Potassium channel kinetics in squid axons with elevated levels of external potassium concentration.

Potassium ion current in squid axons is usually modified by the effects of ion accumulation in the periaxonal space during voltage-clamp depolarization. The time course of potassium channel activation and ion accumulation usually overlap. A widely accepted procedure for circumventing the effects of accumulation in measurements of activation kinetics consists of measuring the difference in the current at the end of a depolarizing pulse and immediately following return of the membrane potential to the holding level. This instantaneous jump procedure is based upon the assumptions that the potassium channel current-voltage relation (IV) is a linear function of the driving force, and that the IV and the potassium channel-gating kinetics are both independent of ion accumulation. The latter assumption appears to be appropriate for activation kinetics. However, both assumptions concerning the IV are incorrect, in general. Consequently, the jump procedure provides a misleading view of gating kinetics for membrane depolarizations that produce net current flow. Jump conductance measurements for depolarizations that produce little or no net current indicate that the Hodgkin-Huxley n4 model of potassium channel kinetics is appropriate for the physiological range of membrane potentials.     

Potassium channel activity recorded from the apical membrane of freshly isolated epithelial cells in rat caudal epididymis.

K+ channels were recorded in excised, inside-out patches from the apical membrane of the freshly isolated tubule of the caudal portion of the rat epididymis. With asymmetric K+ concentrations in bath and pipette (140 mM K+in/6 mM K+out), the channels had a slope conductance of 54.2 pS at 0 mV. The relative permeability of K+ over Na+ was about 171 to 1. The channels were activated by intracellular Ca2+ and by membrane depolarization. These channels belong to a class defined as "intermediate-conductance Ca2+-activated K+ channel. " External tetraethylammonium ions (TEA+) caused a flickery block of the channel with reduction in single-channel current amplitude measured at a range of holding membrane potentials (-40 to 60 mV). Activity of the K+ channels was inhibited by intracellular ATP (KD =1.188 mM). The channel activity was detected only occasionally in patches from the apical membrane (about 1 in 17 patches containing active channels). The presence of the intermediate-conductance Ca2+-activated K+ channels indicates that they could provide a route for K+ secretion in a Ca2+-dependent process responsible for a high luminal K+ concentration found in the epididymal duct of the rat.     

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

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

K channel kinetics during the spontaneous heart beat in embryonic chick ventricle cells.

By averaging the current that passes through cell-attached patches on beating heart cells, while measuring action potentials with a whole-cell electrode, we were able to study K channels during beating. In 7-d chick ventricle in 1.3 mM K physiological solutions at room temperature, delayed-rectifier channels have three linear conductance states: 60, 30, and 15 pS. The 60 and 15 pS conductances can exist alone, but all three states may appear in the same patch as interconverting conductance levels. The delayed-rectifier conductance states have low densities (less than 10 channels per 10-microns diam cell), and all have a reversal potential near -75 mV and the same average kinetics. Outward K current through delayed-rectifier channels follows the upstroke without appreciable delay and lasts throughout the action potential. No inward current flows through delayed-rectifier channels during beating. The early outward channel has a nonlinear conductance of 18-9 pS depending on the potential. It also turns on immediately after the upstroke of the action potential and lasts on average only 50 ms. The early outward channel has an extrapolated reversal potential near -30 mV; no inward current flows during beating. The inward-rectifier has an extrapolated conductance and reversal potential of 2-3 pS and -80 mV in 1.3 mM K. Channel kinetics are independent of external K between 10 and 120 mM, and the channel conducts current only during the late repolarization and diastolic phases of the action potential. No outward current flows through inward-rectifier channels during beating. This work parallels a previous study of Na channels using similar techniques (Mazzanti, M., and L. J. DeFelice. 1987, Biophys. J. 52:95-100).     

Ca channel kinetics during the spontaneous heart beat in embryonic chick ventricle cells.

