ion channels has been shown to be important for certain pharmacological/toxicological effects of cocaine. Here I show that cocaine selectively blocks a calcium-dependent K⁺ channel in hippocampal neurons grown in culture (IC₅₀ = ∼30 μM). Single-channel recordings show that in the presence of cocaine, the channel openings are interrupted with brief closures (flicker block). As the concentration of cocaine is increased the open-time is reduced, whereas the duration of brief closures is independent of concentration. The association and dissociation rate constants of cocaine for the neuronal Ca²⁺-activated K⁺ channels are 261 ± 37 μM⁻¹s⁻¹ and 11451 ± 1467 s⁻¹. The equilibrium dissociation constant (K_B) for cocaine, determined from single-channel parameters, is 43 μM. The lack of voltage dependence of block suggests that cocaine probably binds to a site at the mouth of the pore. Block of Ca²⁺-dependent K⁺ channels by cocaine may be involved in functions that include broadening of the action potential, which would facilitate transmitter release, enhancement of smooth muscle contraction particularly in blood vessels, and modulation of repetitive neuronal firing by altering the repolarization and afterhyperpolarization phases of the action potential.     

K<Superscript>+</Superscript> Channels of Squid Giant Axons Open by an Osmotic Stress in Hypertonic Solutions Containing Nonelectrolytes

In hypertonic solutions made by adding nonelectrolytes, K⁺ channels of squid giant axons opened at usual asymmetrical K⁺ concentrations in two different time courses; an initial instantaneous activation (I _IN) and a sigmoidal activation typical of a delayed rectifier K⁺ channel (I _D). The current–voltage relation curve for I _IN was fitted well with Goldman equation described with a periaxonal K⁺ concentration at the membrane potential above −10 mV. Using the activation–voltage curve obtained from tail currents, K⁺ channels for I _IN are confirmed to activate at the membrane potential that is lower by 50 mV than those for I _D. Both I _IN and I _D closed similarly at the holding potential below −100 mV. The logarithm of I _IN/I _D was linearly related with the osmolarity for various nonelectrolytes. Solute inaccessible volumes obtained from the slope increased with the nonelectrolyte size from 15 to 85 water molecules. K⁺ channels representing I _D were blocked by open channel blocker tetra-butyl ammonium (TBA) more efficiently than in the absence of I _IN, which was explained by the mechanism that K⁺ channels for I _D were first converted to those for I _IN by the osmotic pressure and then blocked. So K⁺ channels for I _IN were suggested to be derived from the delayed rectifier K⁺ channels. Therefore, the osmotic pressure is suggested to exert delayed-rectifier K⁺ channels to open in shrinking rather hydrophilic flexible parts outside the pore than the pore itself, which is compatible with the recent structure of open K⁺ channel pore.     

An ATP-Sensitive K+ Current that Regulates Progression Through Early G1 Phase of the Cell Cycle in MCF-7 Human Breast Cancer Cells

Whole-cell recordings were used to identify in MCF-7 human breast cancer cells the ion current(s) required for progression through G1 phase of the cell cycle. Macroscopic current-voltage curves were fitted by the sum of three currents, including linear hyperpolarized, linear depolarized and outwardly rectifying currents. Both linear currents, but not the outwardly rectifying current, were increased by 1 μm intracellular Ca²⁺ and blocked by 2 mm intracellular ATP. When tested at concentrations previously shown to inhibit proliferation by 50%, linogliride, glibenclamide and quinidine inhibited the linear hyperpolarized current, and quinidine and linogliride inhibited the linear depolarized current; none of these agents affected the outwardly rectifying current. In contrast, tetraethylammonium completely inhibited the outwardly rectifying current, but did not inhibit either linear current. Changing the bath solution to symmetric K⁺ shifted the reversal potential of the linear hyperpolarized current from near the K⁺ equilibrium potential (−84 mV) to −4 mV. Arrest of the cell cycle in early G1 by quinidine was associated with significantly smaller linear hyperpolarized currents, without a change in the linear depolarized or outwardly rectifying currents, but this reduction was not observed with arrest by lovastatin at a site ≈6 hr later in G1. The linear hyperpolarized current was significantly larger in ras-transformed than in untransformed cells. We conclude that the linear hyperpolarized current is an ATP-sensitive K⁺ current required for progression of MCF-7 cells through G1 phase. Received: 22 January 1999/Revised: 11 May 1999     

Membrane Potassium Channels and Human Bladder Tumor Cells. I. Electrical Properties

