Ca2+-activated K+ conductance of human red cell membranes exhibits two different types of voltage dependence

Summary The voltage dependence for outward-going current of the Ca-activated K⁺ conductance (g _k (Ca)) of the human red cell membrane has been examined over a wide range of membrane potentials (V _m) at constant values of [K⁺]_ex, [K⁺]_c and pH_c, the intact cells being preloaded to different concentrations of ionized calcium. Outward-current conductances were calculated from initial net effluxes of K⁺ and the corresponding (V _m-E_k) values. The basic conductance, defined as the outward-current coductance at (V _m-E_k) 20 mV and [K⁺]_ex 3mM (B. Vestergaard-Bogind, P. Stampe and P. Christophersen,J. Membrane Biol. 95:121–130, 1987) was found to be a function of cellular ionized Ca. At all degrees of Ca activationg _K(Ca) was an apparently linear function of voltage (V _m range –40 to +70 mV), the absolute level as well as the slope decreasing with decreasing activation. In a simple two-state model the constant voltage dependence can, at the different degrees of Ca activation, be accounted for by a Boltzmann-type equilibrium function with an equivalent valence of 0.4, assuming chemical equilibrium atV _m=0 mV. Alternatively, the phenomenon might be explained by a voltage-dependent block of the outward current by an intracellular ion. Superimposed upon the basic conductance is the apparently independent inward-rectifying steep voltage function with an equivalent valence of 5 and chemical equilibrium at the givenE _K value.Abbreviations CCCP carbonyl cyanidem-chlorophenylhydrazone - DIDS 4,4-diisothiocyanostilbene-2,2-disul     

Voltage dependence of the Ca2+-activated K+ conductance of human red cell membranes is strongly dependent on the extracellular K+ concentration

Summary The conductance of the Ca²⁺-activated K⁺ channel (g _K(Ca)) of the human red cell membrane was studied as a function of membrane potential (V _m ) and extracellular K⁺ concentration ([K⁺]_ex). ATP-depleted cells, with fixed values of cellular K⁺ (145mm) and pH (7.1), and preloaded with 27 m ionized Ca were transferred, with open K⁺ channels, to buffer-free salt solutions with given K⁺ concentrations. Outward-current conductances were calculated from initial net effluxes of K⁺, correspondingV _m , monitored by CCCP-mediated electrochemical equilibration of protons between a buffer-free extracellular and the heavily buffered cellular phases, and Nernst equilibrium potentials of K ions (E _K) determined at the peak of hyperpolarization. Zero-current conductances were calculated from unidirectional effluxes of⁴²K at (V _m –E _K)0, using a single-file flux ratio exponent of 2.7. Within a [K⁺]_ex range of 5.5 to 60mm and at (V _m –E _K) 20 mV a basic conductance, which was independent of [K⁺]_ex, was found. It had a small voltage dependence, varying linearly from 45 to 70 S/cm² between 0 and –100 mV. As (V _m –E _K) decreased from 20 towards zero mVg _K(Ca) increased hyperbolically from the basic value towards a zero-current value of 165 S/cm². The zero-current conductance was not significantly dependent on [K⁺]_ex (30 to 156mm) corresponding toV _m (–50 mV to 0). A further increase ing _K(Ca) symmetrically aroundE _K is suggested as (V _m –E _K) becomes positive. Increasing the extracellular K⁺ concentration from zero and up to 3mm resulted in an increase ing _K(Ca) from 50 to 70 S/cm². Since the driving force (V _m –E _K) was larger than 20 mV within this range of [K⁺]_ex this was probably a specific K⁺ activation ofg _K(Ca). In conclusion: The Ca²⁺-activated K⁺ channel of the human red cell membrane is an inward rectifier showing the characteristic voltage dependence of this type of channel.     

Ca2+-activated K+ conductance causes membrane hyperpolarizations in a monkey kidney cell line (JTC-12)

Summary We have previously reported hyperpolarizing membrane potential changes in a monkey kidney cell line (JTC-12) which has characteristics resembling proximal tubular cells. These hyperpolarizations could be observed spontaneously or evoked by mechanically touching adjacent cells. In this report, we have shown further evidence that these hyperpolarizations are elicited by an increase in membrane conductance to K⁺ which is caused by an increase in cytosolic Ca²⁺ concentration. In addition, we have found another type of hyperpolarization which is evoked by applying flow of extracellular fluid to the cell. Intracellular injection of Ca²⁺ and Sr²⁺ evoked hyperpolarizations, while intracellular injection of Mn²⁺ and Ba²⁺ did not. Intracellular injection of EGTA suppressed both spontaneous and mechanically evoked hyperpolarizations. In Ca²⁺-free medium, both spontaneous and flow-evoked hyperpolarizations were not observed, while mechanical stimuli consistently evoked hyperpolarization. In Na⁺-free medium, the incidence of cells showing the spontaneous or flow-evoked hyperpolarization increased, and the amplitude and the duration of the mechanically evoked hyperpolarization became greater. Quinidine inhibited all types of hyperpolarization. These data suggest that hyperpolarizations in JTC-12 cells are due to an increase in Ca²⁺-activated K⁺ conductance.     

