Store-operated Ca2+ channel in renal microcirculation and glomeruli

Store-operated Ca2+ channel (SOC) is defined as a channel that opens in response to depletion of the internal Ca2+ stores. During the last decade, many investigators have made a great effort to identify and characterize SOC, and to evaluate its physiologic function and pathophysiologic relevance in a variety of cell lines, primary cultures, and native tissues. To date, accumulating evidence has demonstrated that SOC is an essential Ca2+ entry mechanism in vascular smooth-muscle cells of renal microvasculature and glomerular mesangial cells, both of which tightly control glomerular hemodynamics and filtration. Store-operated Ca2+, combined with other types of Ca2+ entry channels, constitutes a profile of Ca2+ changes in response to physiologic vasoconstrictors and, thereby, regulates renal microcirculation and mesangial function. In addition, SOC is associated with altered Ca2+ signaling occurring in diseased kidneys, such as diabetic nephropathy. Although the gating mechanism and molecular identity of SOC are still enigmatic and may be cell-type and tissue specific, data from several independent groups suggest that protein kinase C plays an important role in SOC activation and that certain isoforms of canonical transient receptor potential (TRPC) proteins are candidates of SOC in renal microvessels and mesangial cells.     

Ultrastructural and immunohistochemical studies in callitrichid renal glomeruli

The “normal” mesangium of callitrichids exhibits certain features pointing to enhanced activity. Protrusions of the mesangial cellular cytoplasm (blebs) into the capillary lumen were observed very frequently as were electron-dense granules in mesangial matrix channels. Histochemical, α-actin expression was invariably observed in the mesangial cells of callitrichids.     

Volume-sensitive outwardly rectifying chloride channels are involved in oxidative stress-induced apoptosis of mesangial cells

Volume-sensitive outwardly rectifying (VSOR) Cl- channels have been electrophysiologically identified in human and mouse mesangial cells, but the functional role of VSOR Cl- channels in mesangial cell apoptosis is not clear. The aim of the present study was to demonstrate the role of VSOR Cl- channels in oxidative stress-induced mesangial cell apoptosis. H2O2-induced Cl- currents showed phenotypic properties of VSOR Cl- channels, including outward rectification, voltage-dependent inactivation at more positive potentials, sensitivity to hyperosmolarity, and inhibition by VSOR Cl- channel blockers. Moreover, blockage of VSOR Cl- channels by DIDS (100 microM), NPPB (10 microM) or niflumic acid (10 microM) rescued mesangial cell apoptosis induced by H2O2. Treatment with 150 microM H2O2 for 2h resulted in significant reduction of cell volume, in contrast, nuclear condensation and/or fragmentation were not observed and the caspase-3 activity was also not increased. The early-phase alterations in cell volume were markedly abolished by pretreatment with VSOR Cl- channel blockers. We conclude that VSOR Cl- channels are involved in H2O2-induced apoptosis in cultured mesangial cells and its mechanism is associated with apoptotic volume decrease processes.     

Ecto-5'-nucleotidase of cultured rat mesangial cells

Because adenosine plays a role in the regulation of glomerular filtration rate and of the release of renin, we examined the possibility of a local source for this mediator. We found that rat cultured glomerular mesangial cells converted 5'-AMP into adenosine. The properties of the enzyme involved in the reaction were those of an ecto-5' nucleotidase: (1) the products of the reaction were generated in the extracellular fluid although no 5'-nucleotidase was released by the cells into the medium; (2) identical activities were found for cultured cells in situ and sonicated cells; (3) the diazonium salt of sulfanilic acid which is a nonpenetrating reagent inhibited up to 75% of the enzyme activity. Ecto-5'-nucleotidase activity of intact cells obeyed Michaelis-Menten kinetics. Apparent Km for 5'-AMP was 0.32 mM. 5'-UMP was a strictly competitive inhibitor. ADP exerted a very powerful inhibitory effect and behaved also as a competitive inhibitor. ATP was inhibitory both by increasing Km and by decreasing Vmax. Ecto-5'-nucleotidase was active in the absence of divalent cations. However, Mg2+, Ca2+, Co2+ and Mn2+ were stimulatory. Zn2+ and Cu2+ suppressed the activity. Concanavalin A, a plant lectin, was markedly inhibitory, suggesting that a glycoprotein moiety was necessary to express enzyme activity. Ecto-5'-nucleotidase activity was not modified during phagocytosis of serum-treated zymosan by mesangial cells. Rat cultured glomerular epithelial cells exhibited a 5'-nucleotidase activity which was 4 times lower than that of the mesangial cells in primary culture.(ABSTRACT TRUNCATED AT 250 WORDS)     

