Quantitative immunoelectron microscopic localization of (Na+,K+)ATPase on rat exocrine pancreatic cells

Distribution of (Na+,K+)ATPase in rat exocrine pancreatic cells was investigated quantitatively by immunoelectron microscopy using the post-embedding protein A-gold technique. We found that in acinar and duct cells (Na+,K+)ATPase exists on both the luminal and the basolateral surfaces, with higher particle density on the luminal surface (4.4 times in the acinar cells and 5.6 times in the duct cells). According to Bolender (J Cell Biol 61:269, 1974), the luminal surface represents only 5% of the total cell surface of an average pancreatic acinar cell. It is roughly estimated, therefore, that approximately 80% of the plasma membrane (Na+,K+)ATPase in the acinar cells exists on the basolateral surface. When the acinar and duct cells were compared, more than twice as many particles were found on acinar cells than on duct cells. The enzyme existed on all the cell surfaces, preferentially on the microvilli or on the cell membrane folds, and no clustering was detected. We suggest that the (Na+,K+)ATPase on the basolateral surface is mainly responsible for the extrusion of a large number of sodium ions that are incorporated into the cytoplasm accompanying the secondary active transport of various organic substances and inorganic ions, whereas that on the luminal surface is responsible for active extrusion of sodium ions that are partially responsible for the fluid secretion of the pancreatic cells.     

Quantitative immunoelectron microscopic localization of (Na+,K+)ATPase in rat parotid gland

Distribution of (Na+,K+)ATPase on the cell membranes of acinar and duct cells of rat parotid gland was investigated quantitatively by immunoelectron microscopy using the post-embedding protein A-gold technique. In acinar cells, ATPase was localized predominantly on the basolateral plasma membranes. A small but significant amount of (Na+,K+)ATPase was, however, detected on the luminal plasma membranes, especially on the microvillar region of the acinar cells; the surface density on the luminal membrane was approximately one third of that on the basolateral membranes. In duct cells, many gold particles were found on the basolateral membrane, especially along the basal infoldings of the plasma membranes, whereas no significant gold particles were found on the luminal plasma membranes, suggesting unilateral distribution of ATPase in duct cells. We suggest that in acinar cells sodium ion is not only transported paracellularly but is also actively transported intracellularly into the luminal space by the (Na+,K+)ATPase located on the luminal plasma membranes, and that water is passively transported to the luminal space to form a plasma-like isotonic primary saliva, while in the duct cells the same ion is selectively re-absorbed intracellularly by (Na+,K+)ATPase found in abundance along the many infoldings of the basal plasma membranes, thus producing the hypotonic saliva.     

Energetics of potassium ion transport in Aplysia gut

Basolateral membranes of Aplysia californica foregut epithelia contain an ATP-dependent Na(+)/K(+) transporter (Na(+)/K(+) pump or Na(+)/K (+) -ATPase). This Na(+)/K(+) pump accounts for both the intracellular Na(+) electrochemical potential (micro) being less than the extracelluar Na(+) micro and the intracellular K(+) micro being more than the extracellular K(+ ) micro. Also, K(+) channel activity resides in both luminal and basolateral membranes of the Aplysia foregut epithelial cells. Increased activity of the Na(+)/K(+) pump, coupled to luminal and basolateral membrane depolarization altered the K(+) transport energetics across the basolateral membrane to a greater extent than the alteration in K(+) transport energetics across the luminal membrane. These results suggest that K(+) transport, either into or out of the Aplysia foregut epithelial cells, is rate-limiting at the basolateral membrane.     

