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Mutations of the transmembrane channel-like 1 ( TMC1) gene can cause dominant and recessive forms of deafness in humans and mice. TMC1 is one of eight mammalian TMC genes of unknown function. The multi-pass transmembrane topologic structure of the proteins they encode suggests roles as a receptor, transporter, channel, or pump. Tmc1 and the closely related Tmc2 gene are expressed in neurosensory hair cells of the auditory and vestibular end organs of the mouse inner ear. Recent studies have demonstrated that Tmc1 and Tmc2 are specifically required for mechanoelectrical transduction in hair cells. The exact role of these proteins in mechanoelectrical transduction is unknown. TMC1 and TMC2 are viable candidates for the mechanoelectrical transduction channel of hair cells, whose component molecules have eluded identification for over 30 years. We expect that studies of TMC proteins will yield insights into molecular components and mechanisms of mechanosensation in auditory and vestibular hair cells, as well as in other tissues and organs. [ABSTRACT FROM AUTHOR]

Polycystic kidney disease 2-like 1 (PKD2L1), previously called transient receptor potential polycystin 3 (TRPP3), forms a voltage-dependent nonselective cation channel that exhibits large tail currents triggered by repolarization after depolarization. Since it has previously been proposed that temperature sensitivity of some TRP channels is linked to the voltage-dependent gating, we here investigated heating effects on PKD2L1 currents in human embryonic kidney HEK293T cells overexpressing mouse PKD2L1. Tail PKD2L1 currents were increased by heating to 32 °C, but decreased at more than 36 °C. Voltage dependency of the PKD2L1 channel was shifted by heating in a bimodal fashion: an increase in temperature to 32 °C and to 36 °C shifted the activation curves toward the left and the right, respectively. In addition, heating accelerated deactivation of tail PKD2L1 currents. To analyze the channel gating kinetics, single-channel events of the PKD2L1 channel were recorded at hyperpolarized potentials under whole-cell configurations. A rise in temperature decreased the open probability of the channel. Dwell-time analysis showed that both open and closed dwell times during heating were shorter than those at room temperature. Interestingly, a rapid temperature drop after heating markedly enhanced the PKD2L1 currents at both single-channel and whole-cell levels. The rebound activation of the PKD2L1 channel was due to an increase in the open probability but not in the single-channel conductance. These results suggest that heating opens but subsequently inactivates PKD2L1 channels, which is essential for the rebound activation of the channel after heating. [ABSTRACT FROM AUTHOR]

Polycystic kidney disease 2-like 1(PKD2L1), previously called transient receptor potential polycystin 3 (TRPP3), forms constitutively active voltage-dependent nonselective cation channels in the plasma membrane. The mechanism of regulation of PKD2L1 channels, however, has been poorly understood. In the present study, we found a bell-shaped alkaline pH dependence of PKD2L1 channel activity at the single-channel and whole-cell levels in patch-clamp recordings in HEK293T cells overexpressing mouse PKD2L1: alkalization to pH 8.0-9.0 increased the PKD2L1 currents, but alkalization to pH 10.0 decreased them. Single-channel analysis revealed that alkalization changed the open probability of PKD2L1 channels, but not their single-channel conductance. In addition, the voltage dependence of PKD2L1 channels was negatively and positively shifted by treatment with solutions of pH 8.0-9.0 and pH 10.0, respectively. These results indicate that the voltage-dependent gating of PKD2L1 channels was modulated by alkalization through two different mechanisms. Interestingly, we observed rebound activation of the PKD2L1 channel on washout of the alkaline solution after PKD2L1 channel inhibition at pH 10.0, suggesting that alkalization to pH 10.0 decreased PKD2L1 currents by inactivating the channels. Consistently, the PKD2L1 tail currents were accelerated by alkalization. These results suggest that alkalization is a bimodal modulator of mouse PKD2L1 channels. [ABSTRACT FROM AUTHOR]