Epidermal growth factor activates store-operated Ca2+ channels through an inositol 1,4,5-trisphosphate-independent pathway in human glomerular mesangial cells

One of the fastest cellular responses following activation of epidermal growth factor receptor is an increase in intracellular Ca2+ concentration. This event is attributed to a transient Ca2+ release from internal stores and Ca2+ entry from extracellular compartment. Store-operated Ca2+ channels are defined the channels activated in response to store depletion. In the present study, we determined whether epidermal growth factor activated store-operated Ca2+ channels and further, whether depletion of internal Ca2+ stores was required for the epidermal growth factor-induced Ca2+ entry in human glomerular mesangial cells. We found that 100 nm epidermal growth factor activated a Ca2+-permeable channel that had identical biophysical and pharmacological properties to channels activated by 1 microm thapsigargin in human glomerular mesangial cells or A431 cells. The epidermal growth factor-induced Ca2+ currents were completely abolished by a selective phospho-lipase C inhibitor, U73122. However, xestospongin C, a specific inositol 1,4,5-trisphosphate receptor inhibitor, did not affect the membrane currents elicited by epidermal growth factor despite a slight reduction in background currents. Following emptying of internal Ca2+ stores by thapsigargin, epidermal growth factor still potentiated the Ca2+ currents as determined by the whole-cell patch configuration. Furthermore, epidermal growth factor failed to trigger measurable Ca2+ release from endoplasmic reticulum. However, another physiological agent linked to phospholipase C and inositol 1,4,5-trisphosphate cascade, angiotensin II, produced a striking Ca2+ transient. These results indicate that epidermal growth factor activates store-operated Ca2+ channels through an inositol 1,4,5-trisphosphate-independent, but phospholipase C-dependent, pathway in human glomerular mesangial cells.     

Calcium channels in smooth muscle cells

In smooth muscle cells, the electrophysiological properties of potential-dependent calcium channels are similar to those described in other excitable cells. The calcium current is dependent on the extracellular calcium concentration; it is insensitive to external sodium removal and tetrodotoxin application. Other ions (Ba2+, Sr2+, Na+) can flow through the calcium channel. This channel is blocked by Mn2+, Co2+, Cd2+ and by organic inhibitors. The inactivation mechanism is mediated by both the membrane potential and the calcium influx. Ca2+ ions can also penetrate into the cell through receptor-operated channels. These channels show a low ionic selectivity and are generally less sensitive to organic Ca-blockers than the potential-dependent calcium channels. The finding of specific channel inhibitors as well as the study of the biochemical pathways between receptor activation and channel opening are prerequisites to further characterization of receptor-operated channels.     

Continuity of the mesangial channel network with the glomerular basement membrane and intraglomerular subendothelial space

The composition and exact structure of the non-cellular mesangial matrix in the glomerulus of the human kidney are a matter of debate. It may appear like a structure similar to the glomerular basement membrane (GBM), it has been described to contain microfilaments. The exact transport route of fluids, solvents and immunocomplexes in the mesangium is not well-known either. We know that in some glomerular diseases immunocomplexes can be found in the GBM and the mesangium at the same time in the same patient. A possible explanation of the above findings could be provided by our hypothesis, i.e. the existence of a well-defined mesangial channel network (MChN). This MChN would consist of intercommunicating channels, which were embedded into the spongy cytoplasm of the mesangial cells (MCs) and surrounded by the plasma membrane of the mesangial cells. The MChN would lead from the subendothelial space through deep mesangium to the vascular pole or the juxtaglomerular apparatus and may transport fluid and other materials such as immunocomplexes into the mesangium. It would be continuous with the GBM. Microfilaments of the MC would be anchored to the walls of the MChN regulating its diameter, thus mesangial fluid transport and pressure. The dilatation of these channels by mechanical obstruction could contribute to glomerular sclerosis. The hypothesis can be challenged by methods like electronmicroscopy, immunoelectronmicroscopy, confocal laser-scanning microscopy, and vital stain studies. We provide some images suggesting the existence of the channel and its connection with the GBM. If the hypothesis was true, it could contribute to understanding of mesangial transport processes, pressure regulation and pathogenesis of glomerular mesangial diseases.     

