Potassium channels in human NK cells are involved in discrete stages of the killing process

Using the whole-cell variation of the patch-clamp technique, we have found a voltage-dependent K+ current in human natural killer (NK) cells. This K+ current is reduced in a dose-dependent manner by a variety of ion-channel blockers (verapamil, quinidine, 4-aminopyridine, Cd2+) at concentrations comparable to those that inhibit natural killing. Pretreatment of target cells with quinidine or verapamil did not significantly reduce their sensitivity to killing, whereas substantial inhibition of killing was observed after pretreatment of effector cells. Both verapamil and quinidine reduced the proportion of effector-target cell conjugates, suggesting that K channels play a role in the "binding" phase of the killing process. By adding EDTA or channel blockers as various times in a Ca-pulse assay system, we have also delineated a blocker-sensitive phase of bound conjugates that strictly corresponds with the Ca-dependent "programming" stage of killing. In contrast, the killer cell-independent stage, which is Ca2+ independent, apparently does not require functioning K channels. Verapamil and quinidine do not affect target cell sensitivity to the putative soluble mediator of natural killing, natural killer cytotoxic factor (NKCF), but inhibit release of NKCF from NK cells. Thus, the data suggest that K channels in NK cells play essential roles in the natural killing process that include events in the "programming-for-lysis" phase leading to release of NKCF.     

Basolateral potassium channels of rabbit colon epithelium: role in sodium absorption and chloride secretion

In order to assess the role of different classes of K(+) channels in recirculation of K(+) across the basolateral membrane of rabbit distal colon epithelium, the effects of various K(+) channel inhibitors were tested on the activity of single K(+) channels from the basolateral membrane, on macroscopic basolateral K(+) conductance, and on the rate of Na(+) absorption and Cl(-) secretion. In single-channel measurements using the lipid bilayer reconstitution system, high-conductance (236 pS), Ca(2+)-activated K(+) (BK(Ca)) channels were most frequently detected; the second most abundant channel was a low-conductance K(+) channel (31 pS) that exhibited channel rundown. In addition to Ba(2+) and charybdotoxin (ChTX), the BK(Ca) channels were inhibited by quinidine, verapamil and tetraethylammonium (TEA), the latter only when present on the side of the channel from which K(+) flow originates. Macroscopic basolateral K(+) conductance, determined in amphotericin-permeabilised epithelia, was also markedly reduced by quinidine and verapamil, TEA inhibited only from the lumen side, and serosal ChTX was without effect. The chromanol 293B and the sulphonylurea tolbutamide did not affect BK(Ca) channels and had no or only a small inhibitory effect on macroscopic basolateral K(+) conductance. Transepithelial Na(+) absorption was partly inhibited by Ba(2+), quinidine and verapamil, suggesting that BK(Ca) channels are involved in basolateral recirculation of K(+) during Na(+) absorption in rabbit colon. The BK(Ca) channel inhibitors TEA and ChTX did not reduce Na(+) absorption, probably because TEA does not enter intact cells and ChTX is 'knocked off' its extracellular binding site by K(+) outflow from the cell interior. Transepithelial Cl(-) secretion was inhibited completely by Ba(2+) and 293B, partly by quinidine but not by the other K(+) channel blockers, indicating that the small (<3 pS) K(V)LQT1 channels are responsible for basolateral K(+) exit during Cl(-) secretion. Hence different types of K(+) channels mediate basolateral K(+) exit during transepithelial Na(+) and Cl(-) transport.     