Ca2+-activated K+ permeability in human erythrocytes: Modulation of single-channel events

Elevated levels of intracellular Ca²⁺ activate a K⁺-selective permeability in the membrane of human erythrocytes. Currents through single channels were analysed in excised inside-out membrane patches. The effects of several ions that are known to inhibit K⁺ fluxes are described with respect to the single-channel events. The results suggest that the blocking ions can partly move into the channels (but cannot penetrate) and interact with other ions inside the pore. The reduction of single-channel conductance by Cs⁺, tetraethylammonium and Ba²⁺ and of single-channel activity by quinine and Ba²⁺ is referred to different rates of access to the channel. The concentration- and voltage-dependent inhibition by ions with measurable permeability (Na⁺ and Rb⁺) can be explained by their lower permeability, with single-file movement and ionic interactions inside the pore.     

Ca2+-activated K+ channels in the apical membrane ofNecturus choroid plexus

Summary The properties of Ca²⁺-activated K⁺ channels in the apical membrane of theNecturus choroid plexus were studied using single-channel recording techniques in the cell-attached and excised-patch configurations. Channels with large unitary conductances clustered around 150 and 220 pS were most commonly observed. These channels exhibited a high selectivity for K⁺ over Na⁺ and K⁺ over Cs⁺. They were blocked by high cytoplasmic Na⁺ concentrations (110mm). Channel activity increased with depolarizing membrane potentials, and with increasing cytoplasmic Ca²⁺ concentrations. Increasing Ca²⁺ from 5 to 500nm, increased open probability by an order of magnitude, without changing single-channel conductance. Open probability increased up to 10-fold with a 20-mV depolarization when Ca²⁺ was 500nm. Lowering intracellular pH one unit, decreased open probability by more than two orders of magnitude, but pH did not affect single-channel conductance. Cytoplasmic Ba²⁺ reduced both channel-open probability and conductance. The sites for the action of Ba²⁺ are located at a distance more than halfway through the applied electric field from the inside of the membrane. Values of 0.013 and 117mm were calculated as the apparent Ba²⁺ dissociation constants (K _d (0 mV) for the effects on probability and conductance, respectively. TEA⁺ (tetraethylammonium) reduced single-channel current. Applied to the cytoplasmic side, it acted on a site 20% of the distance through the membrane, with aK _d (0 mV)=5.6mm. A second site, with a higher affinity,K _d (0 mV)=0.23mm, may account for the near total block of chanel conductance by 2mm TEA⁺ applied to the outside of the membrane. It is concluded that the channels inNecturus choroid plexus exhibit many of the properties of maxi Ca²⁺-activated K⁺ channels found in other tissues.     

The beta subunit increases the Ca2+ sensitivity of large conductance Ca2+-activated potassium channels by retaining the gating in the bursting states

Coexpression of the beta subunit (KV,Cabeta) with the alpha subunit of mammalian large conductance Ca2+- activated K+ (BK) channels greatly increases the apparent Ca2+ sensitivity of the channel. Using single-channel analysis to investigate the mechanism for this increase, we found that the beta subunit increased open probability (Po) by increasing burst duration 20-100-fold, while having little effect on the durations of the gaps (closed intervals) between bursts or on the numbers of detected open and closed states entered during gating. The effect of the beta subunit was not equivalent to raising intracellular Ca2+ in the absence of the beta subunit, suggesting that the beta subunit does not act by increasing all the Ca2+ binding rates proportionally. The beta subunit also inhibited transitions to subconductance levels. It is the retention of the BK channel in the bursting states by the beta subunit that increases the apparent Ca2+ sensitivity of the channel. In the presence of the beta subunit, each burst of openings is greatly amplified in duration through increases in both the numbers of openings per burst and in the mean open times. Native BK channels from cultured rat skeletal muscle were found to have bursting kinetics similar to channels expressed from alpha subunits alone.     