The ability of Ca ions to inhibit Ca channels presents one of the most intriguing problems in membrane biophysics. Because of this negative feedback, Ca channels can regulate the current that flows through them. The kinetics of the channels depend on voltage, and, because the voltage controls the current, a strong interaction exists between voltage dependence and Ca dependence. In addition to this interaction, the proximity of pores and the local concentration of ions also determine how effectively the Ca ions influence channel kinetics. The present article proposes a model that incorporates voltage-dependent kinetics, current-dependent kinetics, and channel clustering. We have based the model on previous voltage-clamp data and on Ca and Ba action currents measured during the action potential in beating heart cells. In general we observe that great variability exists in channel kinetics from patch to patch: Ba or Ca currents have low or high amplitudes and slow or fast kinetics during essentially the same voltage regime, either applied step-protocols or spontaneous cell action potentials. To explain this variability, we have postulated that Ca channels interact through shared ions. The model we propose expands on our previous model for Ba currents. We use the same voltage-dependent rate constants for the Ca currents that we did for the Ba currents. However, we vary the current-dependent rate constants according to the species of the conducting ion. The model reproduces the main features of our data, and we use it to predict Ca channel kinetics under physiological conditions. Preliminary reports of this work have appeared (DeFelice et al., 1991, Biophys. J. 59:551a; Risso et al., 1992, Biophys. J. 61:248a).     

Maintenance of Y receptor dimers in epithelial cells depends on interaction with G-protein heterotrimers

Treatment of CHO cells expressing human Y receptors (Y₁, Y₂ or Y4 subtype) with pertussis toxin results in a large decrease in functional receptors, with a preferential loss of heteropentameric assemblies of receptor dimers and G-protein trimers. This occurs in parallel to inactivation of the nucleotide site of Gi α subunits, with a half period of about 4 h. The loss could be mainly due to proteolysis at the level of recycling/perinuclear endosomes, and of receptor completion in the ER, since it is reduced by co-treatment with ammonium chloride, an inhibitor of particulate proteinases. Antagonists do not strongly decrease the heteropentameric fraction. These findings indicate that the upkeep of Y receptor dimers in epithelial cell lines depends on the association of receptor oligomers with functional Gi α subunits. This interaction could use the juxtamembrane helix 8 in the fourth intracellular domain, and could also be supported by the C-terminal helix of the third intracellular loop, as outlined in the companion review (Parker et al., Amino Acids, doi:, 2010).     

A higher order chromatin structure that is lost during differentiation of mouse neuroblastoma cells

Application of differential scanning calorimetry to nuclei from rapidly growing mouse neuroblastoma cells showed a melting profile with four major thermal transitions: I (60 degrees C), II (76 degrees C), III (88 degrees C), and IV (105 degrees C). When neuroblastoma cells were induced to differentiate by serum withdrawal or treatment with sodium butyrate, transition IV disappeared, while transition III increased in magnitude. Comparison was made to nuclei from several types of nondividing cells as well as a number of samples from mature tissues. In rapidly dividing cells the predominant endotherm was IV (105 degrees C), while in nondividing cells, transition III (88 degrees C) predominated the calorimetric profile. Cellular differentiation thus appeared to be accompanied by a major change in chromatin structure, as evidenced by a shift in melting temperature from 105 to 90 degrees C, and this may serve to distinguish the Go phase of the cell cycle from G1.     

Potassium ion channels of Chlorella viruses cause rapid depolarization of host cells during infection

Previous studies have established that chlorella viruses encode K(+) channels with different structural and functional properties. In the current study, we exploit the different sensitivities of these channels to Cs(+) to determine if the membrane depolarization observed during virus infection is caused by the activities of these channels. Infection of Chlorella NC64A with four viruses caused rapid membrane depolarization of similar amplitudes, but with different kinetics. Depolarization was fastest after infection with virus SC-1A (half time [t(1/2)], about 9 min) and slowest with virus NY-2A (t(1/2), about 12 min). Cs(+) inhibited membrane depolarization only in viruses that encode a Cs(+)-sensitive K(+) channel. Collectively, the results indicate that membrane depolarization is an early event in chlorella virus-host interactions and that it is correlated with viral-channel activity. This suggestion was supported by investigations of thin sections of Chlorella cells, which show that channel blockers inhibit virus DNA release into the host cell. Together, the data indicate that the channel is probably packaged in the virion, presumably in its internal membrane. We hypothesize that fusion of the virus internal membrane with the host plasma membrane results in an increase in K(+) conductance and membrane depolarization; this depolarization lowers the energy barrier for DNA release into the host.     