Plasma membrane of the rat parotid gland: preparation and partial characterization of a fraction containing the secretory surface

A plasma membrane fraction from the rat parotid gland has been prepared by a procedure which selectively enriches for large membrane sheets. This fraction appears to have preserved several ultrastructural features of the acinar cell surface observed in situ. Regions of membrane resembling the acinar luminal border appear as compartments containing microvillar invaginations, bounded by elements of the junctional complex, and from which basolateral membranes extend beyond the junctional complex either to contact other apical compartments or to terminate as free ends. Several additional morphological features of the apical compartments suggest that they are primarily derived from the surface of acinar cells, rather than from the minority of other salivary gland cell types. Enzymatic activities characteristically associated with other cellular organelles are found at only low levels in the plasma membrane fraction. The fraction is highly enriched in two enzyme activities--K+ -dependent p-nitrophenyl phosphatase (K+ -NPPase, shown to be Na+/K+ adenosine triphosphatase; 20-fold) and gamma-glutamyl transpeptidase (GGTPase; 26-fold)--both known to mark plasma membranes in other tissues. These activities exhibit different patterns of recovery during fractionation, suggesting their distinct distributions among parotid cellular membranes. Secretion granule membranes also exhibit GGTPase, but no detectable K+ -NPPase. Since Na+/K+ adenosine triphosphatase and GGTPase, respectively, mark the basolateral and apical cellular surfaces in other epithelia, we hypothesize that these two enzymes mark distinct domains in the parotid plasmalemma, and that GGTPase, as the putative apical marker, may signify a compositional overlap between the two types of membranes which fuse during exocytosis.     

Electrophysiological effects of basolateral [Na+] in Necturus gallbladder epithelium

In Necturus gallbladder epithelium, lowering serosal [Na+] ([Na+]s) reversibly hyperpolarized the basolateral cell membrane voltage (Vcs) and reduced the fractional resistance of the apical membrane (fRa). Previous results have suggested that there is no sizable basolateral Na+ conductance and that there are apical Ca(2+)-activated K+ channels. Here, we studied the mechanisms of the electrophysiological effects of lowering [Na+]s, in particular the possibility that an elevation in intracellular free [Ca2+] hyperpolarizes Vcs by increasing gK+. When [Na+]s was reduced from 100.5 to 10.5 mM (tetramethylammonium substitution), Vcs hyperpolarized from -68 +/- 2 to a peak value of -82 +/- 2 mV (P less than 0.001), and fRa decreased from 0.84 +/- 0.02 to 0.62 +/- 0.02 (P less than 0.001). Addition of 5 mM tetraethylammonium (TEA+) to the mucosal solution reduced both the hyperpolarization of Vcs and the change in fRa, whereas serosal addition of TEA+ had no effect. Ouabain (10(-4) M, serosal side) produced a small depolarization of Vcs and reduced the hyperpolarization upon lowering [Na+]s, without affecting the decrease in fRa. The effects of mucosal TEA+ and serosal ouabain were additive. Neither amiloride (10(-5) or 10(-3) M) nor tetrodotoxin (10(-6) M) had any effects on Vcs or fRa or on their responses to lowering [Na+]s, suggesting that basolateral Na+ channels do not contribute to the control membrane voltage or to the hyperpolarization upon lowering [Na+]s. The basolateral membrane depolarization upon elevating [K+]s was increased transiently during the hyperpolarization of Vcs upon lowering [Na+]s. Since cable analysis experiments show that basolateral membrane resistance increased, a decrease in basolateral Cl- conductance (gCl-) is the main cause of the increased K+ selectivity. Lowering [Na+]s increases intracellular free [Ca2+], which may be responsible for the increase in the apical membrane TEA(+)-sensitive gK+. We conclude that the decrease in fRa by lowering [Na+]s is mainly caused by an increase in intracellular free [Ca2+], which activates TEA(+)-sensitive maxi K+ channels at the apical membrane and decreases apical membrane resistance. The hyperpolarization of Vcs is due to increase in: (a) apical membrane gK+, (b) the contribution of the Na+ pump to Vcs, (c) basolateral membrane K+ selectivity (decreased gCl-), and (d) intraepithelial current flow brought about by a paracellular diffusion potential.     

Calcium-activated membrane depolarization via modulation of chloride efflux from parietal cells during gastrin stimulation

In the isolated parietal cell the following observations were made: gastrin led to an increase in cytoplasmic free Ca2+ concentration ([Ca2+]i) even in the absence of medium Ca2+ and to transient membrane depolarization in both the absence of the medium Ca2+ and the fura-2-loaded cell. The incorporated Ca2+ chelator BAPTA inhibited the gastrin-induced membrane depolarization. The magnitude of depolarization caused by gastrin was unchanged on removal of medium Na+. Furosemide but not tetraethylammonium inhibited the gastrin-induced depolarization. The results suggest that the Ca2+ released from the store(s) induces membrane depolarization, possibly via modulation of a Cl- efflux across the luminal surface during gastrin stimulation.     