These experiments were conducted to determine the membrane K⁺ currents and channels in human urinary bladder (HTB-9) carcinoma cells in vitro. K⁺ currents and channel activity were assessed by the whole-cell voltage clamp and by either inside-out or outside-out patch clamp recordings. Cell depolarization resulted in activation of a Ca²⁺-dependent outward K⁺ current, 0.57 ± 0.13 nS/pF at −70 mV holding potential and 3.10 ± 0.15 nS/pF at 30 mV holding potential. Corresponding patch clamp measurements demonstrated a Ca²⁺-activated, voltage-dependent K⁺ channel (K_Ca) of 214 ± 3.0 pS. Scorpion venom peptides, charybdotoxin (ChTx) and iberiotoxin (IbTx), inhibited both the activated current and the K_Ca activity. In addition, on-cell patch recordings demonstrated an inwardly rectifying K⁺ channel, 21 ± 1 pS at positive transmembrane potential (V _m ) and 145 ± 13 pS at negative V _m . Glibenclamide (50 μm), Ba²⁺ (1 mm) and quinine (100 μm) each inhibited the corresponding nonactivated, basal whole-cell current. Moreover, glibenclamide inhibited K⁺ channels in inside/out patches in a dose-dependent manner, and the IC₅₀= 46 μm. The identity of this K⁺ channel with an ATP-sensitive K⁺ channel (K_ATP) was confirmed by its inhibition with ATP (2 mm) and by its activation with diazoxide (100 μm). We conclude that plasma membranes of HTB-9 cells contain the K_Ca and a lower conductance K⁺ channel with properties consistent with a sulfonylurea receptor-linked K_ATP. Received: 12 June 1997/Revised: 21 October 1997     

The Role of H<Superscript>+</Superscript>/OH<Superscript>−</Superscript> Channels in the Salt Stress Response of <Emphasis Type="Italic">Chara australis</Emphasis>

We investigate the electrophysiological salt stress response of the salt-sensitive charophyte Chara australis as a function of time in saline artificial pond water (saline APW) containing 50 mM NaCl and 0.1 mM CaCl₂. The effects are due to an increase in Na⁺ concentration rather than an increase in Cl⁻ concentration or medium osmolarity. A previous paper (Shepherd et al. Plant Cell Environ 31:1575–1591, 2008) described the rise in the background conductance and inhibition of proton pumping in saline APW in the first 60 min. Here we investigate the shift of membrane potential difference (PD) to levels above −100 mV and the change of shape of the current–voltage (I/V) profiles to upwardly concave. Arguing from thermodynamics, the I/V characteristics can be modeled by channels that conduct H⁺ or OH⁻. OH⁻ was chosen, as H⁺ required an unrealistic increase in the number/permeability of the channels at higher pH levels. Prolonged exposure to saline APW stimulated opening of more OH⁻ channels. Recovery was still possible even at a PD near −50 mV, with partial return of proton pumping and a decrease in OH⁻ current following APW wash. Upon change of pH from 7 to 9, the response was consistent with previously observed I/V characteristics of OH⁻ channels. For a pH change to 6, the response was transient before channel closure but could still be modeled. The consequences of opening of H⁺ or OH⁻ channels while the cell is under salt stress are discussed.     

Large Conductance Ca2+-Activated K+ Channels in Human Meningioma Cells

Cells from ten human meningiomas were electrophysiologically characterized in both living tissue slices and primary cultures. In whole cells, depolarization to voltages higher than +80 mV evoked a large K⁺ outward current, which could be blocked by iberiotoxin (100 nm) and TEA (half blocking concentration IC₅₀= 5.3 mm). Raising the internal Ca²⁺ from 10 nm to 2 mm shifted the voltage of half-maximum activation (V _1/2) of the K⁺ current from +106 to +4 mV. Respective inside-out patch recordings showed a voltage- and Ca²⁺-activated (BK _Ca ) K⁺ channel with a conductance of 296 pS (130 mm K⁺ at both sides of the patch). V _1/2 of single-channel currents was +6, −12, −46, and −68 mV in the presence of 1, 10, 100, and 1000 μm Ca²⁺, respectively, at the internal face of the patch. In cell-attached patches the open probability (P _o ) of BK _Ca channels was nearly zero at potentials below +80 mV, matching the activation threshold for whole-cell K⁺ currents with 10 nm Ca²⁺ in the pipette. Application of 20 μm cytochalasin D increased P _o of BK _Ca channels in cell-attached patches within minutes. These data suggest that the activation of BK _Ca channels in meningioma cells does not only depend on voltage and internal Ca²⁺ but is also controlled by the cytoskeleton. Received 18 June 1999/Revised: 18 January 2000     

Oxygen consumption rate and Na<Superscript>+</Superscript>/K<Superscript>+</Superscript>-ATPase activity in early developmental stages of the sea urchin <Emphasis Type="Italic">Paracentrotus lividus</Emphasis> Lam.

Changes in oxygen consumption rate and Na⁺/K⁺-ATPase activity during early development were studied in the sea urchin Paracentrotus lividus Lam. The oxygen consumption rate increased from 0.12 μmol O₂ mg protein⁻¹ h⁻¹ in unfertilized eggs to 0.38 μmol O₂ mg protein⁻¹ h⁻¹ 25 min after fertilization. Specific activity of the Na⁺/K⁺-ATPase was significantly stimulated after fertilization, ranging up to 1.07 μmol P_i h⁻¹ mg protein⁻¹ in the late blastula stage and slightly lower values in the early and late pluteus stages.     