Thymus medulla formation and central tolerance are restored in IKKalpha-/- mice that express an IKKalpha transgene in keratin 5+ thymic epithelial cells

Medullary thymic epithelial cells (mTECs) play an essential role in establishing central tolerance due to their unique capacity to present a diverse array of tissue restricted Ags that induce clonal deletion of self-reactive thymocytes. One mTEC subset expresses keratin 5 (K5) and K14, but fails to bind Ulex europaeus agglutinin-1 (UEA-1) lectin. A distinct mTEC subset binds UEA-1 and expresses K8, but not K5 or K14. Development of both mTEC subsets requires activation of the noncanonical NF-kappaB pathway. In this study, we show that mTEC development is severely impaired and autoimmune manifestations occur in mice that are deficient in IkappaB kinase (IKK)alpha, a required intermediate in the noncanonical NF-kappaB signaling pathway. Introduction of an IKKalpha transgene driven by a K5 promoter restores the K5(+)K14(+) mTEC subset in IKKalpha(-/-) mice. Unexpectedly, the K5-IKKalpha transgene also rescues the UEA-1 binding mTEC subset even though K5 expression is not detectable in these cells. In addition, expression of the K5-IKKalpha transgene ameliorates autoimmune symptoms in IKKalpha(-/-) mice. These data suggest that 1) medulla formation and central tolerance depend on activating the alternative NF-kappaB signaling pathway selectively in K5-expressing mTECs and 2) the K5-expressing subset either contains immediate precursors of UEA-1 binding cells or indirectly induces their development.     

Potassium channels in primary cultures of seawater fish gill cells. I. Stretch-activated K(+) channels

Previous studies using the patch-clamp technique demonstrated the presence of a small conductance Cl(-) channel in the apical membrane of respiratory gill cells in primary culture originating from sea bass Dicentrarchus labrax. We used the same technique here to characterize potassium channels in this model. A K(+) channel of 123 +/- 3 pS was identified in the cell-attached configuration with 140 mM KCl in the bath and in the pipette. The activity of the channel declined rapidly with time and could be restored by the application of a negative pressure to the pipette (suction) or by substitution of the bath solution with a hypotonic solution (cell swelling). In the excised patch inside-out configuration, ionic substitution demonstrated a high selectivity of this channel for K(+) over Na(+) and Ca(2+). The mechanosensitivity of this channel to membrane stretching via suction was also observed in this configuration. Pharmacological studies demonstrated that this channel was inhibited by barium (5 mM), quinidine (500 microM), and gadolinium (500 microM). Channel activity decreased when cytoplasmic pH was decreased from 7.7 to 6.8. The effect of membrane distension by suction and exposure to hypotonic solutions on K(+) channel activity is consistent with the hypothesis that stretch-activated K(+) channels could mediate an increase in K(+) conductance during cell swelling.     

Mutations of Epstein-Barr virus gH that are differentially able to support fusion with B cells or epithelial cells

The core fusion machinery of all herpesviruses consists of three conserved glycoproteins, gB and gHgL, suggesting a common mechanism for virus cell fusion, but fusion of Epstein-Barr virus (EBV) with B cells and epithelial cells is initiated differently. Fusion with B cells requires a fourth protein, gp42, which complexes with gHgL and interacts with HLA class II, the B-cell coreceptor. Fusion with an epithelial cell does not require gp42 but requires interaction of gHgL with a novel epithelial cell coreceptor. Epithelial cell fusion can be inhibited by gp42 binding to gHgL and by antibodies to gH that fail to block B-cell fusion. This suggests that regions of gHgL initiating fusion with each cell are separable from each other and from regions involved in fusion itself. To address this possibility we mapped the region of gH recognized by a monoclonal antibody to gH that blocks EBV fusion with epithelial cells but not B cells by making a series of chimeras with the gH homolog of rhesus lymphocryptovirus. Proteins with mutations engineered within this region included those that preferentially mediate fusion with B cells, those that preferentially mediate fusion with epithelial cells, and those that mediate fusion with neither cell type. These results support the hypothesis that the core fusion function of gH is the same for B cells and epithelial cells and that it differs only in the way in which it is triggered into a functionally active state.     

Association of the epithelial sodium channel with Apx and alpha-spectrin in A6 renal epithelial cells.