Cell-cycle-dependent expression of the large Ca2+-activated K+ channels in breast cancer cells

In a previous work, we have reported that the ionic nature of the outward current recorded in MCF-7 cells was that of a K+ current. In this study, we have identified a Ca2+-activated K+ channel not yet described in MCF-7 human breast cancer cells. In cells arrested in the early G1 (depolarized cells), increasing [Ca2+]i induced both a shift in the I-V curve toward more negative potentials and an increase in current amplitude at negative and more at positive potential. Currents were inhibited by r-iberiotoxin (r-IbTX, 50 nM) and charybdotoxin (ChTX, 50 nM). These data indicate that human breast cancer cells express large-conductance Ca2+-activated K+ (BK) channels. BK current-density increased in cells synchronized at the end of G1, as compared with those in the early G1 phase. This increased current-density paralleled the enhancement in BK mRNA levels. Blocking BK channels with r-IbTX, ChTX or both induced a slight depolarization in cells arrested in the early G1, late G1, and S phases and accumulated cells in the S phase, but failed to induce cell proliferation. Thus, the expression of the BK channels was cell-cycle-dependent and seems to contribute more to the S phase than to the G1 phase. However, these K+ channels did not regulate the cell proliferation because of their minor role in the membrane potential.     

Rabbit distal colon epithelium: III. Ca2+-activated K+ channels in basolateral plasma membrane vesicles of surface and crypt cells

Summary In the mammalian distal colon, the surface epithelium is responsible for electrolyte absorption, while the crypts are the site of secretion. This study examines the properties of electrical potential-driven⁸⁶Rb⁺ fluxes through K⁺ channels in basolateral membrane vesicles of surface and crypt cells of the rabbit distal colon epithelium. We show that Ba²⁺-sensitive, Ca²⁺-activated K⁺ channels are present in both surface and crypt cell derived vesicles with half-maximal activation at 5×10^–7 m free Ca²⁺. This suggests an important role of cytoplasmic Ca²⁺ in the regulation of the bidirectional ion fluxes in the colon epithelium.The properties of K⁺ channels in the surface cell membrane fraction differ from those of the channels in the crypt cell derived membranes. The peptide toxin apamin inhibits Ca²⁺-activated K⁺ channels exclusively in surface cell vesicles, while charybdotoxin inhibits predominantely in the crypt cell membrane fraction. Titrations with H⁺ and tetraethylammonium show that both high-and low-sensitive⁸⁶Rb⁺ flux components are present in surface cell vesicles, while the high-sensitive component is absent in the crypt cell membrane fraction. The Ba²⁺-sensitive, Ca²⁺-activated K⁺ channels can be solubilized in CHAPS and reconstituted into phospholipid vesicles. This is an essential step for further characterization of channel properties and for identification of the channel proteins in purification procedures.     

The mitochondrial Ca2+-activated K+ channel activator, NS 1619 inhibits L-type Ca2+ channels in rat ventricular myocytes

We examined the effects of the mitochondrial Ca(2+)-activated K(+) (mitoBK(Ca)) channel activator NS 1619 on L-type Ca(2+) channels in rat ventricular myocytes. NS 1619 inhibited the Ca(2+) current in a dose-dependent manner. NS 1619 shifted the activation curve to more positive potentials, but did not have a significant effect on the inactivation curve. Pretreatment with inhibitors of membrane BK(Ca) channel, mitoBK(Ca) channel, protein kinase C, protein kinase A, and protein kinase G had little effect on the Ca(2+) current and did not alter the inhibitory effect of NS 1619 significantly. The application of additional NS 1619 in the presence of isoproterenol, a selective beta-adrenoreceptor agonist, reduced the Ca(2+) current to approximately the same level as a single application of NS 1619. In conclusion, our results suggest that NS 1619 inhibits the Ca(2+) current independent of the mitoBK(Ca) channel and protein kinases. Since NS 1619 is widely used to study mitoBK(Ca) channel function, it is essential to verify these unexpected effects of NS 1619 before experimental data can be interpreted accurately.     