Effect of Organic and Inorganic Calcium Channel Blockers on γ-Amino-n-Butyiic Acid Release Induced by Monensin and Veratrine in the Absence of External Calcium

The effects of two organic Ca2+ antagonists (verapamil and nitrendipine) and of two inorganic Ca2+ channel blockers (Co2+ and ruthenium red) on the Na+-dependent release of gamma-amino-n-butyric acid (GABA) triggered by veratrine and monensin in the absence of external Ca2+ were studied in mouse brain synaptosomes. Ca2+-independent release of GABA stimulated by the Na+ channel activator veratrine was inhibited with micromolar concentrations of verapamil and nitrendipine. In contrast, GABA release induced by the Na+ ionophore monensin was insensitive to the organic Ca2+ antagonists. Verapamil also failed to modify A23187-stimulated release of GABA in the presence of Ca2+ but inhibited high K+-induced release of the transmitter. Co2+ partially diminished veratrine-induced release but did not change monensin-induced release. Releasing responses to monensin and veratrine were insensitive to ruthenium red, which inhibited the Ca2+-dependent component of GABA release evoked by high K+ depolarization. These data demonstrate that the mechanism of inducing GABA release is different for veratrine and monensin, as evidenced by their differing sensitivities to inhibition by Ca2+ channel antagonists and organic Ca2+ blockers. It is concluded that voltage-sensitive Ca2+ channels of the presynaptic membrane are not involved in the inhibitory action of Ca2+ antagonists on the Na+-dependent, Ca2+-independent mechanism of GABA release.     

Modulation of aminopyridine block of potassium currents in squid axon.

Aminopyridines are known to block potassium (K) currents in excitable membranes in a manner dependent upon membrane potential, such that the block is relieved by depolarization and restored upon repolarization. In the present study, the effects of aminopyridines on voltage-dependent potassium (K) channels were examined in internally perfused, voltage-clamped squid giant axons. The time course of block restoration after conditioning depolarization was found to be modulated by membrane electric field, K-channel gating, and external cations. Depolarized holding potentials accelerated block restoration without altering steady-state block levels, suggesting that the voltage dependence of block restoration may be related to K channel gating rather than drug binding per se. In support of this notion, low external calcium concentration, which shifts the voltage dependence of K-channel gating to more negative potentials, also accelerated block restoration. Conversely, the relationship between the rate of block restoration and membrane holding potential was shifted in the depolarizing direction by phloretin, an agent that shifts the dependence of K-channel opening on membrane potential in a similar manner. Modification of K-channel gating also was found to alter the rate of block restoration. Addition of internal zinc or internal treatment with glutaraldehyde slowed the time course of both K-channel activation and aminopyridine block restoration. Aminopyridines also were found to interact in the K channel with external Cs+, NH4+, and Rb+, each of which slowed aminopyridine block restoration. Our results suggest that aminopyridines enter and occlude K channels, and that the availability of the binding site may be modulated by channel gating such that access is limited by the probability of the channel reaching an intermediate closed state at the resting potential.     

Hypoxia-induced inhibition of whole cell membrane currents and ion transport of A549 cells

In excitable cells, hypoxia inhibits K channels, causes membrane depolarization, and initiates complex adaptive mechanisms. It is unclear whether K channels of alveolar epithelial cells reveal a similar response to hypoxia. A549 cells were exposed to hypoxia during whole cell patch-clamp measurements. Hypoxia reversibly inhibited a voltage-dependent outward current, consistent with a K current, because tetraethylamonium (TEA; 10 mM) abolished this effect; however, iberiotoxin (0.1 microM) does not. In normoxia, TEA and iberiotoxin inhibited whole cell current (-35%), whereas the K-channel inhibitors glibenclamide (1 microM), barium (1 mM), chromanol B293 (10 microM), and 4-aminopyridine (1 mM) were ineffective. (86)Rb uptake was measured to see whether K-channel modulation also affected transport activity. TEA, iberiotoxin, and 4-h hypoxia (1.5% O(2)) inhibited total (86)Rb uptake by 40, 20, and 35%, respectively. Increased extracellular K also inhibited (86)Rb uptake in a dose-dependent way. The K-channel opener 1-ethyl-2-benzimidazolinone (1 mM) increased (86)Rb uptake by 120% in normoxic and hypoxic cells by activation of Na-K pumps (+60%) and Na-K-2Cl cotransport (+170%). However, hypoxic transport inhibition was also seen in the presence of 1-ethyl-2-benzimidazolinone, TEA, and iberiotoxin. These results indicate that hypoxia, membrane depolarization, and K-channel inhibition decrease whole cell membrane currents and transport activity. It appears, therefore, that a hypoxia-induced change in membrane conductance and membrane potential might be a link between hypoxia and alveolar ion transport inhibition.     

