Inwardly rectifying K+ currents in fetal alveolar type II cells: regulation by protein kinase A and protein phosphatases

Fetal guinea-pig lung alveolar type II (ATII) cells have inwardly rectifying (IR) K⁺ currents that display Mg²⁺- and G-protein-dependent run-down. We have used the whole-cell patch-clamp technique to investigate further the regulation of these currents. Under control conditions [KCl-rich pipette solution (1 mM free Mg²⁺, 10 nM free Ca²⁺) and KCl-rich bath solution], we found that IR K⁺ currents diminished with a t _1/2 of 7.6 min and were absent by 30 min. Experimental manoeuvres designed to inhibit phosphorylation increased the rate of current run-down. Thus, intracellular addition of 100 μM H-7, a general kinase inhibitor, reduced the t _1/2 to 4.7 min and the currents were absent by 16 min. Similarly, protein kinase A (PKA) inhibitor peptide (50 nM) also accelerated run-down. Agents known to increase phosphorylation, such as db-cAMP (0.5 mM) and forskolin (10 μM), resulted in a significant slowing of run-down (t _1/2>16 min) as did intracellular addition of the catalytic subunit of PKA (100 nM). Similarly, inhibition of dephosphorylation by either 1 μM okadaic acid [protein phosphatase 1/2A (PP-1/2A) inhibitor] or anti-human protein phosphatase 2Cα (PP2C) antiserum decreased the rate of run-down. These results indicate that the phosphorylation-dependent activation state of the fetal ATII cell IR K⁺ channel is regulated by a complex interplay of kinases and phosphatases involving PKA (activation), and PP2C and PP-1/2A (inactivation). Received: 10 February 1999 / Received after revision and accepted: 31 March 1999     

Characterisation of Ca2+-dependent inwardly rectifying K+ currents in HeLa cells

The whole-cell configuration of the patch-clamp technique was used to examine K⁺ currents in HeLa cells. Under quasi-physiological ionic gradients, using an intracellular solution containing 10^–7 mol/l free Ca²⁺, mainly outward currents were observed. Large inwardly rectifying currents were elicited in symmetrical 145 mmol/l KCl. Replacement of all extracellular K⁺ by isomolar Na⁺, greatly decreased inward currents and shifted the reversal potential as expected for K⁺ selectivity. The inwardly rectifying K⁺ currents exhibited little or no apparent voltage dependence within the range of from –120 mV to 120 mV. A square-root relationship between chord conductance and [K⁺]₀ at negative potentials could be established. The inwardly rectifying nature of the currents was unaltered after removal of intracellular Mg²⁺ and chelation with ATP and ethylenediaminetetraacetic acid (EDTA). Permeability ratios for other monovalent cations relative to K⁺ were: K⁺ (1.0)>Rb⁺ (0.86)>Cs⁺ (0.12)>Li⁺ (0.08)>Na⁺ (0.03). Slope conductance ratios measured at –100 mV were: Rb⁺ (1.66)>K⁺ (1.0)>Na⁺ (0.09)>Li⁺ (0.08)>Cs⁺ (0.06). K⁺ conductance was highly sensitive to intracellular free Ca²⁺ concentration. The relationship between conductance at 0 mV and Ca²⁺ concentration was well described by a Hill expression with a dissociation constant, K _D, of 70 nmol/l and a Hill coefficient, n, of 1.81. Extracellular Ba²⁺ blocked the currents in a concentration- and voltage-dependent manner. The dependence of the K _D for the blockade was analysed using a Woodhull-type treatment, locating the ion interaction site at 19 % of the distance across the electrical field of the membrane and a K _D (0 mV) of 7 mmol/l. Tetraethylammonium and 4-aminopyridine were without effect whilst quinine and quinidine blocked the currents with concentrations for half-maximum effects equal to 7 mol/l and 3.5 mol/l, respectively. The unfractionated venom of the scorpion Leiurus quinquestriatus (LQV) blocked the K⁺ currents of HeLa cells. The toxins apamin and scyllatoxin had no detectable effect whilst charybdotoxin, a component of LQV, blocked in a voltage-dependent manner with half-maximal concentrations of 40 nmol/l at –120 mV and 189 nmol/l at 60 mV; blockade by charybdotoxin accounts for the effect of LQV. Application of ionomycin (5–10 mol/l), histamine (1 mmol/l) or bradykinin (1–10 mol/l) to cells dialysed with low-buffered intracellular solutions induced K⁺ currents showing inward rectification and a lack of voltage dependence.     