Protons block BK channels by competitive inhibition with K+ and contribute to the limits of unitary currents at high voltages

Proton block of unitary currents through BK channels was investigated with single-channel recording. Increasing intracellular proton concentration decreased unitary current amplitudes with an apparent pKa of 5.1 without discrete blocking events, indicating fast proton block. Unitary currents recorded at pH(i) 8.0 and 9.0 had the same amplitudes, indicating that 10(-8) M H(+) had little blocking effect. Increasing H(+) by recording at pH(i) 7.0, 6.0, and 5.0 then reduced the unitary currents by 13%, 25%, and 53%, respectively, at +200 mV. Increasing K(+)(i) relieved the proton block in a manner consistent with competitive inhibition of K(+)(i) action by H(+)(i). Proton block was voltage dependent, increasing with depolarization, indicating that block was coupled to the electric field of the membrane. Proton block was not described by the Woodhull equation for noncompetitive voltage-dependent block, but was described by an equation for cooperative competitive inhibition that included voltage-dependent block from the Woodhull equation. Proton block was still present after replacing the eight negative charges in the ring of charge at the entrance to the intracellular vestibule by uncharged amino acids. Thus, the ring of charge is not the site of proton block or of competitive inhibition of K(+)(i) action by H(+)(i). With 150 mM symmetrical KCl, unitary current amplitudes increased with depolarization, reaching 66 pA at +350 mV (pH(i) 7.0). The increase in amplitude with voltage became sublinear for voltages >100 mV. The sublinearity was unaffected by removing from the intracellular solutions Ca(2+) and Ba(2+) ions, the Ca(2+) buffers EGTA and HEDTA, the pH buffer TES, or by replacing Cl(-) with MeSO(3)(-). Proton block accounted for approximately 40% of the sublinearity at +200 mV and pH 7.0, indicating that factors in addition to proton block contribute to the sublinearity of the unitary currents through BK channels.     

Voltage and Ca2+ activation of single large-conductance Ca2+-activated K+ channels described by a two-tiered allosteric gating mechanism

The voltage- and Ca2+-dependent gating mechanism of large-conductance Ca2+-activated K+ (BK) channels from cultured rat skeletal muscle was studied using single-channel analysis. Channel open probability (Po) increased with depolarization, as determined by limiting slope measurements (11 mV per e-fold change in Po; effective gating charge, q(eff), of 2.3 +/- 0.6 e(o)). Estimates of q(eff) were little changed for intracellular Ca2+ (Ca2+(i)) ranging from 0.0003 to 1,024 microM. Increasing Ca2+(i) from 0.03 to 1,024 microM shifted the voltage for half maximal activation (V(1/2)) 175 mV in the hyperpolarizing direction. V(1/2) was independent of Ca2+(i) for Ca2+(i) < or = 0.03 microM, indicating that the channel can be activated in the absence of Ca2+(i). Open and closed dwell-time distributions for data obtained at different Ca2+(i) and voltage, but at the same Po, were different, indicating that the major action of voltage is not through concentrating Ca2+ at the binding sites. The voltage dependence of Po arose from a decrease in the mean closing rate with depolarization (q(eff) = -0.5 e(o)) and an increase in the mean opening rate (q(eff) = 1.8 e(o)), consistent with voltage-dependent steps in both the activation and deactivation pathways. A 50-state two-tiered model with separate voltage- and Ca2+-dependent steps was consistent with the major features of the voltage and Ca2+ dependence of the single-channel kinetics over wide ranges of Ca2+(i) (approximately 0 through 1,024 microM), voltage (+80 to -80 mV), and Po (10(-4) to 0.96). In the model, the voltage dependence of the gating arises mainly from voltage-dependent transitions between closed (C-C) and open (O-O) states, with less voltage dependence for transitions between open and closed states (C-O), and with no voltage dependence for Ca2+-binding and unbinding. The two-tiered model can serve as a working hypothesis for the Ca2+- and voltage-dependent gating of the BK channel.     

Activation by Ca2+ and block by divalent ions of the K+ channel in the membrane of cytoplasmic drops fromChara australis

Summary Patch-clamp studies of cytoplasmic drops from the charophyteChara australis have previously revealed K⁺ channels combining high conductance (170 pS) with high selectivity for K⁺, which are voltage activated. The cation-selectivity sequence of the channel is shown here to be: K⁺>Rb⁺>NH ₄ ⁺ Na⁺ and Cl^–. Divalent cytosolic ions reduce the K⁺ conductance of this channel and alter its K⁺ gating in a voltage-dependent manner. The order of blocking potency is Ba²⁺>Sr²⁺>Ca²⁺>Mg²⁺. The channel is activated by micromolar cytosolic Ca²⁺, an activation that is found to be only weakly voltage dependent. However, the concentration dependence of calcium activation is quite pronounced, having a Hill coefficient of three, equivalent to three bound Ca²⁺ needed to open the channel. The possible role of the Ca²⁺-activated K⁺ channel in the tonoplast ofChara is discussed.     