Effects of Ca2+ Channel Blockers on Ca2+ Translocation Across Synaptosomal Membranes

The binding of [3H]nimodipine to purified synaptic plasma membranes (SPM) isolated from sheep brain cortex was characterized, and the effects of nimodipine, nifedipine, and (+)-verapamil on the [3H]nimodipine binding were compared to the effects on 45Ca2+ translocation under conditions that separate 45Ca2+ fluxes through Ca2+ channels from 45Ca2+ uptake via Na+/Ca2+ exchange. [3H]Nimodipine labels a single class of sites in SPM, with a KD of 0.64 +/- 0.1 nM, a Bmax of 161 +/- 27 fmol X mg-1 protein, and a Hill slope of 1.07, at 25 degrees C. Competition of [3H]nimodipine binding to purified SPM with unlabelled Ca2+ channel blockers shows that: nifedipine and nimodipine are potent competitors, with IC50 values of 4.7 nM and 5.9 nM, respectively; verapamil and (-)-D 600 are partial competitors, with biphasic competition behavior. Thus, (+)-verapamil shows an IC50 of 708 nM for the higher affinity component and the maximal inhibition is 50% of the specific binding, whereas for (-)-verapamil the IC50 is 120 nM, and the maximal inhibition is 30%; (-)-D 600 is even less potent than verapamil in inhibiting [3H]nimodipine binding (IC50 = 430 nM). However, (+)-verapamil, nifedipine, and nimodipine are less potent in inhibiting depolarization-induced 45Ca2+ influx into synaptosomes in the absence of Na+/Ca2+ exchange than in competing for [3H]nimodipine binding. Thus, (+)-verapamil inhibits Ca2+ influx by 50% at about 500 microM, whereas it inhibits 50% of the binding at concentrations 200-fold lower, and the discrepancy is even larger for the dihydropyridines. The Na+/Ca2+ exchange and the ATP-dependent Ca2+ uptake by SPM vesicles are also inhibited by the Ca2+ channel blockers verapamil, nifedipine, and d-cis-diltiazem, with similar IC50 values and in the same concentration range (10(-5)-10(-3) M) at which they inhibit Ca2+ influx through Ca2+ channels. We conclude that high-affinity binding of the Ca2+ blockers by SPM is not correlated with inhibition of the Ca2+ fluxes through channels in synaptosomes under conditions of minimal Na+/Ca2+ exchange. Furthermore, the relatively high concentrations of blockers required to block the channels also inhibit Ca2+ translocation through the Ca2+-ATPase and the Na+/Ca2+ exchanger. In this study, clear differentiation is made of the effects of the Ca2+ channel blockers on these three mechanisms of moving Ca2+ across the synaptosomal membrane, and particular care is taken to separate the contribution of the Na+/Ca2+ exchange from that of the Ca2+ channels under conditions of K+ depolarization.     

Ion channels in the thick ascending limb of Henle's loop.

The thick ascending limb of Henle's loop (TAL) is polarized with respect to its conductances. The luminal membrane contains a K+ conductance which is made up by the synchronous operation of 60- to 80-pS K+ channels. The basolateral membrane contains a chloride conductance. This conductance corresponds most likely to a 30- to 60-pS Cl- channel present in this membrane. Our knowledge on the properties of the K+ channels of these cells has been increased rapidly by patch clamp studies: these K+ channels are inwardly rectifying. They are highly selective for K+ over Na+, Li+ and many other cations. They do not conduct Rb+, Cs+, NH+4 or other larger cations. In fact, all these three cations as well as choline, tetraethylammonium, lidocaine, verapamil, diltiazem, quinine, quinidine and Ba2+ inhibit these K+ channels. As apparent from kinetic studies the mechanisms of inhibition are different for the various blockers. The TAL K+ channels are downregulated by increasing cytosolic Ca2+ activity. Cytosolic adenosine trisphosphate (ATP) has a similar effect. This ATP inhibition is Ca2+ dependent. The affinity to ATP is augmented by increasing Ca2+. Cytosolic alkalinity increases the open probability of these channels, and cytosolic acidification has the opposite effect. This pH dependence is very marked. A change by 0.2 pH units leads to a more than twofold change in the open-channel probability. The basolateral chloride conductance reflects the properties of an outwardly rectifying 30- to 60-pS Cl- channel. This channel behaves, in many respects, like the Cl- channels of a multitude of Cl- transporting epithelia. It is characterized by two open and two closed states. It is highly selective for Cl- as compared with larger anions, and it is inhibited reversibly by Cl- channel blockers such as 5-nitro-2-(3-phenylpropylamino)-benzoate.     