Recent molecular cloning of the epithelial sodium channel (ENaC) provides the opportunity to identify ENaC-associated proteins that function in regulating its cell surface expression and activity. We have examined whether ENaC is associated with Apx (apical protein Xenopus) and the spectrin-based membrane cytoskeleton in Xenopus A6 renal epithelial cells. We have also addressed whether Apx is required for the expression of amiloride-sensitive Na(+) currents by cloned ENaC. Sucrose density gradient centrifugation of A6 cell detergent extracts showed co-sedimentation of xENaC, alpha-spectrin, and Apx. Immunoblot analysis of proteins co-immunoprecipitating under high stringency conditions from peak Xenopus ENaC/Apx-containing gradient fractions indicate that ENaC, Apx, and alpha-spectrin are associated in a macromolecular complex. To examine whether Apx is required for the functional expression of ENaC, alphabetagamma mENaC cRNAs were coinjected into Xenopus oocytes with Apx sense or antisense oligodeoxynucleotides. The two-electrode voltage clamp technique showed there was a marked reduction in amiloride-sensitive current in oocytes coinjected with antisense oligonucleotides when to compared with oocytes coinjected with sense oligonucleotides. These studies indicate that ENaC is associated in a macromolecular complex with Apx and alpha-spectrin in A6 cells and suggest that Apx is required for the functional expression of ENaC in Xenopus epithelia.     

Glycine transport by hemolysed and restored pigeon red cells. Effects of a Donnan-induced electrical potential on entry and exit kinetics.

The influence of a Donnan effect on the transport of glycine by hemolysed and restored pigeon red cells was examined. The Donnan effect was produced by replacing Cl- with 2,4-toluenedisulfonate or glutamate. The effects of the associated membrane potential and inside-outside pH difference on glycine entry and exit rates were examined. The effects of pH on entry and exit rates in the absence of a Donnan effect were also examined. In the absence of a Donnan effect, Na+-dependent glycine entry requires the protonated form of a group with a pKapp of 7.9 and the deprotonated form of another group with a pKapp of 6.8. Neither of these are required for exit but the deprotonated form of a group(s) with a pKapp of 6.2 is required. The pK 7.9 group and pK 6.2 group probably react with H+ at the inner face of the membrane and the pK 6.8 group probably reacts at the outer face. The V for glycine entry was determined for cells with their Cl- largely replaced by toluenedisulfonate and without such replacement. Between pH 6.1 and 7, the ratio of the respective V values, VT/VC1, was 1.5-1.7. VT/VC1 rose above pH 7 to near 4 at pH 8.3. At pH 6.9, with glutamate replacing cell Cl-, the analogous ratio (VGlu/VC1) was 1.7. The increase of VT/VC1 above pH 7 could be quantitatively accounted for by the increase in cell [H+]/medium [H+] caused by the Donnan effect together with the assumption that the pK 7.9 group reacts with H+ at the inner face of the membrane. When cell Cl- was replaced by toluenedisulfonate or glutamate there was a drop in the term in the glycine Km describing Na+ dependence of glycine entry. When cell Cl- was replaced by toluenedisulfonate therewas a rise in the Na+-independent term in the glycine entry Km. By replacing varying amounts of cell Cl- with either toluenedisulfonate or glutamate, plots were obtained of entry rates vs. the cell [Cl-]/ medium [Cl-] ratio consistent with the assumption that the Donnan-induced membrane potential acts on a "moving" charge. Glycine exit was only slightly accelerated by trans-toluenedisulfonate. The ratio, exit rate into toluenedisulfonate medium/exit rate into Cl- medium rose with decreasing pH. This rise could be accounted for by a Donnan-induced inside-outside pH difference which affects a pKapp 6.2 group reacting with internal H+. The observed influences of the Donnan effect on V (glycine entry), on both components of Km (glycine entry), on the shape of the plot of glycine entry rate vs. the cell [Cl-]/medium [Cl-] ratio and on glycine exit all fit the assumptions that when the empty porter reorients, one unit of negative charge accompanies it "across" the membrane and that no other steps involve charge movement. The properties of the system seem inconsistent with a translational ("ferry boat") mobile carrier.     

Single channel kinetics of a glutamate receptor.