Polyamines block Ca2+-activated K+ channels in pituitary tumor cells (GH3)

The effects of the natural polyamines, putrescine, spermidine and spermine on single calcium-activated potassium channels from clonal rat pituitary tumor cells (GH3) were studied. Applied to inside-out patches, polyamines were found to reduce the current amplitude and open probability of the channels in a dose- and voltage-dependent manner, indicating that polyamines act as fast blockers which sense a fraction of the electrical field in the channel pore. The K _d for spermine was 11.2 mm for the reduction of unitary current amplitude and 0.7 mm for the reduction of the open probability. The order of effectiveness was spermine > spermidine > putrescine. From fitting -functions to current amplitude histograms, blocking and unblocking rates were determined as 11.4 × 10⁴ sec^–1 and 21.9 × 10⁴ sec^–1, respectively. The reduction of the channel open probability was relieved by an increase of the Ca²⁺ concentration of the internal solution, indicating that polyamines compete with Ca²⁺ at the Ca²⁺ sensor of the channel. Putrescine antagonized the effect of spermine on the channel current amplitude. The results suggest that polyamines at intracellular millimolar concentrations suppress ion channel activity and therefore may effect electrical discharge behavior of excitable cells.This work was supported in part by the Austrian Fonds zur Förderung der wissenschaftlichen Forschung, P8587.     

Aminoglycoside blockade of Ca2+-activated K+ channel from rat brain synaptosomal membranes incorporated into planar bilayers

Summary Ca²⁺-activated K⁺ channels from rat brain synaptosomal membranes were incorporated into planar lipid bilayers, and the effects of aminoglycoside antibiotics on the single channel conductance (258±13 pS at 100mm K⁺) were investigated. Aminoglycosides reduced the single channel conductance from the cis (cytoplasmic) side in a dose- and voltage-dependent manner. Voltage dependence of the blockade indicated an interaction between positively charged amino residues of aminoglycoside antibiotics and a binding site located within the electric field of the ion-conducting pathway. The order of blocking potency was consistent with that of the number of amino residues of aminoglycosides (neomycin (6)>dibekacin (5)>ribostamycin (4)=kanamycin (4)), while the electrical distance (z=0.46–0.49) of the binding site kept almost constant for each drug. Thesezs were almost the same with those (0.46–0.51) of alkyldiamine blockers with two amino residues (total net charge of +2) and approximately twice of those (0.25–0.26) of alkylmonoamine blockers (total net charge of +1). Assuming that amino residues of aminoglycosides and alkylamines shared the same binding site located at 25% voltage drop from the cytoplasmic surface of the channel, the site would have to be at least large enough to accommodate one diamino sugar residue of the aminoglycoside in order to simultaneously interact with two positively charged amino groups. Dose- and voltage-dependent blockade of the channel by gallamine, an extremely bulky trivalent organic cation, supported the picture that the channel has a wide mouth on the cytoplasmic side and its pore region, where voltage drop occurs, may also be quite wide and nonselective, suddenly tapering to a constriction where most charged cations block the channel by occluding the K⁺-conducting pathway.     

Glucocorticoids stimulate the activity of large-conductance Ca2+ -activated K+ channels in pituitary GH3 and AtT-20 cells via a non-genomic mechanism

The effects of glucocorticoids on ion currents were investigated in pituitary GH3 and AtT-20 cells. In whole-cell configuration, dexamethasone, a synthetic glucocorticoid, reversibly increased the density of Ca2+ -activated K+ current (IK(Ca)) with an EC50 value of 21 +/- 5 microM. Dexamethasone-induced increase in IK(Ca) density was suppressed by paxilline (1 microM), yet not by glibenclamide (10 microM), pandinotoxin-Kalpha (1 microM) or mifepristone (10 microM). Paxilline is a blocker of large-conductance Ca2+ -activated K+ (BKCa) channels, while glibenclamide and pandinotoxin-Kalpha are blockers of ATP-sensitive and A-type K+ channels, respectively. Mifepristone can block cytosolic glucocorticoid receptors. In inside-out configuration, the application of dexamethasone (30 microM) into the intracellular surface caused no change in single-channel conductance; however, it did increase BKCa -channel activity. Its effect was associated with a negative shift of the activation curve. However, no Ca2+ -sensitiviy of these channels was altered by dexamethasone. Dexamethasone-stimulated channel activity involves an increase in mean open time and a decrease in mean closed time. Under current-clamp configuration, dexamethasone decreased the firing frequency of action potentials. In pituitary AtT-20 cells, dexamethasone (30 microM) also increased BKCa -channel activity. Dexamethasone-mediated stimulation of IK(Ca) presented here that is likely pharmacological, seems to be not linked to a genomic mechanism. The non-genomic, channel-stimulating properties of dexamethasone may partly contribute to the underlying mechanisms by which glucocorticoids affect neuroendocrine function.     