Autologous mixed-lymphocyte reaction in man. XVII. In vitro effect of ion channel-blocking agents on the autologous mixed-lymphocyte response

The in vitro effect of ion channel-blocking agents verapamil (V), 4-aminopyridine (4AP), tetraethylammonium (TEA), and quinine (Q) was examined on the proliferative response of human peripheral blood T lymphocytes in the autologous mixed-lymphocyte reaction (AMLR). All the above channel blockers in a dose-dependent manner inhibited the AMLR. Tetramethylammonium (TMA), an analog of TEA that does not block K+ channel currents, did not inhibit the AMLR. 4AP at 1 mM/ml concentration inhibited the expression of IL-2 receptors, as defined by monoclonal antibody anti-Tac, on T-cell activated in the AMLR. In vitro addition of recombinant interleukin 2(rIL-2) completely corrected the inhibition of the AMLR by channel blockers. Furthermore, the concentrations of ion channel blockers required for blocking 50% response of T cells in the AMLR was much lower than that reported for 50% block of T-cell proliferation in response to phytohemagglutinin or in allogeneic mixed-lymphocyte culture (MLC). These data suggest a role of ion channels in T-cell functions and show that the AMLR provides a more sensitive system, as compared to lectin stimulation or MLC, to examine any immunosuppressive effects of ion channel-blocking agents in disease states where they are used as therapeutic modalities.     

Analysis of hyposmolarity-induced taurine efflux pathways in the bullfrog sympathetic ganglia.

Hyposmolarity-induced taurine release was dependent on the decrease in medium osmolarity (5-50%) in the satellite glial cells of the bullfrog sympathetic ganglia. Release of GABA induced by hyposmolarity was much less than that of taurine. Omission of external Cl- replaced with gluconate totally suppressed taurine release, but only slightly suppressed GABA release. Bumetanide and furosemide, blockers of the Na+/K+/2Cl- cotransport system, inhibited taurine release by about 40%. Removal of external Na+ by replacement with choline, or omission of K+, suppressed taurine release by 40%. Antagonists of the Cl-/HCO3 exchange system, SITS, DIDS and niflumic acid, significantly reduced taurine release. The carbonic anhydrase inhibitor, acetazolamide, reduced the taurine release by 34%. Omission of external HCO3 by replacement with HEPES caused a 40% increase in the hyposmolarity-induced taurine release. Hyposmolarity-induced GABA release was not affected by bumetanide or SITS. Chloride channel blockers, 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) and N-phenylanthranilic acid (DPC), practically abolished taurine release. Blockers of K+ channels, clofilium and quinidine, had no effect on the taurine release. The hyposmolarity-induced taurine release was considerably enhanced by a simultaneous increase in external K+. GABA was not mediated by the same transport pathway as that of taurine. These results indicate that Cl- channels may be responsible for the hyposmolarity-induced taurine release, and that Na+/K+/2Cl- cotransporter and Cl-/HCO3 exchanger may contribute to maintain the intracellular Cl- levels higher than those predicted for a passive thermodynamic distribution in the hyposmolarity-induced taurine release.     