Characterization of whole-cell currents in mucosal and connective tissue rat mast cells using amphotericin-B-perforated patches and temperature control

 Rat mucosal type mast cells are thought to possess only a K⁺-selective inwardly rectifying (IR_K) current in the resting state. We used rat-bone-marrow-derived mast cells (BMMCs) as a model of mucosal mast cells and recorded whole-cell membrane currents from cells perforated with amphotericin B. Under these conditions, both inwardly rectifying (IR) and outwardly rectifying (OR) currents were observed. The reversal potential and conductance of the IR current depended on the extracellular K⁺ concentration, indicating that the channel was K⁺ selective. The OR current was not affected by changes in extracellular K⁺ concentration, but lowering extracellular Cl^–concentration reduced the conductance and shifted the reversal potential in a positive direction. The OR current was not affected by K⁺ channel blockers, but was reversibly blocked by the chloride channel blocker 4,4’-diisothiocyanato-2,2’-stilbenedisulphonate (DIDS), again indicating a Cl^–conductance. The IR_K current was also detected in the majority of cells using the conventional whole-cell recording configuration at room temperature. In contrast, the OR_Cl current was only observed in 7% of recordings made at room temperature with the conventional whole-cell voltage-clamp mode, but was detected in 66% of cells if the bath temperature was increased and the integrity of the cell’s cytoplasm was preserved by using the perforated-patch technique. Under similar conditions, the OR_Cl current was also present in rat peritoneal mast cells, a connective tissue phenotype previously thought to have no whole-cell currents in the resting state. The role of this current and factors affecting its activation are discussed. Received: 10 May 1996 / Received after revision: 4 July 1996 / Accepted: 8 July 1996     

Blockade of the delayed rectifier K+ currents, I Kr, in rabbit sinoatrial node cells by external divalent cations

We investigated the actions of various divalent cations on the delayed rectifier K⁺ currents (I _Kr) in rabbit sinoatrial node cells using the whole-cell voltage-clamp technique in isotonic K⁺ solutions. External divalent cations decreased the amplitude of currents, accelerated the time course of deactivation and shifted the activation to positive potentials in a dose-dependent manner. The concentrations for half-maximum inhibition of the steady-state currents (K _M) obtained at 0 mV were 0.63, 1.36, 1.65 and 2.16 mM for Ni²⁺, Co²⁺, Mn²⁺ and Ba²⁺, respectively. The effect was voltage dependent (K _M decreased e-fold for 12.2–16.8 mV hyperpolarization), but the dependence did not vary significantly among different cations. Acceleration of the time course of current deactivation by the increase of divalent cation concentration was well fitted by the voltage-dependent block model, and the binding rate constant (k ₁) was obtained. The binding rates for the ions took the following order: Ni²⁺ >Co²⁺ >Mn²⁺ >Ba²⁺. The degree of the shift of activation occurred in the same order: Ni²⁺ >Co²⁺ >Mn²⁺ >Ba²⁺. From these results, we conclude that I _Kr channels are non-selectively blocked by most divalent cations from the external side and that the binding site is located deep inside the channel, resulting in a steep voltage dependence of the blockade. Received: 26 January 1999 / Received after revision: 16 March 1999 / Accepted: 18 March 1999     

Opioid potentiation of N-type Ca2+ channel currents via pertussis-toxin-sensitive G proteins in NG108-15 cells

Opioids have both inhibitory and stimulatory effects on neurotransmitter release. While the inhibitory effect has been ascribed to presynaptic inhibition of Ca²⁺ channels, the cellular mechanism underlying the stimulatory effect is not clear. In order to address this issue, we analyzed the effects of [d-Ala², d-Leu⁵]-enkephalin (DADLE) on whole-cell Ba²⁺ currents (I _Ba) through voltage-gated Ca²⁺ channels in NG108–15 neuroblastoma × glioma hybrid cells. Application of DADLE inhibited and washout of DADLE transiently potentiated I _Ba. Furthermore, potentiation of I _Ba was elicited even in the presence of DADLE, when inhibition was relieved by a large depolarizing prepulse. DADLE-induced potentiation, as well as inhibition, had both voltage-sensitive and -insensitive components and was abolished by treatment with ICI174864, a δ-opioid antagonist, pertussis toxin (PTX) and ω-conotoxin GVIA. Potentiation developed over @3 min and took 5–20 min to recover, whereas inhibition was complete within 30 s and recovered within 1 min. Although this potentiation should contribute to DADLE-induced desensitization of Ca²⁺ channel inhibition, it was not the sole mechanism for desensitization. We conclude that the δ-opioid receptor exerts a dual action on N-type Ca²⁺ channels via PTX-sensitive G proteins, i.e., rapid inhibition followed by slowly developing potentiation. Received: 31 March 1999 / Received after revision: 27 April 1999 / Accepted: 28 April 1999     