Single-file diffusion through the Ca2+-activated K+ channel of human red cells

Summary We have examined the effect of internal and external pH on Na⁺ transport across toad bladder membrane vesicles. Vesicles prepared and assayed with a recently modified procedure (Garty & Asher, 1985) exhibit large, rheogenic, amiloridesensitive fluxes. Of the total²²Na uptake measured 0.5–2.0 min after introducing tracer, 80±4% (mean±se,n=9) is blocked by the diuretic with aK ₁ of 2×10^–8 m. Thus, this amiloridesensitive flux is mediated by the apical sodium-selective channels. Varying the internal (cytosolic) pH over the physiologic range 7.0–8.0 had no effect on sodium transport; this result suggests that variation of intracellular pHin vivo has no direct apical effect on modulating sodium uptake. On the other hand,²²Na was directly and monotonically dependent on external pH. External acidification also reduced the amiloride-sensitive efflux across the walls of the vesicles. This inhibition of²²Na efflux was noted at external Na⁺ concentrations of both 0.2 m and 53mm.These results are different from those reported with whole toad bladder. A number of possible bases for these differences are considered and discussed. We suggest that the natriferic response induced by mucosal acidification of whole toad urinary bladder appears to operate indirectly through one or more factors, presumably cytosolic, present in whole cells and absent from the vesicles.     

pH i -Dependent membrane conductance of proximal tubule cells in culture (OK): Differential effects on K+- and Na+-conductive channels

Summary Confluent monolayers of the established opossum kidney cell line were exposed to NH₄Cl pulses (20 mmol/liter) during continuous intracellular measurements of pH, membrane potential (PD _m ) and membrane resistance (R _m) in bicarbonate-free Ringer. The removal of extracellular NH₄Cl leads to an intracellular acidification from a control value of 7.33±0.08 to 6.47±0.03 (n=7). This inhibits the absolute K conductance (g _K+), reflected by a decrease of K⁺ transference number from 71±3% (n=28) to 26±6% (n=5), a 2.6±0.2-fold rise ofR _m, and a depolarization by 24.2±1.5 mV (n=52). In contrast, intracellular acidification during a block ofg _K+ by 3 mmol/liter BaCl₂ enhances the total membrane conductance, being shown byR _m decrease to 68±7% of control and cell membrane depolarization by 9.8±2.8 mV (n=17). Conversely, intracellular alkalinization under barium elevatesR _m and hyperpolarizes PD _m . The replacement of extracellular sodium by choline in the presence of BaCl₂ significantly hyperpolarizes PD _m and increasesR _m, indicating the presence of a sodium conductance. This conductance is not inhibited by 10^–4 mol/liter amiloride (n=7). Patch-clamp studies at the apical membrane (excised inside-out configuration) revealed two Na⁺-conductive channels with 18.8±1.4 pS (n=10) and 146 pS single-channel conductance. Both channels are inwardly rectifying and highly selective towards Cl^–. The low-conductive channel is 4.8 times more permeable for Na⁺ than for K⁺. Its open probability rises at depolarizing potentials and is dependent on the pH of the membrane inside (higher at pH 6.5 than at pH 7.8).     

Electrogenic behavior of the human red cell Ca2+ pump revealed by disulfonic stilbenes

Summary A systematic study was made of the action of 4-acetamido-4-isothiocyanostilbene-2,2-disulfonic acid (SITS) and 4,4-diisothiocyanostilbene-2,2-disulfonic acid (DIDS) on active Ca²⁺ transport of human erythrocytes. Pumping activity was estimated in inside-out vesicles (IOV's) by means of Ca²⁺-selective electrodes or use of tracer⁴⁵Ca²⁺. The stilbenes exhibited an approximately equal inhibitory potency and their action could be overcome by carbonyl cyanidep-trifluoromethoxyphenylhydrazone (FCCP) at low but not at high stilbene concentrations. In the absence of DIDS. Ca²⁺ transport was not affected upon addition of valinomycin, but it was appreciably reduced when vesicles were preincubated with low DIDS concentrations. Such an effect was strictly dependent on the external K⁺ concentration and it was abolished when valinomycin was added together with FCCP. Similar results were obtained using IOV's prepared from intact cells which had been previously exposed to the stilbene. The findings clearly demonstrate the presence in human red cells of a partially electrogenic Ca²⁺ pump, exchanging one Ca²⁺ ion for one proton.     