Quantitative immunocytochemical localization of Na+,K+-ATPase alpha-subunit in the lateral wall of rat cochlear duct

Ultrastructural localization of the alpha-subunit of Na+,K+-ATPase on the lateral wall of rat cochlear duct was investigated quantitatively by the protein A-gold method, using affinity-purified antibody against the alpha-subunit of rat kidney Na+,K+-ATPase. In the stria vascularis, gold particles were sparse over the endolymphatic luminal surface of the marginal cells but were numerous over the basolateral membrane. The labeling density of the basolateral membrane was almost equal to that of the same domain of the distal tubule cells of kidney. The intermediate cells were studded with a large number of gold particles on the plasma membrane domain facing the basolateral domain of the marginal cells. On the luminal surfaces of the other epithelial cells, including those of Reissner's membrane, no significant amount of gold particles was found. Many gold particles were localized on all the plasma membranes of the spiral prominence stromal cells and on the intracellular membrane domain of the external sulcus cells.     

Transport energetics of the Na+ pump in Aplysia californica gut

Basolateral membranes of Aplysia californica foregut epithelia contain an ATP-dependent Na+ transporter (Na+ pump). Increased activity of the Na+ pump, coupled to luminal Na+/AIB symporter activity and basolateral membrane depolarization, changed the Na+ transport energetics across the basolateral membrane to a greater extent than the change in Na+ transport energetics across the luminal membrane.     

The surface membrane of the small intestinal epithelial cell. I. Localization of adenyl cyclase.

The subcellular distribution of adenyl cyclase was investigated in small intestinal epithelial cells. Enterocytes were isolated, disrupted and the resulting membranes fractionated by differential and sucrose gradient centrifugation. Separation of luminal (brush border) and contra-luminal (basolateral) plasma membrane was achieved on a discontinuous sucrose gradient. The activity of adenyl cyclase was followed during fractionation in relation to other enzymes, notably those considered as markers for luminal and contraluminal plasma membrane. The luminal membrane was identified by the membrane-bound enzymes sucrase and alkaline phosphatase and the basolateral region by (Na+ + K+)-ATPase. Enrichment of the former two enzymes in purified luminal plasma membrane was 8-fold over cells and that of (Na+ + K+)-ATPase in purified bisolateral plasma membranes was 13-fold. F--activated adenyl cyclase co-purified with (Na+ + K+)-ATPase, suggesting a common localization on the plasma membrane. The distribution of K+-stimulated phosphatase and 5'-nucleotidase also followed (Na+ + K+)-ATPase during fractionation.     

Role of Ca2+-activated K+ channel in regulation of NaCl reabsorption in thick ascending limb of Henle's loop

1. Reabsorption of NaCl in the thick ascending limb of Henle's loop involves the integrated function of the Na+,K+,Cl- -cotransport system and a Ca2+-activated K+ channel in the luminal membrane with the Na+,K+-pump and a net Cl- conductance in the basolateral membrane. 2. Assay of K+ channel activity after reconstitution into phospholipid vesicles shows that the K+ channel is stimulated by Ca2+ in physiological concentrations and that its activity is regulated by calmodulin and phosphorylation from cAMP dependent protein kinase. 3. For purification luminal plasma membrane vesicles are isolated and solubilized in CHAPS. K+ channel protein is isolated by affinity chromatography on calmodulin columns. The purified protein has high Ca2+-activated K+ channel activity after reconstitution into vesicles. 4. The purified K+ channel consists of two proteins of 51 and 36 kDa. Phosphorylation from cAMP dependent protein kinase stimulates K+ channel activity and labels the 51 kDa band. The 36 kDa band is rapidly cleaved by trypsin and may be involved in Ca2+ stimulation. 5. Opening of the K+ channel by Ca2+ in physiological concentrations and regulation by calmodulin and phosphorylation by protein kinase may mediate kinetic and hormonal regulation of NaCl transport across the tubule cells in TAL.     