Distinction of two components of passive Ca2+ transport into human erythrocytes by Ca2+ entry blockers

The nature of downhill Ca2+ net-transport into human erythrocytes was investigated using the experimental models of Ca2+ pump inhibition by vanadate and of intracellular chelation of Ca2+ by quin2. Ca2+ uptake by erythrocytes loaded with 0.5 mM vanadate and suspended in 145 mM Na+ -5 mM K+ media was reduced by about 60% when medium K+ was raised to 80 mM. Organic and inorganic Ca2+ entry blockers such as nifedipine (10(-5) M), verapamil (10(-4) M), diltiazem (10(-4) M), Co2+ (1.5 mM) and Cu2+ (0.1 mM) as well as the K+ channel blocker quinidine (1mM) inhibited Ca2+ uptake in 145 mM Na+ -5 mM K+ media by 60-75%. Flunarizine was less effective. In vanadate-loaded cells suspended in 70 mM Na+ -80 mM K+ media, in contrast, flunarizine exerted a dose-dependent inhibition of Ca2+ uptake by up to 80% at 10(-5) M, the other blockers being ineffective (except for verapamil at 10(-4) M). A similar pattern of inhibition was seen in quin2-loaded erythrocytes. The different susceptibility towards inhibitors may indicate that passive Ca2+ uptake by vanadate-loaded erythrocytes suspended in 145 mM Na+ -5 mM K+ media, on the one hand, and by vanadate-loaded erythrocytes suspended in 70 mM Na+ -80 mM K+ media as well as by quin2-loaded erythrocytes, on the other hand, is mediated by two different transport components.     

Autoregulation of apical membrane Na+ permeability of tight epithelia. Noise analysis with amiloride and CGS 4270

Noise analysis of the Na+ channels of the apical membranes of frog skin bathed symmetrically in a Cl-HCO3 Ringer solution was done with amiloride and CGS 4270. Tissues were studied in their control states and after inhibition of transepithelial Na+ transport (Isc) by addition of quinine or quinidine to the apical solution. A critical examination of the amiloride-induced noise indicated that the single channel Na+ currents (iNa) were decreased by quinine and quinidine, probably because of depolarization of apical membrane voltage. Despite considerable statistical uncertainty in the methods of estimation of the Na+ channel density with amiloride-induced noise (NA, see text), the striking observation was a large increase of NA with amiloride inhibition of the rate of Na+ entry into the cells. NA was increased to 406% of control, whereas Isc was inhibited to 8.6% of control by 6 microM amiloride. Studies were done also with the Na+ channel blocker CGS 4270. Noise analysis with this compound was advantageous, permitting iCGSNa and NCGS to be measured in individual tissues with a relatively small inhibition of Isc. As with amiloride, inhibition of Isc with CGS 4270 caused large increases of the Na+ channel density (approximately 200% at approximately 35% inhibition of the Isc). Quinine and quinidine caused an approximately 50% increase of Na+ channel density while inhibiting iNa by approximately 60-70%. As inhibition of Na+ entry leads to an increase of Na+ channel density, a mechanism of autoregulation appears to be a major factor in adjusting the apical membrane Na+ permeability of the cells.     

Voltage-gated potassium channels in brown fat cells

We studied the membrane currents of isolated cultured brown fat cells from neonatal rats using whole-cell and single-channel voltage-clamp recording. All brown fat cells that were recorded from had voltage-gated K currents as their predominant membrane current. No inward currents were seen in these experiments. The K currents of brown fat cells resemble the delayed rectifier currents of nerve and muscle cells. The channels were highly selective for K+, showing a 58-mV change in reversal potential for a 10-fold change in the external [K+]. Their selectivity was typical for K channels, with relative permeabilities of K+ greater than Rb+ greater than NH+4 much greater than Cs+, Na+. The K currents in brown adipocytes activated with a sigmoidal delay after depolarizations to membrane potentials positive to -50 mV. Activation was half maximal at a potential of -28 mV and did not require the presence of significant concentrations of internal calcium. Maximal voltage-activated K conductance averaged 20 nS in high external K+ solutions. The K currents inactivated slowly with sustained depolarization with time constants for the inactivation process on the order of hundreds of milliseconds to tens of seconds. The K channels had an average single-channel conductance of 9 pS and a channel density of approximately 1,000 channels/cell. The K current was blocked by tetraethylammonium or 4-aminopyridine with half maximal block occurring at concentrations of 1-2 mM for either blocker. K currents were unaffected by two blockers of Ca2+-activated K channels, charybdotoxin and apamin. Bath-applied norepinephrine did not affect the K currents or other membrane currents under our experimental conditions. These properties of the K channels indicate that they could produce an increase in the K+ permeability of the brown fat cell membrane during the depolarization that accompanies norepinephrine-stimulated thermogenesis, but that they do not contribute directly to the norepinephrine-induced depolarization.     