Role of Na+/Ca2+ exchange and the plasma membrane Ca2+ pump in hormone-mediated Ca2+ efflux from pancreatic acini

Summary The relative contributions of the Na⁺/Ca²⁺ exchange and the plasma membrane Ca²⁺ pump to active Ca²⁺ efflux from stimulated rat pancreatic acini were studied. Na⁺ gradients across the plasma membrane were manipulated by loading the cells with Na⁺ or suspending the cells in Na⁺-free media. The rates of Ca²⁺ efflux were estimated from measurements of [Ca²⁺] _i using the Ca²⁺-sensitive fluorescent dye Fura 2 and⁴⁵Ca efflux. During the first 3 min of cell stimulation, the pattern of Ca²⁺ efflux is described by a single exponential function under control, Na⁺-loaded, and Na⁺-depleted conditions. Manipulation of Na⁺ gradients had no effect on the hormone-induced increase in [Ca²⁺] _i . The results indicate that Ca²⁺ efflux from stimulated pancreatic acinar cells is mediated by the plasma membrane Ca²⁺ pump. The effects of several cations, which were used to substitute for Na⁺, on cellular activity were also studied. Choline⁺ and tetramethylammonium⁺ (TMA⁺) released Ca²⁺ from intracellular stores of pancreatic acinar, gastric parietal and peptic cells. These cations also stimulated enzyme and acid secretion from the cells. All effects of these cations were blocked by atropine. Measurements of cholecystokinin-octapeptide (CCK-OP)-stimulated amylase release from pancreatic acini, suspended in Na⁺, TMA⁺, choline⁺, or N-methyl-d-glucamine⁺ (NMG⁺) media containing atropine, were used to evaluate the effect of the cations on cellular function. NMG⁺, choline⁺, and TMA⁺ inhibited amylase release by 55, 40 and 14%, respectively. NMG⁺ also increased the Ca²⁺ permeability of the plasma membrane. Thus, to study Na⁺ dependency of cellular function, TMA⁺ is the preferred cation to substitute for Na⁺. The stimulatory effect of TMA⁺ can be blocked by atropine.     

