The Influences of I h on Temporal Summation in Hippocampal CA1 Pyramidal Neurons: A Modeling Study

Recent experimental and theoretical studies have found that active dendritic ionic currents can compensate for the effects of electrotonic attenuation. In particular, temporal summation, the percentage increase in peak somatic voltage responses invoked by a synaptic input train, is independent of location of the synaptic input in hippocampal CA1 pyramidal neurons under normal conditions. This independence, known as normalization of temporal summation, is destroyed when the hyperpolarization-activated current, I _h, is blocked [Magee JC (1999a), Nature Neurosci. 2: 508–514]. Using a compartmental model derived from morphological recordings of hippocampal CA1 pyramidal neurons, we examined the hypothesis that I _h was primarily responsible for normalization of temporal summation. We concluded that this hypothesis was incomplete. With a model that included I _h, the persistent Na⁺ current (I _NaP), and the transient A-type K⁺ current (I _A), however, we observed normalization of temporal summation across a wide range of synaptic input frequencies, in keeping with experimental observations.     

The vicissitudes of the pacemaker current <Emphasis Type="Italic">I</Emphasis><Subscript>Kdd</Subscript> of cardiac purkinje fibers

Summary The mechanisms underlying the pacemaker current in cardiac tissues is not agreed upon. The pacemaker potential in Purkinje fibers has been attributed to the decay of the potassium current I _Kdd. An alternative proposal is that the hyperpolarization-activated current I _f underlies the pacemaker potential in all cardiac pacemakers. The aim of this review is to retrace the experimental development related to the pacemaker mechanism in Purkinje fibers with reference to findings about the pacemaker mechanism in the SAN as warranted. Experimental data and their interpretation are critically reviewed. Major findings were attributed to K⁺ depletion in narrow extracellular spaces which would result in a time dependent decay of the inward rectifier current I _K1. In turn, this decay would be responsible for a “fake” reversal of the pacemaker current. In order to avoid such a postulated depletion, Ba²⁺ was used to block the decay of I _K1. In the presence of Ba²⁺ the time-dependent current no longer reversed and instead increased with time and more so at potentials as negative as −120 mV. In this regard, the distinct possibility needs to be considered that Ba²⁺ had blocked I _Kdd (and not only I _K1). That indeed this was the case was demonstrated by studying single Purkinje cells in the absence and in the presence of Ba²⁺. In the absence of Ba²⁺, I _Kdd was present in the pacemaker potential range and reversed at E _K. In the presence of Ba²⁺, I _Kdd was blocked and I _f appeared at potentials negative to the pacemaker range. The pacemaker potential behaves in a manner consistent with the underlying I _Kdd but not with I _f. The fact that I _f is activated on hyperpolarization at potential negative to the pacemaker range makes it suitable as a safety factor to prevent the inhibitory action of more negative potentials on pacemaker discharge. It is concluded that the large body of evidence reviewed proves the pacemaker role of I _Kdd (but not of I _f) in Purkinje fibers.     

Dendritic I h Selectively Blocks Temporal Summation of Unsynchronized Distal Inputs in CA1 Pyramidal Neurons

The active dendritic conductances shape the input-output properties of many principal neurons in different brain regions, and the various ways in which they regulate neuronal excitability need to be investigated to better understand their functional consequences. Using a realistic model of a hippocampal CA1 pyramidal neuron, we show a major role for the hyperpolarization-activated current, Ih, in regulating the spike probability of a neuron when independent synaptic inputs are activated with different degrees of synchronization and at different distances from the soma. The results allowed us to make the experimentally testable prediction that the Ih in these neurons is needed to reduce neuronal excitability selectively for distal unsynchronized, but not for synchronized, inputs.     