Calcium-dependent potassium channels are implicated in electrolyte transport, cell volume regulation and mechanical responses in epithelia, although the pathways for calcium entry and their coupling to the activation of potassium channels are not fully understood. We now show molecular evidence for the presence of TRPV4, a calcium permeable channel sensitive to osmotic and mechanical stress, and its functional coupling to the large conductance calcium-dependent potassium channel (BKCa) in a human bronchial epithelial cell line (HBE). Reverse transcriptase polymerase chain reaction, intracellular calcium imaging and whole-cell patch–clamp experiments using HBE cells demonstrated the presence of TRPV4 messenger and Ca2+ entry, and outwardly rectifying cationic currents elicited by the TRPV4 specific activator 4α-phorbol 12,13-didecanoate (4αPDD). Cell-attached and whole-cell patch–clamp of HBE cells exposed to 4αPDD, and hypotonic and high-viscosity solutions (related to mechanical stress) revealed the activation of BKCa channels subsequent to extracellular Ca2+ influx via TRPV4, an effect lost upon antisense-mediated knock-down of TRPV4. Further analysis of BKCa modulation after TRPV4 activation showed that the Ca2+ signal can be generated away from the BKCa location at the plasma membrane, and it is not mediated by intracellular Ca2+ release via ryanodine receptors. Finally, we have shown that, unlike the reported disengagement of TRPV4 and BKCa in response to hypotonic solutions, cystic fibrosis bronchial epithelial cells (CFBE) preserve the functional coupling of TRPV4 and BKCa in response to high-viscous solutions. [ABSTRACT FROM AUTHOR]

Voltage-dependent, outwardly rectifying anion channels have been described in various cells including lymphocytes. In this study, we found that murine B cells express the voltage-dependent slowly activating anion channels (VSACs). Using a whole-cell configuration, I VSAC in Bal-17 was induced by a sustained depolarization (>0 mV) which was remarkably facilitated at 35°C ( Q 10=23 at 30 mV of clamp voltage). Substitution of extracellular Cl− with gluconate shifted the reversal potential to the right (35.7 mV). Gd3+ (IC50=0.11 μM) significantly attenuated I VSAC, but DIDS partially blocked I VSAC. In addition, extracellular acidification suppressed I VSAC whereas alkalinization facilitated the channel activation. I VSAC was decreased by 90% at pH 6.35 and increased by 180% at pH 8.0. In cell-attached and inside-out patch clamps, depolarization slowly activated the anion channels of large conductance (~270 pS) with multiple levels of subconductances. The single channel currents were also blocked by Gd3+ and acidic pH. Furthermore, I VSAC was also observed in WEHI-231 (an immature B cell line) and freshly isolated splenic B cells of mice. In summary, murine B cells express unique voltage-dependent anion channels that show a strong sensitivity to both temperature and extracellular pH. Further investigation is required to understand the physiological roles of VSAC and its molecular identity. [ABSTRACT FROM AUTHOR]

The Human Genome Organisation (HUGO) Nomenclature Committee Database provides a list of transporter families of the solute carrier (SLC) gene series (see http://www.gene.ucl.ac.uk/nomenclature/). Currently, it includes 43 families and 298 transporter genes. This special issue features mini-reviews on each of these SLC families written by the experts in each field. A WEB site has been established (http://www.pharmaconference.org/slctable.asp) that gives the latest updates for the SLC families and their members as well as relevant links to gene databases and reviews in the literature. A list of all currently known SLC families, a discussion of additional SLC families and family members as well as a brief summary of non-SLC transporter genes is included in this introduction. [ABSTRACT FROM AUTHOR]

