Potassium currents in cultured rabbit retinal pigment epithelial cells

Membrane potential and ionic currents were studied in cultured rabbit retinal pigment epithelial (RPE) cells using whole-cell patch clamp and perforated-patch recording techniques. RPE cells exhibited both outward and inward voltage-dependent currents and had a mean membrane capacitance of 26±12 pF (sd, n=92). The resting membrane potential averaged ?31±15 mV (n=37), but it was as high as ?60 mV in some cells. When K⁺ was the principal cation in the recording electrode, depolarization-activated outward currents were apparent in 91% of cells studied. Tail current analysis revealed that the outward currents were primarily K⁺ selective. The most frequently observed outward K⁺ current was a voltage- and time-dependent outward current (I _K) which resembled the delayed rectifier K⁺ current described in other cells. I _K was blocked by tetraethylammonium ions (TEA) and barium (Ba²⁺) and reduced by 4-aminopyridine (4-AP). In a few cells (3–4%), depolarization to ?50 mV or more negative potentials evoked an outwardly rectifying K⁺ current (I _Kt) which showed more rapid inactivation at depolarized potentials. Inwardly rectifying K⁺ current (I _KI) was also present in 41% of cells. I _KI was blocked by extracellular Ba²⁺ or Cs⁺ and exhibited time-dependent decay, due to Na⁺ blockade, at negative potentials. We conclude that cultured rabbit RPE cells exhibit at least three voltage-dependent K⁺ currents. The K⁺ conductances reported here may provide conductive pathways important in maintaining ion and fluid homeostasis in the subretinal space.     

Veratridine blocks voltage-gated potassium current in human T lymphocytes and in mouse neuroblastoma cells

(i) Effects of veratridine on ionic conductances of human peripheral blood T lymphocytes have been investigated using the whole-cell patch-clamp technique, (ii) Veratridine reduces the net outward current evoked by membrane depolarizations. The reduction originates from block of a 4-aminopyridine-sensitive, voltage-gated K⁺ current, (iii) Human T lymphocytes do not appear to express voltage-gated Na⁺ channels, since inward currents are observed neither in control nor in veratridine- and bretylium-exposed lymphocytes. (iv) The effect of veratridine consists of an increase in the rate of decay of the voltage-gated K⁺ current and a reduction of the peak current amplitude. Both effects depend on veratridine concentration. Halfmaximum block occurs at 97 m and the time constant of decay is reduced by 50% at 54 m of veratridine. (v) Possible mechanisms of veratridine action are discussed. The increased rate of K⁺ current decay is most likely due to open channel block. The decrease of current amplitude may involve an additional mechanism. (vi) In cultured mouse neuroblastoma N1E-115 cells, veratridine blocks a component of voltage-gated K⁺ current, in addition to its effect on voltage-gated Na⁺ current. This result shows that the novel effect of veratridine is not confined to lymphocytes.We thank Jacobien Künzel of the Wilhelmina Hospital for Children, Utrecht, for providing the blood samples and Aart de Groot for technical assistance. The research was supported by a fellowship of the Royal Netherlands Academy of Arts and Sciences to M. Oortgiesen.     

Characterization of ion channels in the plasma membrane of epidermal cells of expanding pea (Pisum sativum arg) leaves

Ion channels in isolated patches of the plasma membrane of pea (Pisum sativum arg) epidermal cells were studied with the patch-clamp technique. One anion and one cation channel were dominantly present in most trials. The anion channel conducts nitrate, halides and malate, with a conductance in symmetrical 100 mm Cl^– of 300 pS and can be blocked by SITS when applied to the cytoplasmic side of the membrane. The cation channel poorly discriminates between potassium, sodium and lithium, is not blocked by either TEA or Ba²⁺, and has a conductance of 35 pS in symmetrical 100 mm K⁺. The open probability of the cation channel increases with increase of the Ca²⁺ concentration on the cytoplasmic side of the membrane from 0.1 to 1 m. The possible role of these two channels in the physiology of epidermal cells is discussed.This work was supported by NSF grant DCB-890 3744 to E.V.     

Plasma membrane Ca2+ channels in roots of higher plants and their role in aluminium toxicity

Single Ca²⁺ channel records were obtained from plasma membrane-enriched fractions of wheat roots incorporated into artificial planar lipid bilayers. The channel had a unitary conductance of 15 pS for a 10 to 95 mM CaCl₂ gradient (cytoplasm: outside of the cell). The voltage dependence displayed by the channel agreed with that expected for Ca²⁺ channels in the plasma membrane. The channel gating was strongly modified by addition of 20 M extracellular verapamil (a Ca²⁺ channel antagonist). Extracellular AlCl₃ (70 M, pH 4.9) almost completely blocked the channel.     