Atrial proarrhythmia due to increased inward rectifier current (IK1) arising from KCNJ2 mutation – A simulation study

Atrial fibrillation (AF) has been linked to increased inward rectifier potassium current, I_K1, either due to AF-induced electrical remodelling, or from functional changes due to the Kir2.1 V93I mutation. The aim of this simulation study was to identify at cell and tissue levels' mechanisms by which increased I_K1 facilitates and perpetuates AF. The Courtemanche et al. human atrial cell action potential (AP) model was modified to incorporate reported changes in I_K1 induced by the Kir2.1 V93I mutation in both heterozygous (Het) and homozygous (Hom) mutant forms. The modified models for wild type (WT), Het and Hom conditions were incorporated into homogeneous 1D, 2D and 3D tissue models. Restitution curves of AP duration (APD), effective refractory period (ERP) and conduction velocity (CV) were computed and both the temporal and the spatial vulnerability of atrial tissue to re-entry were measured. The lifespan and tip meandering pattern of re-entry were also characterised. For comparison, parallel simulations were performed by incorporating into the Courtmanche et al. model a linear increase in maximal I_K1 conductance. It was found that the gain-in-function of V93I ‘mutant’ I_K1 led to abbreviated atrial APs and flattened APD, ERP and CV restitution curves. It also hyperpolarised atrial resting membrane potential and slowed down intra-atrial conduction. V93I ‘mutant’ I_K1 reduced the tissue's temporal vulnerability but increased spatial vulnerability to initiate and sustain re-entry, resulting in an increased overall susceptibility of atrial tissue to arrhythmogenesis. In the 2D model, spiral waves self-terminated for WT (lifespan < 3.3 s) tissue, but persisted in Het and Hom tissues for the whole simulation period (lifespan > 10 s). The tip of the spiral wave meandered more in WT tissue than in Het and Hom tissues. Increased I_K1 due to augmented maximal conductance produced similar results to those of Het and Hom Kir2.1 V93I mutant conditions. In the 3D model the dynamic behaviour of scroll waves was stabilized by increased I_K1. In conclusion, increased I_K1 current, either by the Kir2.1 V93I mutation or by augmented maximal conductance, increases atrial susceptibility to arrhythmia by increasing the lifespan of re-entrant spiral waves and the stability of scroll waves in 3D tissue, thereby facilitating initiation and maintenance of re-entrant circuits.     

心室细胞生成的生物起搏器的仿真研究

植入电子起搏器可以治疗因窦房结功能失常引起的猝死等心脏疾病.但是,电子起搏器存在很多弊端,比如电池寿命有限、容易感染等.因此,生物起搏器被期待能够取代电子起搏器.为了探讨在心室内诱导心室细胞生成生物起搏器的可行性,我们首先抑制内向整流钾电流(I_(K1)),使心室肌细胞产生起搏行为,然后基于理想心室组织和真实人体心室切片数据,构建2D生物起搏器模型.基于该模型,我们研究细胞间的电偶联和起搏电流I_f对起搏器功能的影响.发现起搏电流I_f对起搏器功能有增强作用,但细胞间的弱电偶联对起搏器的起搏有更为关键的影响.     

Bifurcation Analysis of Genetically Engineered Pacemaking in Mammalian Heart

Genetically engineered pacemaking in ventricular cells has been achieved by down-regulation of the time independent inward rectifying current (I _K1), or insertion of the hyperpolarisation-activated funny current (I _f). We analyse the membrane system (i.e. ionic concentrations clamped) of an epicardial Luo-Rudy dynamic cell model using continuation algorithms with the maximum conductance () of I _K1 and I _f as bifurcation parameters. Pacemaker activity can be induced either via Hopf or homoclinic bifurcations. As _K1 is decreased by ≈74%, autorhythmicity emerged via a homoclinic bifurcation, i.e., the periodicity first appear with infinitely large periods. In contrast, the insertion of _f induced periodicity via a subcritical Hopf bifurcation at _f≈ 0.25 mSμF⁻¹. Stable autorhythmic action potentials occurred at _f > 0.329 mSμF⁻¹.     