The glutamate receptor-channel of locust muscle membrane was studied using the patch-clamp technique. Muscles were pretreated with concanavalin A to block receptor-channel desensitization, thus facilitating analysis of receptor-channel gating kinetics. Single channel kinetics were analyzed to aid in identification of the molecular basis of channel gating. Channel dwell-time distributions and dwell-time autocorrelation functions were calculated from single channel data recorded in the precence of 10-4M glutamate. Analysis of the dwell time distributions in terms of mixtures of exponential functions revealed there to be at least three open states of the receptor-channel and at least four closed states. Autocorrelation function analysis showed there to be at least three pathways linking the open states with the closed. This results in a minimal scheme for gating of the glutamate receptor-channel, which is suggestive of allosteric models of receptor-channel gating.     

Identification of sequences of chromosome 7 that are expressed in sweat gland epithelial cells

Summary This paper describes an approach that can be used to identify specifically expressed coding sequences in defined regions of genomic DNA. We developed this method to identify expressed sequences from chromosome 7 located at or near the cystic fibrosis (CF) locus. Radioactively labelled single-stranded cDNAs derived from sweat gland epithelial cells and from fibroblasts were used to screen a genomic library constructed from flow-sorted chromosomes. Differential screening of phage lifts with these two probes yielded 36 different DNA segments. By using somatic cell hybrids containing different portions of chromosome 7, four of the clones were mapped to the 7q31 region in which the CF locus is located. These four clones and two others that gave strong differential epithelial signals but that were not within 7q31 were studied further. Restriction fragment length polymorphisms (RFLPs) were identified for two of the DNA segments within 7q31 and used for linkage analysis using a panel of CF families. One DNA segment was assigned to a location centromeric to the met locus. The other marker did not show recombination with CF but was subsequently excluded from the CF region by physical mapping. Three of the six DNA segments were found to hybridize to various RNAs using the Northern technique and therefore contain portions of genes. One of the clones showed strong differential expression when epithelial tissues were compared to fibroblasts and may represent an epithelium-specific gene.     

Filament formation of MSF-A,a mammalian septin,in human mammary epithelial cells depends on interactions with microtubules

Septins are a family of conserved proteins implicated in a variety of cellular functions such as cytokinesis and vesicle trafficking, but their properties and modes of action are largely unknown. Here we now report findings of immunocytochemical and biochemical characterization of a mammalian septin, MSF-A. Using an antibody specific for MSF subfamily proteins, MSF-A was found to be expressed predominantly in mammary human mammary epithelial cells (HMEC). MSF-A was associated with microtubules in interphase HMEC cells as it localized with the mitotic spindle and the bundle of microtubule at midzone during mitosis. Biochemical analysis revealed direct binding of MSF-A with polymerized tubulin through its central region containing guanine nucleotide-interactive motifs. GTPase activity, however, was not required for the association. Conditions that disrupt the microtubule network also disrupted the MSF-A-containing filament structure, resulting in a punctate cytoplasmic pattern. Depletion of MSF-A using small interfering RNAs caused incomplete cell division and resulted in the accumulation of binucleated cells. Unlike Nedd5, an MSF mutant deficient in GTPase activity forms filament indistinguishable from that of the wild type in COS cells. These results strongly suggest that septin filaments may interact not only with actin filaments but also with microtubule networks and that GTPase activity of MSF-A is not indispensable to incorporation of MSF-A into septin filaments.     

Open channel noise. IV. Estimation of rapid kinetics of formamide block in gramicidin A channels.

Blocking events in currents through biological ion channels occur over a wide range of characteristic times. The interruptions in single-channel currents from blocking events may be characterized by the direct measurement of gap durations or by analyzing open-channel current histograms, provided that the events are not much shorter than the time resolution of single-channel recordings (approximately 10 microseconds). Here we present a method for the characterization of channel block on a much faster time scale by combining open-channel noise measurements with subsequent model fits according to a theoretical approach (Frehland, E. 1978. Biophysical Chemistry. 8:255-265). Although the bandwidth limitations in open-channel noise experiments are the same as in conventional single-channel experiments, from the dependence of the mean current and the spectral density of the noise on the concentration of the blocking agent, kinetics of very brief blocking events can be estimated. As an example we have analyzed the open-channel noise of K+ currents through the gramicidin A channel in the presence of various concentrations of formamide, a weak blocker, at neutral pH. We estimate the blocking and unblocking rates to be approximately 10(7)s-1 at 1 M formamide and discuss possible mechanisms for the blocking process.