K+ secretion across frog skin. Induction by removal of basolateral Cl-

We examined the development of K+ secretion after removing Cl- from the basolateral surface of isolated skins of Rana temporaria using noise analysis. K+ secretion was defined by the appearance of a Lorentzian component in the power density spectrum (PDS) when Ba2+ was present in the apical bath (0.5 mM). No Lorentzians were observed when tissues were bathed in control, NaCl Ringer solution. Replacement of basolateral Cl- by gluconate, nitrate, or SO4- (0-Clb) yielded Lorentzians with corner frequencies near 25 Hz, and plateau values (So) that were used to estimate the magnitude of K+ secretion through channels in the apical cell membranes of the principal cells. The response was reversible and reproducible. In contrast, removing apical Cl- did not alter the PDS. Reduction of basolateral Cl- to 11.5 mM induced Lorentzians, but with lower values of So. Inhibition of Na+ transport with amiloride or by omitting apical Na+ depressed K+ secretion but did not prevent its appearance in response to 0-Clb. Using microelectrodes, we observed depolarization of the intracellular voltage concomitant with increased resistance of the basolateral membrane after 0-Clb. Basolateral application of Ba2+ to depolarize cells also induced K+ secretion. Because apical conductance and channel density are unchanged after 0-Clb, we conclude that K+ secretion is "induced" simply by an increase of the electrical driving force for K+ exit across this membrane. Repolarization of the apical membrane after 0-Clb eliminated K+ secretion, while further depolarization increased the magnitude of the secretory current. The cell depolarization after 0-Clb is most likely caused directly by a decrease of the basolateral membrane K+ conductance. Ba2(+)-induced Lorentzians also were elicited by basolateral hypertonic solutions but with lower values of So, indicating that cell shrinkage per se could not entirely account for the response to 0-Clb and that the effects of 0-Clb may be partly related to a fall of intracellular Cl-.     

Extracellular ATP activates receptor-operated cation channels in mouse lacrimal acinar cells to promote calcium influx in the absence of phosphoinositide metabolism.

In exocrine acinar cells a variety of neurotransmitters (e.g. acetylcholine) stimulate phosphatidylinositol 4,5-bisphosphate hydrolysis elevating intracellular calcium to activate calcium-dependent membrane currents (outward K+ and inward Cl-). This study shows that in lacrimal acinar cells extracellular application of ATP is also associated with outward and inward current responses; these, however, are not the result of phosphoinositide metabolism. ATP directly activates receptor-operated cation channels which permit influx of Na+ and Ca+ (the inward current). The elevation in [Ca2+]i which results is sufficient to activate the outward K+ current. ATP thus promotes Ca+ influx in the absence of phosphoinositide metabolism.     

Autoradiographic and cytochemical localization of (Na+-K+)-activated adenosine triphosphatase in the acini of dog salivary gland (author's transl)

The distribution of Na pump sites (Na+-K+ ATPase) in the acinar cells of dog submandibular gland was demonstrated by light and electron microscopical radioautography of 3H-ouabain binding sites and electron microscopical ATPase cytochemistry. The grains of 3H-ouabain by light microscopical radioautography were localized to the basal parts of acini and/or the striated ducts, and a small quantity of the grains was also present on the luminal parts of acini. The grains of 3H-ouabain by electron microscopical radioautography and the reaction products of ATPase were found to be localized on the basolateral plasma membrane of both serous and mucous cells, while slightly on the microvilli of the luminal plasma membranes. The present evidence that the distribution of ATPase on the acinar cells determined by the cytochemistry is well concomitant with that of 3H-ouabain binding sites on the acinar cells by the radioautography, suggests that the above mentioned ATPase is Na+-K+ ATPase, a Na pump. The relationship of the distribution of the Na+-K+ ATPase and the cation transport of the plasma membranes in the acinar cells of the dog submandibular gland are discussed.     