Effects of pressure overload-induced hypertrophy on TTX-sensitive inward currents in guinea pig left ventricle

We investigated the effects of pressure overload hypertrophy on inward sodium (I _Na) and calcium currents (I _Ca) in single left ventricular myocytes to determine whether changes in these current systems could account for the observed prolongation of the action potential. Hypertrophy was induced by pressure overload caused by banding of the abdominal aorta. Whole-cell patch clamp experiments were used to measure tetrodotoxin (TTX)-sensitive inward currents. The main findings were that I _Ca density was unchanged whereas I _Na density after stepping from –80 to –30 mV was decreased by 30% (–9.0 ± 1.16 pA pF^–1 in control and –6.31 ± 0.67 pA pF^–1 in hypertrophy, p < 0.05, n= 6). Steady-state activation/inactivation variables of I _Na, determined by using double-pulse protocols, were similar in control and hypertrophied myocytes, whereas the time course of fast inactivation of I _Na was slowed (p < 0.05) in hypertrophied myocytes. In addition, action potential clamp experiments were carried out in the absence and presence of TTX under conditions where only Ca²⁺ was likely to enter the cell via TTX-sensitive channels. We show for the first time that a TTX-sensitive inward current was present during the plateau phase of the action potential in hypertrophied but not control myocytes. The observed decrease in I _Na density is likely to abbreviate rather than prolong the action potential. Delayed fast inactivation of Na⁺ channels was not sustained throughout the voltage pulse and may therefore merely counteract the effect of decreased I _Na density so that net Na⁺ influx remains unaltered. Changes in the fast I _Na do not therefore appear to contribute to lengthening of the action potential in this model of hypertrophy. However, the presence of a TTX-sensitive current during the plateau could potentially contribute to the prolongation of the action potential in hypertrophied cardiac muscle. (Mol Cell Biochem 261: 217–226, 2004)     

In silico risk assessment for drug-induction of cardiac arrhythmia

The main components of repolarization reserve for the ventricular action potential (AP) are the rapid (I_Kr) and slow (I_Ks) delayed outward K⁺ currents. While many drugs block I_Kr and cause life-threatening arrhythmias including torsades de pointes, the frequency of arrhythmias varies between different I_Kr-blockers. Different types of block of I_Kr cause distinct phenotypes of prolongation of action potential duration (APD), increase in transmural dispersion of repolarization (TDR) and, accordingly, occurrence of torsades de pointes. Therefore the assessment of a drug's proarrhythmic risk requires a method that provides quantitative and comprehensive comparison of the effects of different forms of I_Kr-blockade upon APDs and TDR. However, most currently available methods are not adapted to such an extensive comparison. Here, we introduce I_Kr–I_Ks two-dimensional maps of APD and TDR as a novel risk-assessment method. Taking the kinetics of I_Kr-blockade into account, APDs can be calculated upon a ventricular AP model which systematically alters the magnitudes of I_Kr and I_Ks. The calculated APDs are then plotted on a map where the x axis represents the conductance of I_Kr while the y axis represents that of I_Ks. TDR is simulated with models corresponding to APs in epicardial, midcardial and endocardial myocardium. These two-dimensional maps of APD and TDR successfully account for differences in the risk resulting from three distinct types of I_Kr-blockade which correspond to the effects of dofetilide, quinidine and vesnarinone. This method may be of use to assess the arrhythmogenic risk of various I_Kr-blockers.     