Whole-cell and single channel K+ and Cl- currents in epithelial cells of frog skin.

Whole-cell and single channel currents were studied in cells from frog (R. pipiens and R. catesbiana) skin epithelium, isolated by collagenase and trypsin treatment, and kept in primary cultures up to three days. Whole-cell currents did not exhibit any significant time-dependent kinetics under any ionic conditions used. With an external K gluconate Ringer solution the currents showed slight inward rectification with a reversal potential near zero and an average conductance of 5 nS at reversal. Ionic substitution of the external medium showed that most of the cell conductance was due to K and that very little, if any, Na conductance was present. This confirmed that most cells originate from inner epithelial layers and contain membranes with basolateral properties. At voltages more positive than 20 mV outward currents were larger with K in the medium than with Na or N-methyl-D-glucamine. Such behavior is indicative of a multi-ion transport mechanism. Whole-cell K current was inhibited by external Ba and quinidine. Blockade by Ba was strongly voltage dependent, while that by quinidine was not. In the presence of high external Cl, a component of outward current that was inhibited by the anion channel blocker diphenylamine-2-carboxylate (DPC) appeared in 70% of the cells. This component was strongly outwardly rectifying and reversed at a potential expected for a Cl current. At the single channel level the event most frequently observed in the cell-attached configuration was a K channel with the following characteristics: inward-rectifying I-V relation with a conductance (with 112.5 mM K in the pipette) of 44 pS at the reversal potential, one open and at least two closed states, and open probability that increased with depolarization. Quinidine blocked by binding in the open state and decreasing mean open time. Several observations suggest that this channel is responsible for most of the whole-cell current observed in high external K, and for the K conductance of the basolateral membrane of the intact epithelium. On a few occasions a Cl channel was observed that activated upon excision and brief strong depolarization. The I-V relation exhibited strong outward rectification with a single channel conductance of 48 pS at 0 mV in symmetrical 112 mM Cl solutions. Kinetic analysis showed the presence of two open and at least two closed states. Open time constants and open probability increased markedly with depolarization.(ABSTRACT TRUNCATED AT 400 WORDS)     

Dihydropyridine Binding Sites Regulate Calcium Influx Through Specific Voltage-Sensitive Calcium Channels in Cerebellar Granule Cells

In primary cultures of cerebellar granule cells, [3H]nitrendipine binds with high affinity to a single site (KD 1 nM and Bmax 20 fmol/mg protein). The 1,4-dihydropyridine (DHP) class of compounds such as nitrendipine, nifedipine, and BAY K 8644 displace [3H]nitrendipine binding at nanomolar concentrations. Verapamil partially inhibits whereas diltiazem slightly increases the [3H]nitrendipine binding. In these cells, the calcium influx that is induced by depolarization is very rapid and is blocked by micromolar concentrations of inorganic calcium blockers such as cadmium, cobalt, and manganese. The calcium influx resulting from cell depolarization is potentiated by BAY K 8644 and partially inhibited (approximately 40%) by nitrendipine and nifedipine. Other non-DHP voltage-sensitive calcium channel (VSCC) antagonists, such as verapamil and diltiazem, completely blocked the depolarization-induced calcium influx. This suggested that nitrendipine and nifedipine block only a certain population of VSCCs. In contrast, verapamil and diltiazem do not appear to be selective and block all of VSCCs. Perhaps some VSCCs can be allosterically modulated by the binding site for the DHPs, whereas verapamil and diltiazem may block completely the function of all VSCCs by occupying a site that differs from the DHP binding site.     