Localization of Na+,K+-ATPase alpha-subunit to the sinusoidal and lateral but not canalicular membranes of rat hepatocytes

Controversy has recently developed over the surface distribution of Na+,K+-ATPase in hepatic parenchymal cells. We have reexamined this issue using several independent techniques. A monoclonal antibody specific for the endodomain of alpha-subunit was used to examine Na+,K+-ATPase distribution at the light and electron microscope levels. When cryostat sections of rat liver were incubated with the monoclonal antibody, followed by either rhodamine or horseradish peroxidase-conjugated goat anti-mouse secondary, fluorescent staining or horseradish peroxidase reaction product was observed at the basolateral surfaces of hepatocytes from the space of Disse to the tight junctions bordering bile canaliculi. No labeling of the canalicular plasma membrane was detected. In contrast, when hepatocytes were dissociated by collagenase digestion, Na+,K+-ATPase alpha-subunit was localized to the entire plasma membrane. Na+,K+-ATPase was quantitated in isolated rat liver plasma membrane fractions by Western blots using a polyclonal antibody against Na+,K+-ATPase alpha-subunit. Plasma membranes from the basolateral domain of hepatocytes possessed essentially all of the cell's estimated Na+,K+-ATPase catalytic activity and contained a 96-kD alpha-subunit band. Canalicular plasma membrane fractions, defined by their enrichment in alkaline phosphatase, 5' nucleotidase, gamma-glutamyl transferase, and leucine aminopeptidase had no detectable Na+,K+-ATPase activity and no alpha-subunit band could be detected in Western blots of these fractions. We conclude that Na+,K+-ATPase is limited to the sinusoidal and lateral domains of hepatocyte plasma membrane in intact liver. This basolateral distribution is consistent with its topology in other ion-transporting epithelia.     

Ion channels in mammalian proximal renal tubules.

In the plasma membranes of mammalian proximal renal tubules single ion channels were investigated mainly in isolated tubules perfused on one side, in isolated nonperfused (collapsed) tubules and in primary cell cultures. With these techniques, the following results were obtained: in the luminal membrane of isolated one-sided perfused tubules of rabbit and mouse S3 segments, K(+)-selective channels with single-channel conductance (g) of 33 pS and 63 pS, respectively, were recorded. In primary cultures of rabbit S1 segments, a small-conductance (42 pS) as well as a large-conductance (200 pS) K+ channel were observed. The latter was Ca2(+)- and voltage-sensitive. In cultured cells a Ca2(+)-activated, nonselective cation channel with g = 25 pS was also recorded. On the other hand, an amiloride-sensitive channel with g = 12 pS, which was highly selective for Na+ over K+, was observed in the isolated perfused S3 segment. In the basolateral membrane of isolated perfused S3 segments, two types of K+ channels with g = 46 pS and 36 pS, respectively, were observed. The latter channel was not dependent on cytosolic Ca2+ in cell-excised patches. A K+ channel with g = 54 pS was recorded in isolated nonperfused S1 segments. This channel showed inward rectification and was more active at depolarizing potentials. In isolated perfused S3 segments, in addition to the K+ channels also a nonselective cation channel with g = 28 pS was observed. This channel was highly dependent on cytosolic Ca2+ in cell-free patches. It can be concluded that the K+ channels both in the luminal and contraluminal cell membrane are involved in the generation of the cell potential. Na+ channels in the luminal membrane may participate in Na+ reabsorption, whereas the function of a basolateral cation channel remains unclear. Recently, single anion-selective channels were recorded in membranes of endocytotic vesicles, isolated from rat proximal tubules. Vesicles were enlarged by the dehydration/rehydration method and investigated with the patch clamp technique. The Cl- channel had a conductance of 73 pS, the current-voltage curve was linear and the channel inactivated at high negative clamp potentials. It is suggested that this channel is responsible for charge neutrality during active H+ uptake into the endosomes.     