Hyperpolarization-Activated Current (Ih) Is Reduced in Hippocampal Neurons from Gabra5?/? Mice

Changes in the expression of γ-aminobutyric acid type A (GABA_A) receptors can either drive or mediate homeostatic alterations in neuronal excitability. A homeostatic relationship between α5 subunit-containing GABA_A (α5GABA_A) receptors that generate a tonic inhibitory conductance, and HCN channels that generate a hyperpolarization-activated cation current (I_h) was recently described for cortical neurons, where a reduction in I_h was accompanied by a reciprocal increase in the expression of α5GABA_A receptors resulting in the preservation of dendritosomatic synaptic function. Here, we report that in mice that lack the α5 subunit gene (Gabra5−/−), cultured embryonic hippocampal pyramidal neurons and ex vivo CA1 hippocampal neurons unexpectedly exhibited a decrease in I_h current density (by 40% and 28%, respectively), compared with neurons from wild-type (WT) mice. The resting membrane potential and membrane hyperpolarization induced by blockade of I_h with ZD-7288 were similar in cultured WT and Gabra5−/− neurons. In contrast, membrane hyperpolarization measured after a train of action potentials was lower in Gabra5−/− neurons than in WT neurons. Also, membrane impedance measured in response to low frequency stimulation was greater in cultured Gabra5−/− neurons. Finally, the expression of HCN1 protein that generates I_h was reduced by 41% in the hippocampus of Gabra5−/− mice. These data indicate that loss of a tonic GABAergic inhibitory conductance was followed by a compensatory reduction in I_h. The results further suggest that the maintenance of resting membrane potential is preferentially maintained in mature and immature hippocampal neurons through the homeostatic co-regulation of structurally and biophysically distinct cation and anion channels.     

The Serotonin 1A A Receptor: A Representative Member of the Serotonin Receptor Family

1. Serotonin is an intrinsically fluorescent biogenic amine that acts as a neurotransmitter and is found in a wide variety of sites in the central and peripheral nervous system. Serotonergic signaling appears to play a key role in the generation and modulation of various cognitive and behavioral functions.2. Serotonin exerts its diverse actions by binding to distinct cell surface receptors which have been classified into many groups. The serotonin_1A (5-HT_1A) receptor is the most extensively studied of the serotonin receptors and belongs to the large family of seven transmembrane domain G-protein coupled receptors.3. The tissue and sub-cellular distribution, structural characteristics, signaling of the serotonin_1A receptor and its interaction with G-proteins are discussed.4. The pharmacology of serotonin_1A receptors is reviewed in terms of binding of agonists and antagonists and sensitivity of their binding to guanine nucleotides.5. Membrane biology of 5-HT_1A receptors is presented using the bovine hippocampal serotonin_1A receptor as a model system. The ligand binding activity and G-protein coupling of the receptor is modulated by membrane cholesterol thereby indicating the requirement of cholesterol in maintaining the receptor organization and function. This, along with the reported detergent resistance characteristics of the receptor, raises important questions on the role of membrane lipids and domains in the function of this receptor.     

Different photoreceptors within the same retina express unique combinations of potassium channels

Single electrode current and voltage clamp recordings in Calliphora, and whole-cell voltage clamp recordings in Drosophila were used to characterise the voltage-gated K channels in both major classes of photoreceptors, R7/8 (long visual fibres, LVFs) and R1-6 (short visual fibres, SVFs). R7/8 were identified by their unique spectral properties, ca. 3–4 fold higher input resistances and 3–4 fold lower cell capacitance. In Calliphora SVFs possess both fast and slow activating delayed rectifier potassium conductances. Drosophila SVFs possess a slowly inactivating delayed rectifier (I_Ks), a very rapidly inactivating A channel encoded by the Shaker gene (I_A), and, in a minority of cells, a third K conductance with intermediate kinetics (I_Kf). In both specs the LVFs lack the slowest component, but exhibit the faster K conductance(s) with properties indistinguishable from those in the SVFs. These findings add to established evidence demonstrating the significant role played by potassium channels in tuning the photoreceptor membrane. The results also suggest that R1-6 photoreceptors and R7/8 form inputs to visual subsystems tuned to different temporal frequencies.Abbreviations LVF long visual fibre - SVF short visual fibre - R1-6 retinular cells 1 to 6 inclusive - R7/8 retinular cell 7 and 8 - I _A rapidly inactivating A type potassium conductance; channel coded by Shaker gene - I _Kf rapidly activating, slowly inactivating delayed rectifier-like potassium conductance - I _Ks slowly activating, slowly inactivating delayed rectifier-like potassium conductance - I _KDs slowly activating delayed rectifier potassium conductance - I _KDf rapidly activating delayed rectifier potassium conductance     