Ionic composition of endolymph and perilymph in the inner ear of the oyster toadfish, Opsanus tau

The concentrations of free Na+, K+, Ca(+, and Cl(-)in endolymph and perilymph from the inner ear of the oyster toadfish, Opsanus tau, were measured in vivo using double-barreled ion-selective electrodes. Perilymph concentrations were similar to those measured in other species, while endolymph concentrations were similar to those measured previously in elasmobranch fish, though significantly different from concentrations reported in mammals. Perilymph concentrations (mean +/- std. dev.) were as follows: Na+, 129 mmol l(-1) +/- 20; K+, 4.96 mmol l(-1) +/- 2.67; Ca2+, 1.83 mmol l(-1) +/- 0.27; and Cl(-), 171 mmol l(-1) +/- 20. Saccular endolymph concentrations were Na+, 166 mmol l(-1) +/- 22; K+, 51.4 mmol l(-1) +/- 16.7; Ca2+, 2.88 mmol l(-1) +/- 0.27; and Cl(-), 170 mmol l(-1) +/- 12; and semicircular canal (utricular vestibule) endolymph concentrations were Na+, 122 mmol l(-1) +/- 15; K+, 47.7 mmol l(-1) +/- 13.2; Ca2+, 1.78 mmol l(-1) +/- 0.48; Cl(-), 176 mmol l(-1) +/- 27. The relatively high concentrations of Ca2+ and Na+ in the endolymph may have significant implications for the physiological function of the mechanoelectrical transduction channels in the vestibular hair cells of fish compared to those of their mammalian counterparts.     

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.     

Inhibition of prostaglandin E2 synthesis by a blocker of epithelial chloride channels

Arginine-vasopressin (AVP) elicits a variety of responses in cultured rat mesangial cells, among them stimulation of prostaglandin biosynthesis and activation of Cl- channels. AVP produced an 11-fold increase over basal levels in prostaglandin E2 release from cultured mesangial cells. This response was completely inhibited by 25 microM indomethacin and 82 +/- 5% inhibited by 25 microM 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) which is a potent blocker of epithelial Cl- channels. The IC50 for NPPB inhibition of prostaglandin E2 release was 8 microM. Indomethacin and NPPB at 25 microM also inhibited AVP-stimulated cellular accumulation of prostaglandin E2 by 98% and 79 +/- 7% respectively. The inhibitory effect of NPPB was not due to interference with the cellular response to AVP since at 50 microM it did not block AVP-stimulated release of arachidonate metabolites from cells metabolically labeled with [3H]-arachidonic acid. It is suggested that NPPB inhibition of prostaglandin E2 synthesis is at the cyclooxygenase level on the basis of its structural similarity to the fenamic acid type of cyclooxygenase inhibitors.     

Chromaffin cell calcium channel kinetics measured isotopically through fast calcium, strontium, and barium fluxes