Rapid Ca2+ extrusion via the Na+/Ca2+ exchanger of the human platelet

Summary This communication reports the kinetics of the Na⁺/ Ca²⁺ exchanger and of the plasma membrane (PM) Ca²⁺ pump of the intact human platelet. The kinetic properties of these two systems were deduced by studying the rate of Ca²⁺ extrusion and its Na⁺ dependence for concentrations of cytoplasmic free Ca²⁺ ([Ca²⁺]_cyt) in the 1–10-m range. The PM Ca²⁺ATPase was previously characterized (Johansson, J.S. Haynes, D.H. 1988. J. Membrane Biol. 104:147–163) for [Ca²⁺]_cyt] 1.5 m with the fluorescent Ca²⁺ indicator quin2 (K _d= 115 nm). That study determined that the PM Ca²⁺ pump in the basal state has a V _max = 0.098 mm/min, a K _m= 80 nm and a Hill coefficient = 1.7. The present study extends the measurable range of [Ca²⁺]_cyt with the intracellular Ca²⁺ probe, rhod2 (K _d= 500 nm), which has almost a fivefold lower affinity for Ca²⁺. An Appendix also describes the Mg²⁺ and pH dependence of the K _dand fluorescence characteristics of the commercially available dye, which is a mixture of two molecules. Rates of active Ca²⁺ extrusion were determined by two independent methods which gave good agreement: (i) by measuring Ca²⁺ extrusion into a Ca²⁺-free medium (above citation) or (ii) by the newly developed ionomycin short-circuit method, which determines the ionomycin concentration necessary to short circuit the PM Ca²⁺ extrusion systems. Absolute rates of extrusion were determined by knowledge of how many Ca²⁺ ions are moved by ionomycin per minute. The major findings are as follows: (i) The exchanger is saturable with respect to Ca²⁺ with a K _m= 0.97 ± 0.31 m and V_max = 1.0 ± 0.6 mm/ min. (ii) At high [Ca²⁺]_cyt, the exchanger works at a rate 10 times as large as the basal V _max of the PM Ca²⁺ extrusion pump. (iii) The exchanger can work in reverse after Na⁺ loading of the cytoplasm by monensin. (iv) The PM Ca²⁺ extrusion pump is activated by exposure to [Ca²⁺]_cyt 1.5 m for 20–50 sec. Activation raises the pump V _max to 1.6 ± 0.6 mm/min and the K _mto 0.55 ± 0.24 m. (v) The Ca²⁺ buffering capacity of the cytoplasm is 3.6 mm in the 0.1 to 3 m range of [Ca²⁺]_cyt. In summary, the results show that the human platelet can extrude Ca²⁺ very rapidly at high [Ca²⁺]_cyt. Both the Na⁺/Ca²⁺ exchanger and Ca²⁺ pump activation may prevent inappropriate platelet activation by marginal stimuli.Abbreviations cAMP cyclic adenosine 3,5-monophosphate - cGMP cyclic guanosine 3,5,-monophosphate - Ca-CAM calcium calmodulin; - DT dense tubules - B intrinsic cytoplasmic Ca²⁺ binding sites - R rhod2 or 5-(3,6-bis(dimethylamino)xanth-9-yl)-1-(2-amino-4-hy droxy lphenoxy)-2-(2-amino-5-methylphen- oxy)ethane-N,N,NN-tetraacetic acid - [Ca²⁺]_cyt cytoplasmic Ca²⁺ activity - quin2 2-[[2-bis[(carboxymethyl)amino]-5-methyl-phenoxy]methyl]-6-methoxy-8-[bis(carboxymethyl)amino]quinoline - V or V_extrusion true rate of Ca²⁺ extrusion - fura-2 1-[2-(5-carboxyoxazol-2-yl)-6-aminobenzofuran-5-oxy]-2-(2-amino-5-methylphenoxy)-ethane-N,N,NN-tetraacetic acid - AM acetoxymethyl ester - DMSO dimethylsulfoxide - CTC chlortetracycline - EGTA ethyleneglycol-bis(-aminoethyl ether) N,N,N,N- tetraacetic acid - HEPES 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid - NMDG N-methyl-d-glucamine - PIPES 1,4-piperazine-bis-(ethanesulfonic acid) - HPLC high performance liquid chromatography - _I fraction of high-affinity rhod2 complexed with Ca²⁺ - F the observed fluorescence - F_min the minimal fluorescence observed in the absence of Ca²⁺ - F_max the maximal fluorescence observed when the dye is saturated with Ca²⁺ - X₁ the fraction of high-affinity dye - K _d,1 dissociation constant of high-affinity dye - K _d,2 dissociation constant of the low-affinity dye - -d₁/dt rate of Ca²⁺ removal from the rhod2-Ca complex; - -dF/dt the slope representing the absolute rate of fluorescence decrease in a progress curve - F_max (F_max — F_min)_cyt difference between maximal and minimal fluorescence for cytoplasmic high affinity form of rhod2 - F₅₀ fluorescence of the high-affinity form ofrhod2for[Ca²⁺]_cyt=50 nM - [Ca²⁺]₀ external Ca²⁺concentration - K _p proportionality constant between the total number of Ca²⁺ ions moved and the change in high-affinity rhod2 complexation to Ca² - (d[Ca²⁺]_{cyt, T})/dt rate of Ca²⁺ influx obtained with maximal levels of ionomycin - k_leak rate constant for passive inward Ca²⁺ leakage - k_inno rate constant for ionomycin-mediated Ca²⁺ influx - T total - [rhod2]_cyt,T total intracellular rhod2 concentration - [quin2]_cyt,T total intracellular quin2 concentration - [B]_T total cytoplasmic buffering capacity - A[Ca²⁺]_cyt,T total number of Ca²⁺ ions moved into the cytoplasm - [rhod2-Ca]_{cyt, T} change in concentration of total intracellular high-affinity rhod2 complexed to Ca²⁺ - [B-Ca]_T change in concentration of total cytoplasmic binding sites complexed to Ca²⁺ - [quin2]_{cyt, T} change in concentration of total intracellular quinl complexed to Ca²⁺ - change in the degree of intracellular quin2 saturation - ₁ change in degree of saturation of cytoplasmic high-affinity rhod2 - ₁-/t rate of change in degree of saturation of cytoplasmic high affinityrhod2 - V_obs observed rate of Ca²⁺ removal from the rhod2-Ca complex - V_{8.3 m} the rate of Ca²⁺ removal from the high affinity rhod2-Ca complex at [Ca²⁺]_cyt = 8.3 m - /t rate of change in of the degree of quin2 saturation - [Ca²⁺]_cytT/_t initial linear rate of ionomycin-mediated Ca²⁺ influx - EC₅₀ effective concentration giving a half-maximal effect - [Na⁺]_cyt cytoplasmic Na⁺ activity - CAM calmodulin - ACN acetonitrile - TFA trifuloroacetic acid