Peculiarities of 4-aminopyridine-induced blockade of the A-type potassium current in rat hippocampal neurons

We studied the effect of 4-aminopyridine (4-AP) on the channels responsible for transient rapidly inactivating potassium A-type current (I _A ) in the somatic membrane of cultured rat hippocampal neurons. External application of 4-AP in different concentrations (from 100 μM to 5mM) was made to the locus of the neuron under study using a fast local superfusion technique. TheI _A blockade was dose-dependent and voltage-independent. Interaction of the blocker with theI _A -conducting channels at a test potential of +40 mV could be approximated by Hill isotherm with the cooperativity coefficient of 2 and EC₅₀ equal to 2.1 mM. The action of 4-AP accelerated temporal inactivation ofI _A . The intensity ofI _A blockade became higher as the frequency of test membrane potential shifts increased.     

Spatial correlograms of soil cover as an indicator of landscape heterogeneity

Soil cover, which is one of the most informative and integrative landscape factors, can be used for the analysis of landscape patterns. We studied the spatial autocorrelation (Moran's I) of raster format soil maps (1:10,000; 10 m pixel size) in 35 study areas representing all landscape regions in Estonia. The carbonate concentration of soils, volumetric soil moisture (%) and the depth of the groundwater table were taken into consideration in compiling a scale of contrast of 17 soil groups. We introduce a simple characteristic based on spatial correlograms: a half-value distance lag, h_I = 0.5—a distance where Moran's I drops below 0.5. Spatial autocorrelation decreased very rapidly in the case of heights with a very heterogeneous landscape composition, showing low values of h_I = 0.5 (<100 m in all 6 study areas). In uplands and depressions, the spatial autocorrelation also decreased relatively rapidly (h_I = 0.5 < 200 m). In most of the plains, coastal lowlands, sea islands and inland paludified lowlands, the values of Moran's I did decrease slowly with increasing lag, being >200 m in all forest and bog areas with complex topographical conditions due to the variety of glacial landforms and peatlands. All of the eight FRAGSTATS landscape metrics studied demonstrated significant correlations with h_I = 0.5, whereas five of them – Contrast Weighted Edge Density (CWED); Percentage of Like Adjacencies (PLADJ), Edge Density (ED), Patch Density (PD) and Mean Patch Area Distribution (AREA_MN) – had Spearman Rank Order Correlation values higher than 0.8. Landscapes with high ED, PD, and CWED values have a low autocorrelation: PD, ED, and CWED correlated negatively with h_I = 0.5. PD, ED, and CWED decreased and PLADJ increased with the power-law relationship with increasing h_I = 0.5. Spatial autocorrelation is lower in landscapes with complex structure and high contrast. The positive relationship with PLADJ indicates the same. Thus, spatial correlograms of potential landscape structure based on soil cover analysis can be used for the characterization of human-influenced landscape (land use) structure.     

Understanding effects on excitability of simulated <Emphasis Type="Italic">I</Emphasis><Subscript>h</Subscript> modulation in simple neuronal models

The hyperpolarization-activated, inward, mixed cation current, I _h, appears in a wide variety of cells in the nervous system, contributes to diverse neuronal properties, and is up-regulated by a number of important neurotransmitters. Up-regulation of I _h is usually associated with an excitability-enhancing depolarization of resting membrane potential and an excitability-depressing shunting effect caused by a decrease in input resistance. In order to gain a better understanding of the interaction of these effects and their influence on excitability with I _h modulation, we systematically analyze changes in neuronal properties associated with excitability during I _h modulation in simplified, yet, biophysical neuron models based on a hippocampal pyramidal neuron. We simulate I _h modulation by varying both its maximal conductance and its half-activation voltage, mimicking the effects of cAMP-linked neurotransmitters, through ranges of physiologically realistic parameter regimes. Of particular interest is the contribution of the different effects of I _h up-regulation when membrane potentials are held at common levels and neuronal excitability is probed. Our modeling results suggest that, although holding potentials at common levels may compensate for changes in resting membrane potentials, this protocol may exaggerate the excitability-depressing influences of changes in input resistances with I _h up-regulation.     