Fast Ca2+ uptake into K+-depolarized cultured bovine adrenal chromaffin cells has been isotopically measured in a time scale of 1-10 s. Depolarized cells retained as much as 80-fold 45Ca2+ taken up by resting cells; Ca2+ was not taken up by fibroblasts or endothelial-like cells. Because Ca2+ entry was inhibited by inorganic (La3+, Co2+, Mg2+) and organic (nifedipine) Ca2+ channel antagonists and enhanced by the Ca2+ channel activator Bay-K-8644, it seems clear that Ca2+ gains access to the chromaffin cell cytosol mainly through specific voltage-dependent Ca2+ channels. Ca2+ uptake evoked by 59 mM K+ was linear during the first 5 s of stimulation and continued to rise at a much slower rate up to 60 s. The rate of Ca2+ entry became steeper as the external [Ca2+] increased; initial rates of Ca2+ uptake varied from 0.06 fmol/cells . s at 0.125 mM Ca2+ to 2.85 fmol/cell . s at 7.5 mM Ca2+. The early 90Sr2+ uptake was linear but faster than Ca2+ uptake and later on was also saturated; 133Ba2+ was taken up still at a much faster rate and was linear for the entire depolarization period (2-60 s). Increased [K+] gradually depolarized chromaffin cells; Ca2+ and Sr2+ uptakes were not apparent below 30 mM K+ but were linear for 30 to 60 mM K+. In contrast, substantial Ba2+ uptake was seen even in K+-free solutions; and in 5.9 mM K+, Ba2+ uptake was as high as Ca2+ uptake obtained in 60 mM K+. Five to ten-second pulses of 45Ca2+, 90Sr2+, or 133Ba2+ given at different times after pre-depolarization of chromaffin cells served to analyze the kinetics of inactivation of the rates of entry of each divalent cation. Inactivation of Ca2+ uptake was faster than Sr2+, and Ba2+ uptake inactivated very little. Neither voltage changes nor Ca2+ ions passing through the channels seems to cause their inactivation; however, experiments aimed to manipulate the levels of internal Ca2+ using the cell-permeable chelator Quin-2 or the ionophore A23187 strongly suggest that intracellular Ca2+ levels determine the rates of inactivation of these channels.     

High extracellular Ca2+ hyperpolarizes human parathyroid cells via Ca(2+)-activated K+ channels

Membrane potential has a major influence on stimulus-secretion coupling in various excitable cells. The role of membrane potential in the regulation of parathyroid hormone secretion is not known. High K+-induced depolarization increases secretion from parathyroid cells. The paradox is that increased extracellular Ca2+, which inhibits secretion, has also been postulated to have a depolarizing effect. In this study, human parathyroid cells from parathyroid adenomas were used in patch clamp studies of K+ channels and membrane potential. Detailed characterization revealed two K+ channels that were strictly dependent of intracellular Ca2+ concentration. At high extracellular Ca2+, a large K+ current was seen, and the cells were hyperpolarized (-50.4 +/- 13.4 mV), whereas lowering of extracellular Ca2+ resulted in a dramatic decrease in K+ current and depolarization of the cells (-0.1 +/- 8.8 mV, p < 0.001). Changes in extracellular Ca2+ did not alter K+ currents when intracellular Ca2+ was clamped, indicating that K+ channels are activated by intracellular Ca2+. The results were concordant in cell-attached, perforated patch, whole-cell and excised membrane patch configurations. These results suggest that [Ca2+]o regulates membrane potential of human parathyroid cells via Ca2+-activated K+ channels and that the membrane potential may be of greater importance for the stimulus-secretion coupling than recognized previously.     

A mechanosensitive K+ channel in heart cells. Activation by arachidonic acid

Mechanosensitive ion channels have been described in many types of cells. These channels are believed to transduce pressure signals into intracellular biochemical and physiological events. In this study, the patch-clamp technique was used to identify and characterize a mechanosensitive ion channel in rat atrial cells. In cell-attached patches, negative pressure in the pipette activated an ion channel in a pressure-dependent manner. The pressure to induce half-maximal activation was 12 +/- 3 mmHg at +40 mV, and nearly full activation was observed at approximately 20 mmHg. The probability of opening was voltage dependent, with greater channel activity at depolarized potentials. The mechanosensitive channel was identical to the K+ channel previously shown to be activated by arachidonic acid and other lipophilic compounds, as judged by the outwardly rectifying current-voltage relation, single channel amplitude, mean open time (1.4 +/- 0.3 ms), bursty openings, K+ selectivity, insensitivity to any known organic inhibitors of ion channels, and pH sensitivity. In symmetrical 140 mM KCl, the slope conductance was 94 +/- 11 pS at +60 mV and 64 +/- 8 pS at -60 mV. Anions and cations such as Cl-, glutamate, Na+, Cs+, Li+, Ca2+, and Ba2+ were not permeant. Extracellular Ba2+ (1 mM) blocked the inward K+ current completely. GdCl3 (100 microM) or CaCl2 (100 microM) did not alter the K+ channel activity or amplitude. Lowering of intracellular pH increased the pressure sensitivity of the channel. The K+ channel could be activated in the presence of 5 mM intracellular [ATP] or 10 microM glybenclamide in inside-out patches. In the absence of ATP, when the ATP-sensitive K+ channel was active, the mechanosensitive channel could further be activated by pressure, suggesting that they were two separate channels. The ATP-sensitive K+ channel was not mechanosensitive. Pressure activated the K+ channel in the presence of albumin, a fatty acid binding protein, suggesting that pressure and arachidonic acid activate the K+ channel via separate pathways.     