Gating current and potassium channels in the giant axon of the squid.

Gating current (Ig) underlying Na-channel activation is large enough to enable resolution of components both preceding and paralleling Na conductance (gNa) turn-on. For large depolarizations (beyond +20 mV), an additional "slow phase" of Ig is observed during a time when Na activation is already complete, but when K-channel opening is just becoming detectable. If Na- and K-channel gating are similar, the slow kinetics and long delay for K activation predict that K channel Ig must be relatively small and slow. Externally applied dibucaine almost totally blocks gNa and greatly reduces the fast (Na channel) Ig without altering gK or the Ig slow phase. The slow phase of Ig depends in part of the presence of functional K channels. Selective diminution in amplitude of the slow phase is consistently observed after a 30-min perfusion with both external and internal K-free media, a procedure which destroys nearly all K channels. This decrease of Ig amounts to approximately 10% of the total charge movements at +40 to +80 mV, with gating charge and K channels disappearing in a ratio of less than 1 e- per picosiemens of gK. These findings are consistent with the idea that part of the Ig slow phase represents gating current generated by the early steps in K-channel activation.     

Extracellular Ca2+ ameliorates NaCl-induced K+ loss from Arabidopsis root and leaf cells by controlling plasma membrane K+ -permeable channels

Calcium can ameliorate Na+ toxicity in plants by decreasing Na+ influx through nonselective cation channels. Here, we show that elevated external [Ca2+] also inhibits Na+ -induced K+ efflux through outwardly directed, K+ -permeable channels. Noninvasive ion flux measuring and patch-clamp techniques were used to characterize K+ fluxes from Arabidopsis (Arabidopsis thaliana) root mature epidermis and leaf mesophyll under various Ca2+ to Na+ ratios. NaCl-induced K+ efflux was not related to the osmotic component of the salt stress, was inhibited by the K+ channel blocker TEA+, was not mediated by inwardly directed K+ channels (tested in the akt1 mutant), and resulted in a significant decrease in cytosolic K+ content. NaCl-induced K+ efflux was partially inhibited by 1 mm Ca2+ and fully prevented by 10 mm Ca2+. This ameliorative effect was at least partially attributed to a less dramatic NaCl-induced membrane depolarization under high Ca2+ conditions. Patch-clamp experiments (whole-cell mode) have demonstrated that two populations of Ca2+ -sensitive K+ efflux channels exist in protoplasts isolated from the mature epidermis of Arabidopsis root and leaf mesophyll cells. The instantaneously activating K+ efflux channels showed weak voltage dependence and insensitivity to external and internal Na+. Another population of K+ efflux channels was slowly activating, steeply rectifying, and highly sensitive to Na+. K+ efflux channels in roots and leaves showed different Ca2+ and Na+ sensitivities, suggesting that these organs may employ different strategies to withstand salinity. Our results suggest an additional mechanism of Ca2+ action on salt toxicity in plants: the amelioration of K+ loss from the cell by regulating (both directly and indirectly) K+ efflux channels.     

Ca2+ channel blockers inhibit secretory C1-channels in intestinal epithelial cells.

Outwardly rectifying Cl- channels are present in the human colonic cell line (HT29D4). The classical Cl- channel blocker 5-nitro-2(3-phenylpropylamino)benzoate inhibits Cl- channel activity with a K0.5 value of 20 microM. Epithelial Cl- channel activity is inhibited by Ca2+ channel blockers. Phenylalkylamines are the most effective inhibitors. (+/-)Verapamil and (-)desmethoxyverapamil induce flickering and then the complete blockade of Cl- channels recorded from outside-out patches. K0.5 values are 60 microM and 100 microM for (-)desmethoxyverapamil and (+/-)verapamil, respectively. Other classes of L-type Ca2+ channel blockers have also been studied but they are less active.     