Modulation of Antagonist Binding to Serotonin1A Receptors from Bovine Hippocampus by Metal Ions

1. The serotonin_1A(5-HT_1A) receptors are members of a superfamily of seven transmembrane domain receptors that couple to G-proteins. They appear to be involved in various behavioral and cognitive functions. Although specific 5-HT_1Aagonists have been discovered more than a decade back, the development of selective 5-HT_1Aantagonists has been achieved only recently.2. We have examined the modulation of the specific antagonist [³H]p-MPPF binding to 5-HT_1Areceptors from bovine hippocampal membranes by monovalent and divalent metal ions. Our results show that the antagonist binding to 5-HT_1Areceptors is inhibited by both monovalent and divalent cations in a concentration-dependent manner. This is accompanied by a concomitant reduction in binding affinity.3. Our results also show that the specific antagonist p-MPPF binds to all available receptors in the bovine hippocampal membrane irrespective of their state of G-protein coupling and other serotonergic ligands such as 5-HT and OH-DPAT effectively compete with the specific antagonist [³H]p-MPPF.4. These results are relevant to ongoing analyses of the overall modulation of ligand binding in G-protein-coupled seven transmembrane domain receptors.     

A computational study of the role of spike broadening in synaptic facilitation of Hermissenda

Pavlovian conditioning in Hermissenda produces a decrease in voltage-dependent (I_K,A and I_Ca) and Ca²⁺-dependent (I_K,Ca) currents, and an increase in the action potential (AP) duration in type B-photoreceptors. In addition, synaptic connections between B and A photoreceptors and B photoreceptor and type I interneurons are facilitated. The increase in AP duration, produced by decreasing one or more K⁺ currents, may account for synaptic facilitation. The present study examined this issue by using a mathematical model of the B-photoreceptor and the neurosimulator SNNAP. In the model, decreasing g_K,A by 70% increased the duration of the AP in the terminal by 41% and Ca²⁺ influx by 30%. However, if the decrease in g_K,A was combined with a decrease in g_Ca, similar to what has been reported experimentally, the Ca²⁺ influx decreased by 54%. Therefore, the concomitant change in I_Ca counter-acted the broadening-induced increase in Ca²⁺ influx in the synaptic terminal. This result suggests that a spike-duration independent process must contribute to the synaptic facilitation observed following Pavlovian conditioning.     

Computational study of enhanced excitability in Hermissenda: membrane conductances modulated by 5-HT

Serotonin (5-HT) applied to the exposed but otherwise intact nervous system results in enhanced excitability of Hermissenda type-B photoreceptors. Several ion currents in the type-B photoreceptors are modulated by 5-HT, including the A-type K⁺ current (I_K,A), sustained Ca²⁺ current (I_Ca,S), Ca-dependent K⁺ current (I_K,Ca), and a hyperpolarization-activated inward rectifier current (I_h). In this study, we developed a computational model that reproduces physiological characteristics of type B photoreceptors, e.g. resting membrane potential, dark-adapted spike activity, spike width, and the amplitude difference between somatic and axonal spikes. We then used the model to investigate the contribution of different ion currents modulated by 5-HT to the magnitudes of enhanced excitability produced by 5-HT. Ion currents were systematically varied within limits observed experimentally, both individually and in combinations. A reduction of I_K,A or I_K,Ca, or an increase in I_h enhanced excitability by 20–50%. Decreasing I_Ca,S produced a dramatic decrease in excitability. Reductions of I_K,V produced only minimal increases in excitability, suggesting that I_K,V probably plays a minor role in 5-HT induced enhanced excitability. Combinations of changes in I_K,A, I_K,Ca, I_h and I_Ca,S produced increases in excitability comparable to experimental observations. After 5-HT application, the cell's depolarization force is shifted from the I_h–I_Ca,S combination to predominantly I_h.     