Modulation of large conductance Ca2+-activated K+ channel by Gαh (transglutaminase II) in the vascular smooth muscle cell

 Among G-proteins, G_h is unique in structural differences in the GTP-binding domain and possessing transglutaminase activity. We have studied the role of G protein in modulation of large conductance Ca²⁺-activated K⁺ (Maxi-K⁺) channel by the inside-out mode of patch clamp in smooth muscle cells from superior mesenteric artery of the rabbit. When the non-hydrolyzable GTP analogue, GTPγS, was applied, the channel activity was increased about 2.5-fold. Addition of GDPβS resulted in reversal of the GTPγS effect. When the Gα_h7 antibody was applied, the GTPγS-stimulated channel activity was significantly inhibited to control level, suggesting that Gα_h is involved in activation of the Maxi-K⁺ channel in smooth muscle cells. Received: 23 September 1996 / Received after revision: 26 November 1996 / Accepted: 3 December 1996     

Regulation of ionic currents by protein kinase A and intracellular calcium in outer hair cells isolated from the guinea-pig cochlea

 Two prominent potassium currents, termed I _K and I _K,n, and a cation current are found in outer hair cells (OHCs) of the guinea-pig cochlea. We report here whole-cell recordings which indicate that the currents are regulated by intracellular factors. 8-bromo-cAMP (500 μM), a membrane-permeable cAMP analogue, activated potassium currents in OHCs in both apical and basal turns of the cochlea. In OHCs from the cochlear apex, the drug effect was largest at potentials positive to –40 mV, indicating I _K as the target. In short cells from the cochlear base, both I _K and I _K,n were affected. The effects of 8-bromo-cAMP could be blocked by the presence of 1 μM H-89 (a protein kinase A inhibitor) in the patch pipette solution. Extracellular application of 10 nM okadaic acid, a protein phosphatase inhibitor, also activated both potassium currents. Currents were also modulated by intracellular calcium. I _K was activated in long cells by photorelease of calcium from the caged compound nitr5. Cation current activation required calcium release by photolysis of DM-nitrophen, a compound releasing more calcium. The results show that OHC potassium channels are regulated by background phosphorylation through protein kinase A and dephosphorylation by protein phosphatase. Cellular calcium also activates I _K and the cation channel, but with different sensitivities. Received: 1 September 1998 / Received after revision: 21 October 1998 / Accepted: 22 October 1998     

Na+-dependent regulation of the free Mg2+ concentration in neuropile glial cells and P neurones of the leech Hirudo medicinalis

 To investigate the Mg²⁺ regulation in neuropile glial (NG) cells and pressure (P) neurones of the leech Hirudo medicinalis the intracellular free Mg²⁺ ([Mg²⁺]_i) and Na⁺ ([Na⁺]_i) concentrations, as well as the membrane potential (E _m), were measured using Mg²⁺- and Na⁺-selective microelectrodes. The mean steady-state values of [Mg²⁺]_i were found to be 0.91 mM (mean E _m=–63.6 mV) in NG cells and 0.20 mM (mean E _m=–40.6 mV) in P neurones with a [Na⁺]_i of 6.92 mM (mean E _m=–61.6 mV) and 7.76 mM (mean E _m=–38.5 mV), respectively. When the extracellular Mg²⁺ concentration ([Mg²⁺]_o) was elevated, [Mg²⁺]_i in P neurones increased within 5–20 min whereas in NG cells a [Mg²⁺]_i increase occurred only after long-term exposure (6 h). After [Mg²⁺]_o was reduced back to 1 mM, a reduction of the extracellular Na⁺ concentration ([Na⁺]_o) decreased the inwardly directed Na⁺ gradient and reduced the rate of Mg²⁺ extrusion considerably in both NG cells and P neurones. In P neurones Mg²⁺ extrusion was reduced to 15.4% in Na⁺-free solutions and to 6.0% in the presence of 2 mM amiloride. Mg²⁺ extrusion from NG cells was reduced to 6.2% in Na⁺-free solutions. The results suggest that the major [Mg²⁺]_i-regulating mechanism in both cell types is Na⁺/ Mg²⁺ antiport. In P neurones a second, Na⁺-independent Mg²⁺ extrusion system may exist. Received: 11 August 1998 / Received after revision: 14 October 1998 / Accepted: 15 October 1998     