Background osmolyte current involved in cell volume regulation of neuroblastoma x glioma hybrid NG108-15 cells.

Recently, we showed that at constant extracellular osmolarity, the volume of NG108-15 cells was dependent on the external NaCl concentration and we assumed that the responsible mechanism was mediated by background channels (Rouzaire-Dubois et al. 1999). In order to confirm this view, the mean cell volume and the background current of NG108-15 cells were measured under different experimental conditions, after blockade of specific volume regulating mechanisms and ion channels. When the external NaCl concentration was decreased, the reversal potential of the background current was shifted toward negative values and the membrane conductance decreased. Opposite effects were observed when the NaCl concentration was increased. Substitution of external Na+ with various monovalent cations altered the mean cell volume by: Rb+, +17%; Cs+, +15%; K+, +10%; Li+, -6%; choline, -9%; N-methylglucamine, -25% . The reversal potential of the background current and the membrane conductance were altered by these Na+ substitutes in such a way that the cell volume increased linearly with the background current at -60 mV. Substitution of external Cl- with various monovalent anions altered the mean cell volume by: I-, +4%; Br-, 0%; NO-, -3%; F-, -5%; isethionate, -30%; gluconate, -50%. Cl- substitutes did not significantly alter the background current at -60 mV, except F- which increased it by 39%. These results suggest that 1. the cell volume is dependent on ion fluxes through background channels; 2. electrogenic cation fluxes are larger than anionic ones and the background current is proportional to the difference between these fluxes; 3. whereas external cations do not interfere with anion fluxes, external anions alter cation fluxes.     

Effect of amiloride on the activity of membrane-bound and soluble Na+-,K+-ATPase preparations

Amiloride (8 X 10(-4), an inhibitor of sodium channels of nonexcited membranes, inhibits the activity of Na+,K+-ATPase in the kidney cortex homogenate as well as that of the partially purified membrane-bound and lubrol-soluble Na+,K+-ATPase preparations from the cattle brain. Inhibition of Na+,K+-ATPase from different organs of various animals by amiloride, a blocker of sodium channels, indicates similarity of the molecular organization of the Na+-recognizing component both of sodium channels and sodium centres of Na+,K+-ATPase.     

Extracellular polyvalent cation block of slow Na+ channels in Xenopus laevis oocytes

Sustained depolarization of the Xenopus oocyte membrane elicits a slowly activating Na+ current, thought to be due to the opening of sodium selective channels. These channels are induced to become voltage gated by the depolarization. They show unconventional gating properties and are insensitive to tetrodotoxin (TTX). The present study was undertaken to evaluate the effect of extracellular multivalent cations (Ca2+, Co2+, Cd2+, La3+, Mg2+, Mn2+, Ni2+, Sr2+ and Zn2+) on these TTX-resistant channels, either on membrane potential responses or on current responses. Our data show that all the polyvalent cations used blocked Na+ channels in a concentration-dependent manner. The order of potency of the most efficient cations was Co2+ < Ni2+ < Cd2+ < Zn2+, the respective concentration required to cause a 50% decrease of Na+ current was 0.9+/-0.29; 0.47+/-0.15; 0.36+/-0.09 and 0.06+/-0.02 mmol/l. The comparison of the activation curves from controls and after treatment with the cations indicated a shift towards more positive voltages. These results can be interpreted as due to the screening effect of divalent cations together with an alteration of the Na+ channel gating properties. We also show that divalent cations blocked Na+ channels in an open state without interfering with the induction mechanism of the channels. The possibility that cation block was due to a possible interaction between cations and SH-groups was investigated, but a sulphydryl alkylating reagent failed to abolish Zn2+ block.     