On the endothelial cell ISOC

Ca²⁺ store depletion activates both Ca²⁺ selective and non-selective currents in endothelial cells. Recently, considerable progress has been made in understanding the molecular make-up and regulation of an endothelial cell thapsigargin-activated Ca²⁺ selective current, I_SOC. Indeed, I_SOC is a relatively small inward Ca²⁺ current that exhibits an approximate +40 mV reversal potential and is strongly inwardly rectifying. This current is sensitive to organization of the actin-based cytoskeleton. Transient receptor potential (TRP) proteins 1 and 4 (TRPC1 and TRPC4, respectively) each contribute to the molecular basis of I_SOC, although it is TRPC4 that appears to be tethered to the cytoskeleton through a dynamic interaction with protein 4.1. Activation of I_SOC requires association between protein 4.1 and the actin-based cytoskeleton (mediated through spectrin), suggesting protein 4.1 mediates the physical communication between Ca²⁺ store depletion and channel activation. Thus, at present findings indicate a TRPC4–protein 4.1 physical linkage regulates I_SOC activation following Ca²⁺ store depletion.     

Synthesis and pharmacological evaluation of novel 1- and 8-substituted-3-furfuryl xanthines as adenosine receptor antagonists

The synthesis of an important set of 3-furfurylxanthine derivatives is described. Binding affinities were determined for rat A₁ and human A_2A, A_2B and A₃ receptors. Several of the 3-furfuryl-7-methylxanthine derivatives showed moderate-to-high affinity at human A_2B receptors, the most active compound (10d) having a K_i of 7.4 nM for hA_2B receptors, with selectivities over rA₁ and hA_2A receptors up to 14-fold and 11-fold, respectively. Affinities for hA₃ receptors were very low for all members of the set.     

DIDS increases K+ secretion through an I sKchannel in apical membrane of vestibular dark cell epithelium of gerbil

Vestibular dark cell epithelium secretes K⁺ via I _sKchannels in the apical membrane. The previous observation that disulfonic stilbenes increased the equivalent short circuit current (I _sc) suggested that these agents might be useful investigative tools in this tissue. The present experiments were conducted to determine if the increase in I _scwas associated with an increase in K⁺ flux and if the effect was directly on the I _sKchannel or indirectly via a cytosolic intermediary. Measurements of transepithelial K⁺ flux with the K⁺-selective vibrating probe and of changes in net cellular solute flux by measurements of epithelial cell height showed that 4,4-diisothiocyanatostilbene-2,2-disulfonic acid (DIDS) increased K⁺ flux by a factor of 1.96±0.71 and caused net solute efflux. The apical membrane was partitioned with a macropatch pipette and DIDS was applied either to the membrane outside the pipette, inside the pipette or to the entire apical membrane. DIDS inside the pipette increased the current across the patch, the membrane conductance, the slowly-inactivating (I _sK) component of the membrane current and shifted the reversal voltage toward the equilibrium potential for K⁺. DIDS outside the patch decreased the patch current and conductance, consistent with shunting of current away from the membrane patch. These findings strongly support the notion that DIDS increases K⁺ secretion through I _sKchannels in the apical membrane of vestibular dark cell epithelium by acting directly on the channels or on a tightly colocalized membrane component.We thank Dr. Peter J.S. Smith and Alan Shipley of the National Vibrating Probe Facility at the Marine Biological Laboratory at Woods Hole, MA for their support and assistance in the measurements of K⁺ flux. This work was supported by National Institutes of Health grants R01-DC00212, R29-DC1098 and P41-RR01395.