Pronounced differences between the native K+ channels and KAT1 and KST1 α-subunit homomers of guard cells

Stomatal opening is the result of K⁺-salt accumulation in guard cells. Potassium uptake in these motor cells is mediated by voltage-dependent, K⁺-selective ion channels. Here we compare the in-vitro properties of two guard-cell K⁺-channel α-subunits from Arabidopsis thaliana (L.) Heynh. (KAT1) and Solanum tuberosum L. (KST1) after heterologous expression with the respective K⁺-transport characteristics in their mother cell. The KAT1 and KST1 subunits when expressed in Xenopus oocytes shared the basic features of the K⁺-uptake channels in the corresponding guard cells, including voltage dependence and single-channel conductance. Besides these similarities, the electrophysiological comparison of K⁺ channels in the homologous and the heterologous expression systems revealed pronounced differences with respect to modulation and block by extracellular cations. In the presence of 1 mM Cs⁺, 50% of the guard-cell K⁺-uptake channels (GCKC1_in) in A. thaliana and S. tuberosum, were inhibited upon hyperpolarization to −90 mV. For a similar effect on KAT1 and KST1 in oocytes, voltages as negative as −155 mV were required. In contrast, compared to the K⁺ channels in vivo the functional α-subunit homomers almost lacked a voltage-dependent block by extracellular Ca²⁺. Similar to the block by Cs⁺ and Ca²⁺, the acid activation of the α-homomers was less pronounced in oocytes. Upon acidification the voltage-dependence shifted by 82 and 90 mV for GCKCL_in in A. thaliana and S. tuberosum, respectively, but only by 25 mV for KAT1 and KST1. From the differences in K⁺-channel modulation in vivo and after heterologous expression we conclude that the properties of functional guard-cell K⁺-uptake channels result either from the heterometric assembly of different α-subunits or evolve from cell-type-specific posttranslational modification. Received: 6 March 1998 / Accepted: 9 July 1998     

Pannexin1 channels act downstream of P2X7 receptors in ATP-induced murine T-cell death

Death of murine T cells induced by extracellular ATP is mainly triggered by activation of purinergic P2X₇ receptors (P2X₇Rs). However, a link between P2X₇Rs and pannexin1 (Panx1) channels, which are non-selective, has been recently demonstrated in other cell types. In this work, we characterized the expression and cellular distribution of pannexin family members (Panxs 1, 2 and 3) in isolated T cells. Panx1 was the main pannexin family member clearly detected in both helper (CD4⁺) and cytotoxic (CD8⁺) T cells, whereas low levels of Panx2 were found in both T-cell subsets. Using pharmacological and genetic approaches, Panx1 channels were found to mediate most ATP-induced ethidium uptake since this was drastically reduced by Panx1 channel blockers (¹⁰Panx1, Probenecid and low carbenoxolone concentration) and absent in T cells derived from Panx1^?/? mice. Moreover, electrophysiological measurements in wild-type CD4⁺ cells treated with ATP unitary current events and pharmacological sensitivity compatible with Panx1 channels were found. In addition, ATP release from T cells treated with 4Br-A23187, a calcium ionophore, was completely blocked with inhibitors of both connexin hemichannels and Panx1 channels. Panx1 channel blockers drastically reduced the ATP-induced T-cell mortality, indicating that Panx1 channels mediate the ATP-induced T-cell death. However, mortality was not reduced in T cells of Panx1^?/? mice, in which levels of P2X₇Rs and ATP-induced intracellular free Ca²⁺ responses were enhanced suggesting that P2X₇Rs take over Panx1 channels lose-function in mediating the onset of cell death induced by extracellular ATP.     

Voltage-dependent Conductance Changes in a Nonvoltage-activated Sodium Current from a Mast Cell Line

Nonexcitable cells do not express voltage-activated Na⁺ channels. Instead, selective Na⁺ influx is accomplished through GTP-activated Na⁺ channels, the best characterized of which are found in renal epithelia. We have described recently a GTP-dependent Na⁺ current in rat basophilic leukemia (RBL) cells that differs from previous reported Na⁺ channels in several ways including selectivity, pharmacology and mechanism of activation. In this report, we have investigated the biophysical properties of the RBL cell Na⁺ current using the whole cell patch-clamp technique. Following activation by 250–500 μm GTPγS, hyperpolarizing steps to a fixed potential (−100 mV) from a holding potential of 0 mV evoked transient inward Na⁺ currents that declined during the pulse. If the holding potential was made more positive (range 0 to +100 mV), then the amplitude of the transient inward current evoked by the hyperpolarization increased steeply, demonstrating that the conductance of the channels was voltage-dependent. Using a paired pulse protocol (500 msec pulses to −100 mV from a holding potential of 0 mV), it was found that the peak amplitude of the current during the second pulse became larger as the interpulse potential became more positive. In addition, increasing the time at which the cells were held at positive potentials also resulted in larger currents, indicating a time-dependent conductance change. With symmetrical Na⁺ solutions, outward currents were recorded at positive potentials and these demonstrated both a time- and voltage-dependent increase in conductance. The results show that a nonvoltage activated Na⁺ channel in an electrically nonexcitable cell undergoes prominent voltage-dependent transitions. Possible mechanisms underlying this voltage dependency are discussed. Received: 12 March 1998/Revised: 5 June 1998