Evidence against parallel operation of sodium/calcium antiport and ATP-driven calcium transport in plasma membrane vesicles from kidney tubule cells

The present study aimed to clarify the existence of a Na+/Ca2+ antiport device in kidney tubular epithelial cells discussed in the literature to represent the predominant mechanistic device for Ca2+ reabsorption in the kidney. Inside-out oriented plasma membrane vesicles from tubular epithelial cells of guinea-pig kidney showed an ATP-driven Ca2+ transport machinery similar to that known to reside in the plasma membrane of numerous cell types. It was not affected by digitalis compounds which otherwise are well-documented inhibitors of Ca2+ reabsorption. The vesicle preparation contained high, digitalis-sensitive (Na+ + K+)-ATPase activities indicating its origin from the basolateral portion of plasma membrane. The operation of a Na+/Ca2+ antiport device was excluded by the findings that steep Ca2+ gradients formed by ATP-dependent Ca2+ accumulation in the vesicles were not discharged by extravesicular Na+, and did not drive 45Ca2+ uptake into the vesicles via a Ca2+-45Ca2+ exchange. The ATP-dependent Ca2+ uptake into the vesicles became increasingly depressed with time by extravesicular Na+. This was not due to an impairment of the Ca2+ pump itself, but caused by Na+/Ca2+ competition for binding sites on the intravesicular membrane surface shown to be important for high Ca2+ accumulation in the vesicles. Earlier observations on Na+-induced release of Ca2+ from vesicles pre-equilibrated with Ca2+, seemingly favoring the existence of a Na+/Ca2+ antiporter in the basolateral plasma membrane, were likewise explained by the occurrence of Na+/Ca2+ competition for binding sites. The weight of our findings disfavors the transcellular pathway of Ca2+ reabsorption through tubule epithelium essentially depending on the operation of a Na+/Ca2+ antiport device.     

Purinergic stimulation induces Ca2+-dependent activation of Na+-K+-2Cl- cotransporter in human nasal epithelia

Increasing evidence suggests that P2 receptors (P2Rs) in airway epithelial cells perform critical functions in auto- or paracrine regulation of fluid and mucus secretion. In the present study, we characterized the effects of P2R stimulation on Na(+)-K(+)-2Cl(-) cotransporter (NKCC) activity in normal human nasal epithelial (NHNE) cells. [Ca(2+)](i) and pH(i) were measured in primary cultures of NHNE cells using a double perfusion chamber, which enabled us to analyze membrane-specific transporter activities. NKCC activities were estimated by the pH(i) reduction due to Na(+)-dependent and bumetanide-sensitive intracellular uptake of NH(4)(+). NKCC activities were observed in the basolateral membrane, but not in the luminal membrane, of NHNE cells. Interestingly, P2Rs were expressed in both membranes, and the stimulation of either luminal or basolateral P2R increased NKCC activity. Blockades of luminal Cl(-) channels, basolateral K(+) channels, or protein kinase C did not affect the activation of NKCC by basolateral P2R stimulation. The effects of luminal P2R stimulation were partially reduced by Cl(-) channel blockers. However, chelation of intracellular Ca(2+) by 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) treatment completely blocked the stimulatory effects of luminal and basolateral P2Rs on NKCC. In addition, increasing [Ca(2+)](i) by treatment with ionomycin-stimulated NKCC activity. These results provide evidence that stimulation of P2Rs directly activates basolateral NKCC by Ca(2+)-dependent pathways in NHNE cells, which is an important aspect of the purinergic regulation of ion and fluid secretions in human airway epithelia under physiologic and pathologic conditions.     

Mechanisms of activated Ca2+ entry in the rat pancreatoma cell line, AR4-2J.