An ultrastructural study of proliferative nephritis induced experimentally by a monoclonal antibody against mesangial cells: replacement of mesangial cells by cells of the monocyte-macrophage system

An experimental nephritis accompanied by transient proteinuria can be produced by an intravenous injection of the monoclonal antibody, 1-22-3, raised against isolated rat glomeruli. The present study deals with the ultrastructural changes in the glomeruli in rats after the injection of this antibody. At 2 h after injection, all the mesangial cells had completely degenerated and neutrophils invaded most mesangial areas. Monocytes occupied the vacant mesangial areas at 24 h and gradually increased in number over the next 4 days. At 4 and 6 days, macrophage-like cells, possibly derived from monocytes, underwent frequent mitosis, resulting in a remarkable proliferation of these cells. The interpretation of these cells as macrophages was strongly supported by the fact that they contained previously injected latex particles in large numbers. From 2 to 4 weeks after injection, the macrophage-like cells gradually transformed into cells indistinguishable from normal mesangial cells. In the present experimental nephritis where all mesangial cells were initially destroyed, cells of the monocyte-macrophage system appear to play a leading role in the pathogenesis of the ensuing proliferative glomerulonephritis, and represent the source of the replacing mesangial cells.     

Distinct metal ion binding sites on Ca(2+)-activated K+ channels in inside-out patches of human erythrocytes.

Effects of Cd2+, Co2+, Pb2+, Fe2+ and Mg2+ (1-100 microM) on single-channel properties of the intermediate conductance Ca(2+)-activated K+ (CaK) channels were investigated in inside-out patches of human erythrocytes in a physiological K+ gradient. Cd2+, Co2+ and Pb2+, but not Fe2+ and Mg2+, were able to induce CaK channel openings. The potency of the metals to open CaK channels in human erythrocytes follows the sequence Pb2+, Cd2+ > Ca2+ > or = Co2+ > Mg2+, Fe2+. At higher concentrations Pb2+, Cd2+ and Co2+ block the CaK channel by reducing the opening frequency and the single-channel current amplitude. The potency of the metals to reduce CaK channel opening frequency follows the sequence Pb2+ > Cd2+, Co2+ > Ca2+, which differs from the potency sequence Cd2+ > Pb2+, Co2+ > Ca2+ to reduce the unitary single-channel current amplitude. Fe2+ reduced the channel opening frequency and enhanced the two open times of CaK channels activated by Ca2+, whereas up to 100 microM Mg2+ had no effect on any of the measured single-channel parameters. It is concluded that the activation of CaK channels of human erythrocytes by various metal ions occurs through an interaction with the same regulatory site at which Ca2+ activates these channels. The different potency orders for the activating and blocking effects suggest the presence of at least one activation and two blocking sites. A modulatory binding site for Fe2+ exists as well. In addition, the CaK channels in human erythrocytes are distinct from other subtypes of Ca(2+)-activated K+ channels in their sensitivity to the metal ions.     

Voltage-dependent slowing of K channel closing kinetics by Rb+

We have studied the effect of Rb+ on K channel closing kinetics in toadfish pancreatic islet cells. These channels are voltage dependent, activating at voltages positive to -10 mV. The channels also inactivate upon prolonged depolarizations, and the inactivation time course is best fit by the sum of two exponentials. Instantaneous current-voltage relationships show that external Rb+ enters the channel as easily as K+, but carries less current. In the voltage range from -140 to -50 mV, the closing time course of the channels can be fit with a single exponential. When Rb+ is present in the external solution the channels close more slowly. The magnitude of this Rb+ effect is voltage dependent, decreasing at more negative voltages. Similarly, when the internal solution contains Rb+ instead of K+ the closing time constants are increased. The effect of internal Rb+ is also voltage dependent; at voltages positive to -80 mV the closing time constant in internal Rb+ is slower than in K+, whereas at more negative voltages the difference is negligible. With internal Rb+, the relationship between the closing time constant and voltage is best fit with two exponential components, suggesting the presence of two distinct voltage-dependent processes. The results are discussed in terms of a model of the K channel with two internal binding sites, and we conclude that Rb+ produces its effects on channel gating by binding to a site in the pore.