Effects of D2O on permeation and gating in the Ca2+-activated potassium channel fromChara

We studied the effects of H₂O/D₂O substitution on the permeation and gating of the large conductance Ca²⁺-activated K⁺ channels inChara gymnophylla droplet membrane using the patchclamp technique. The selectivity sequence of the channel was: K⁺>Rb⁺≫Li⁺, Na⁺, Cs⁺ and Cl⁻. The conductance of this channel in symmetric 100mm KCl was found to be 130 pS. The single channel conductance was decreased by 15% in D₂O as compared to H₂O. The blockade of channel conductance by cytosolic Ca²⁺ weakened in D₂O as a result of a decrease in zero voltage Ca²⁺ binding affinity by a factor of 1.4. Voltage-dependent channel gating was affected by D₂O primarily due to the change in Ca²⁺ binding to the channel during the activation step. The Hill coefficient for Ca²⁺ binding was 3 in D₂O and around 1 in H₂O. The values of the Ca²⁺ binding constant in the open channel conformation were 0.6 and 6 μm in H₂O and D₂O, respectively, while the binding in the closed conformation was much less affected by D₂O. The H₂O/D₂O substitution did not produce a significant change in the slope of channel voltage dependence but caused a shift as large as 60 mV with 1mm internal Ca²⁺.     

ATP-inhibited and Ca2+-dependent K+ channels in the soma membrane of cultured leech Retzius neurons

The properties of one ATP-inhibited and one Ca²⁺-dependent K⁺ channel were investigated by the patch-clamp technique in the soma membrane of leech Retzius neurons in primary culture. Both channels rectify at negative potentials. The ATP-inhibited K⁺ channel with a mean conductance of 112 pS is reversibly blocked by ATP (K _i = 100 m), TEA (K _i =0.8 mm) and 10 mm Ba²⁺ and irreversibly blocked by 10 nm glibenclamide and 10 m tolbutamide. It is Ca²⁺ and voltage independent. Its open state probability (P _o) decreases significantly when the pH at the cytoplasmic face of inside-out patches is altered from physiological to acid pH values. The Ca²⁺-dependent K⁺ channel with a mean conductance of 114 pS shows a bell-shaped Ca²⁺ dependence of P _o with a maximum at pCa 7–8 at the cytoplasmic face of the membrane. The P _o is voltage independent at the physiologically relevant V range. Ba²⁺ (10 mm) reduces the single channel amplitude by around 25% (ATP, TEA, glibenclamide, tolbutamide, and Ba²⁺ were applied to the cytoplasmic face of the membrane).We conclude that the ATP-dependent K⁺ channel may play a role in maintaining the membrane potential constant—independently from the energy state of the cell. The Ca²⁺-dependent K⁺ channel may play a role in generating the resting membrane potential of leech Retzius neurons as it shows maximum activity at the physiological intracellular Ca²⁺ concentration.This study was supported by the Deutsche Forschungsgemeinschaft (W.-R. Schlue) and by a fellowship of the Konrad-Adenauer-Stiftung (G. Frey). We thank Dr. Draeger (Hoechst AG) for the gift of glibenclamide. The data are part of a future Ph.D. thesis of G. Frey.     

Tachykinins Potentiate N-Methyl-D-Aspartate Responses in Acutely Isolated Neurons from the Dorsal Horn

Abstract: Substance P and neurokinin A both potentiated N -methyl- d -aspartate (NMDA)-induced currents recorded in acutely isolated neurons from the dorsal horn of the rat. To elucidate the mechanism underlying this phenomenon, we measured the effects of tachykinins and glutamate receptor agonists on [Ca²⁺]_i in these cells. Substance P, but not neurokinin A, increased [Ca²⁺]_i in a subpopulation of neurons. The increase in [Ca²⁺]_i was found to be due to Ca²⁺ influx through voltage-sensitive Ca²⁺ channels. Substance P and neurokinin A also potentiated the increase in [Ca²⁺]_i produced by NMDA, but not by α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, kainate, or 50 m M K⁺. Phorbol esters enhanced the effects of NMDA and staurosporine inhibited the potentiation of NMDA effects by tachykinins. It is concluded that activation of protein kinase C may mediate the enhancement of NMDA effects by tachykinins in these cells. However, the effects of tachykinins on [Ca²⁺]_i can be dissociated from their effects on NMDA receptors.