The characteristics of Ca2+ entry activated by surface receptor agonists and membrane depolarization were studied in the rat pancreatoma cell line, AR4-2J. Ca2+ mobilization activated by substance P, bombesin, or muscarinic receptor stimulation was found to involve both Ca2+ release and entry. In addition, depolarization of the surface membrane of AR4-2J cells with elevated concentrations of K+ activated Ca2+ entry. Ca2+ entry induced by membrane depolarization was inhibited by the L-channel antagonist, nimodipine, while that due to surface receptor agonists was not inhibited by this agent. The microsomal Ca(2+)-ATPase inhibitor, thapsigargin, caused both depletion of the agonist-sensitive intracellular Ca2+ pool and sustained Ca2+ influx indistinguishable from that produced by bombesin or methacholine. These results confirm that, unlike the pancreatic acinar cells from which they are presumably derived, AR4-2J cells express voltage-sensitive, dihydropyridine-inhibitable Ca2+ channels. However, in contrast to previous reports with this cell line, in the AR4-2J cells in use in our laboratory, and under our experimental conditions, surface receptor agonists (including substance P) do not cause Ca2+ influx through voltage-sensitive Ca2+ channels. Instead, we conclude that agonist-activated Ca2+ mobilization is initiated by (1,4,5)IP3-mediated intracellular Ca2+ release and that Ca2+ influx is regulated primarily, if not exclusively, by the state of depletion of the (1,4,5)IP3-sensitive intracellular Ca2+ pool.     

The relationship between mitogen-induced membrane potential changes and intracellular free calcium in human T-lymphocytes

We have investigated the effects of mitogenic lectins on human T-lymphocytes, isolated from peripheral blood, and cells from the T-cell clone, HPB-ALL, using the fluorescent dyes, bis-thiobarbiturate tri-methineoxonol (bisoxonol) and quin2 to sense changes in membrane potential and intracellular free [Ca2+], respectively. The resting potential of both cell types is close to the K+ equilibrium potential. Changes from the resting level occur when mitogenic concentrations of either concanavalin A or phytohaemagglutinin are added. T-lymphocytes undergo a decrease in emission, maximal at 1 to 2 min, corresponding to a small membrane hyperpolarization. This is followed by a depolarization to approximately the resting level. HPB-ALL cells, on the other hand, respond to the mitogens by a sustained increase in fluorescence, denoting a depolarization, that is maximal at 4 to 5 min and 7 to 9 min, respectively. The Ca2+-dependence of these phenomena indicates that the membrane potential response, in both cell types, is the resultant of two opposing effects: a Ca2+-sensitive ion movement tending to hyperpolarize the cells and a Ca2+-insensitive effect that generates a depolarization. Our results suggest that Ca2+-activated K+ channels are responsible for the first effect and that an inward Na+ movement accounts for the depolarization signal in T-lymphocytes. In HPB-ALL cells only part of the depolarization is Na+-dependent. Although the effects elicited by phytohaemagglutinin occur more slowly than those produced by concanavalin A, similar membrane potential and [Ca2+]i changes occur.     

Maxi K+ channels and their relationship to the apical membrane conductance in Necturus gallbladder epithelium

Using the patch-clamp technique, we have identified large-conductance (maxi) K+ channels in the apical membrane of Necturus gallbladder epithelium, and in dissociated gallbladder epithelial cells. These channels are more than tenfold selective for K+ over Na+, and exhibit unitary conductance of approximately 200 pS in symmetric 100 mM KCl. They are activated by elevation of internal Ca2+ levels and membrane depolarization. The properties of these channels could account for the previously observed voltage and Ca2+ sensitivities of the macroscopic apical membrane conductance (Ga). Ga was determined as a function of apical membrane voltage, using intracellular microelectrode techniques. Its value was 180 microS/cm2 at the control membrane voltage of -68 mV, and increased steeply with membrane depolarization, reaching 650 microS/cm2 at -25 mV. We have related maxi K+ channel properties and Ga quantitatively, relying on the premise that at any apical membrane voltage Ga comprises a leakage conductance and a conductance due to maxi K+ channels. Comparison between Ga and maxi K+ channels reveals that the latter are present at a surface density of 0.09/microns 2, are open approximately 15% of the time under control conditions, and account for 17% of control Ga. Depolarizing the apical membrane voltage leads to a steep increase in channel steady-state open probability. When correlated with patch-clamp studies examining the Ca2+ and voltage dependencies of single maxi K+ channels, results from intracellular microelectrode experiments indicate that maxi K+ channel activity in situ is higher than predicted from the measured apical membrane voltage and estimated bulk cytosolic Ca2+ activity. Mechanisms that could account for this finding are proposed.