Modulation of voltage-dependent K+ channel by redox potential in pulmonary and ear arterial smooth muscle cells of the rabbit

 It has been suggested that hypoxic pulmonary vasoconstriction (HPV) results from the depolarization that is induced by the suppression of K⁺ current in pulmonary arterial smooth muscle cells (PASMC). We tested the hypothesis that the effect of the cellular redox potential on voltage-sensitive K⁺ (Kv) current is involved in HPV as a primary sensing mechanism. Kv current in PASMC and ear arterial smooth muscle cells (EASMC) of the rabbit was recorded using the whole-cell patch-clamp technique, and the effect of redox agents [dithiothreitol, DTT and 2,2’-dithio-bis(5-nitropyridine), DTBNP] was tested. Kv current was decreased by DTT, but increased by DTBNP. DTT accelerated the inactivation kinetics, but did not affect steady-state activation and inactivation, whereas DTBNP accelerated activation kinetics. Kv current has a non-inactivating window in the range of from –40 mV to +10 mV. The resting membrane potential measured using the nystatin-perforated-patch method, however, lay between –50 mV and –30 mV and was not depolarized by 5 mM 4-aminopyridine. The membrane-impermeable oxidizing agent DTNB has no effect on Kv current, suggesting that redox modulation sites are intracellular sulphydryl groups. In EASMC, Kv current was decreased by DTT, but increased by DTBNP, indicating that the redox-potential-induced modulation of Kv current in EASMC and in PASMC is the same. It is therefore concluded that Kv current is modulated by the cellular redox potential, but that this modulation is not involved in HPV as a primary sensing mechanism. Received: 8 May 1997 / Received after revision: 20 June 1997 / Accepted: 23 June 1997     

Hypoxic inhibition of K+ currents in isolated rat type I carotid body cells: evidence against the involvement of cyclic nucleotides

 Whole-cell patch-clamp recordings were used to evaluate the effects of the cyclic nucleotides adenosine 3’,5’-cyclic monophosphate (cAMP) and guanosine 3’,5’-cyclic monophosphate (cGMP) on ionic currents in type I carotid body cells isolated from rat pups, and to investigate whether cyclic nucleotides are involved in K⁺ current inhibition by hypoxia. In the presence of 500 μM isobutylmethylxanthine, currents were not significantly modified by 8-bromo-cAMP (2 mM), dibutyryl-cAMP (5 mM) or 8-bromo-cGMP (2 mM). Currents were also unaffected by the phosphodiesterase (PDE)-resistant protein kinase A activators Sp-cyclic adenosine-3′,5′-monophosphorothioate (Sp-cAMPS) and Sp-8-bromoadenosine-3′,5′-monophosphorothioate (Sp-8-bromo-cAMPS) (50 μM), or by β-phenyl-1,N ²-ethenoguanosine-3′,5′-cyclic monophosphate (PET-cGMP) (100 μM) or the nitric oxide donor S-nitroso-N-acetylpenicillamine (SNAP; 500 μM). Ca²⁺ channel currents were also unaffected by Sp-8-Br-cAMPS, PET-cGMP and SNAP at the same concentrations. In the absence of cyclic nucleotide analogues, hypoxia (P_O2 17–23 mmHg) reversibly inhibited K⁺ currents. This degree of hypoxic inhibition was not significantly altered by the PDE-resistant protein kinase A inhibitors Rp-cyclic adenosine-3′,5′-monophosphorothioate (Rp-cAMPS) (50 μM) or Rp-8-bromoadenosine-3′,5′-monophosphorothioate (Rp-8-bromo-cAMPS) (200 μM). Similarly, PET-cGMP (100 μM) and SNAP (500 μM) did not alter the degree of inhibition caused by hypoxia. At the same concentrations used in type I cell experiments, Sp-8-bromo-cAMPS, PET-cGMP and SNAP completely relaxed isolated guinea-pig basilar arteries preconstricted with 20 mM K⁺-containing solutions. Our results indicate that cyclic nucleotides alone are not an important factor in the regulation by O₂ tension of K⁺ currents in rat type I carotid body cells. Received: 12 June 1996 / Accepted: 14 August 1996