Ligand-activated signal transduction in the 2-cell embryo

Platelet-activating factor (PAF) is an autocrine trophic/survival factor for the preimplantation embryo. PAF induced an increase in intracellular calcium concentration ([Ca2+]i) in the 2-cell embryo that had an absolute requirement for external calcium. L-type calcium channel blockers (diltiazem, verapamil, and nimodipine) significantly inhibited PAF-induced Ca2+ transients, but inhibitors of P/Q type (omega-agatoxin; omega-conotoxin MVIIC), N-type (omega-conotoxin GVIA), T-type (pimozide), and store-operated channels (SKF 96365 and econazole) did not block the transient. mRNA and protein for the alpha1-C subunit of L-type channels was expressed in the 2-cell embryo. The L-type calcium channel agonist (+/-) BAY K 8644 induced [Ca2+]i transients and, PAF and BAY K 8644 each caused mutual heterologous desensitization of each other's responses. Depolarization of the embryo (75 mM KCl) induced a [Ca2+]i transient that was inhibited by diltiazem and verapamil. Whole-cell patch-clamp measurements detected a voltage-gated channel (blocked by diltiazem, verapamil, and nifedipine) that was desensitized by prior responses of embryos to exogenous or embryo-derived PAF. Replacement of media Ca2+ with Mn2+ allowed Mn2+ influx to be observed directly; activation of a diltiazem-sensitive influx channel was an early response to PAF. The activation of a voltage-gated L-type calcium channel in the 2-cell embryo is required for normal signal transduction to an embryonic trophic factor.     

Molecular proximity of Kv1.3 voltage-gated potassium channels and beta(1)-integrins on the plasma membrane of melanoma cells: effects of cell adherence and channel blockers

Tumor cell membranes have multiple components that participate in the process of metastasis. The present study investigates the physical association of beta1-integrins and Kv1.3 voltage-gated potassium channels in melanoma cell membranes using resonance energy transfer (RET) techniques. RET between donor-labeled anti-beta1-integrin and acceptor-labeled anti-Kv1.3 channels was detected on LOX cells adherent to glass and fibronectin-coated coverslips. However, RET was not observed on LOX cells in suspension, indicating that molecular proximity of these membrane molecules is adherence-related. Several K(+) channel blockers, including tetraethylammonium, 4-aminopyridine, and verapamil, inhibited RET between beta1-integrins and Kv1.3 channels. However, the irrelevant K(+) channel blocker apamin had no effect on RET between beta1-integrins and Kv1.3 channels. Based on these findings, we speculate that the lateral association of Kv1.3 channels with beta1-integrins contributes to the regulation of integrin function and that channel blockers might affect tumor cell behavior by influencing the assembly of supramolecular structures containing integrins.     

Modulation of Dopamine Transporter Activity by Nicotinic Acetylcholine Receptors and Membrane Depolarization in Rat Pheochromocytoma PC12 Cells

To elucidate the regulation of the rat dopamine transporter (rDAT), we established several PC12 variants overexpressing the rDAT. Treating these cells with a nicotinic agonist (1,1-dimethyl-4-phenylpiperazinium iodide, 30 microM) depolarized the plasma membrane potential from -31 +/- 2 to 43 +/- 5 mV and inhibited rDAT activity significantly in a calcium- and protein kinase C-independent manner. Membrane depolarization by a high external K+ concentration or two K+ channel blockers (tetraethylammonium hydroxide and BaCl2) also resulted in a marked inhibition of rDAT activity. Such inhibition of dopamine uptake is due to a reduction in Vmax, with no marked effect on the Km for dopamine. The potency of cocaine in inhibiting dopamine uptake was not significantly altered, whereas that of amphetamine was slightly enhanced by membrane depolarization. Removing extracellular Ca2+ or blocking the voltage-sensitive L-type calcium channels using nifedipine did not exert any significant effect on the inhibition of rDAT activity by depolarization. These data confirm that calcium influx on depolarization is not required for inhibition of the rDAT. Collectively, our data suggest that rDAT activity can be altered by a neurotransmitter that modulates the membrane potential, thus suggesting an exquisite mechanism for the fine-tuning of dopamine levels in the synapse.