Regulation of Na+-K+ pump current (Ip) and cystic fibrosis transmembrane conductance regulator (CFTR) Cl– current (ICFTR) by protein kinases A and C (PKA and PKC) was compared under identical experimental conditions by simultaneous measurement of the two currents in guinea-pig ventricular myocytes whole-cell voltage-clamped at 30–32 °C. Membrane current (I) was monitored at a holding potential (V) of –20 mV. I/V relationships were obtained by hyperpolarizing voltage ramps. Phorbol 12,13-dibutyrate (PDBu, 0.1–1 µM) and chelerythrine (10 µM) were used to stimulate and inhibit, respectively, PKC activity. PKA was stimulated by forskolin (4 µM) and inhibited by H-89 (50 µM). At –20 mV, stimulation of PKC by PDBu increased Ip to 121–123% of control. Addition of chelerythrine completely reversed this effect. The PDBu-induced augmentation of Ip was voltage dependent. The ratio Ip(PDBu)/Ip(control) increased from 1.10 at –100 mV to ~1.35 at positive membrane potentials. Stimulation of PKA by forskolin also increased Ip voltage dependently (128% of control at –20 mV). The effects of PKC and PKA stimulation on Ip were additive. The maximum Ip observed in the presence of PDBu and forskolin was 141% of control. Application of either H-89 or chelerythrine reversibly decreased Ip by 40% and 24%, respectively, suggesting that basal PKA and PKC activities were involved in the regulation of Ip. In the presence of H-89, PDBu was unable to increase Ip. Likewise, pre-application of chelerythrine abolished the forskolin-induced augmentation of Ip. In contrast to Ip, ICFTR (measured simultaneously) was absent under basal conditions. Stimulation of PKA by forskolin activated a pronounced ICFTR. Stimulation of PKC by PDBu, on the other hand, neither activated the Cl– current significantly nor increased ICFTR pre-activated by forskolin. Inhibition of PKC by chelerythrine, however, attenuated the PKA-mediated activation of ICFTR. The results reveal a complex interplay between PKA and PKC in regulating cardiac Ip and ICFTR with some similarities but also important differences. Ip is increased voltage dependently and additively by stimulation of both kinases. The steady-state activity of each of the kinases is involved in the modulation of basal Ip and obligatory for the augmentation of Ip induced by stimulation of the other kinase. In contrast, there appears to be no basal ICFTR. ICFTR is activated significantly only after stimulation of PKA. PKC activity, however, appears to facilitate this activation. [ABSTRACT FROM AUTHOR]

ICln is an ion channel cloned from renal epithelial cells. The reconstitution of the protein in 1,2-diphytanoyl-sn-glycero-3-phosphocholine (Diph-PC) bilayer membranes reveals potassium-selective channels, which become more chloride selective in the presence of calcium. Here we show that the ion selectivity of ICln also depends on the lipid environment in which the channels are reconstituted. Diph-PC is a synthetic lipid commonly used for reconstituting ion channels. However, since this lipid is not found in native membranes, we reconstituted the ICln ion channels in a polar heart-lipid extract. Using this lipid mixture the reconstituted ICln ion channels are chloride selective in the presence of calcium and an acidic pH. The relative ion selectivity of ICln under these conditions is similar to the cation versus anion selectivity of native ion channels activated by cell swelling. [ABSTRACT FROM AUTHOR]

There is indirect evidence that the plasmalemma-integrated eukaryotic porin (the voltage-dependent anion-selective channel, VDAC) functions as the outwardly rectifying chloride channel (ORCC). The channel, which is believed to play a role in cell volume regulation, appears to be relevant for cystic fibrosis (CF) therapy, in that it may function as an alternative Cl– channel. In the present study we showed first that Gd3+ altered the voltage dependence of human type-1 porin incorporated into artificial planar lipid bilayers. Next, using a light-scattering approach on transformed normal or CF human B-lymphocytes in hypotonic Ringer solution, we found slightly differing regulatory volume decrease (RVD) curves for the cell lines under study. Addition of 15–60 µM GdCl3 in hypotonic Ringer increased light scattering, pointing to cell swelling beyond normal values. RVD was not observed in those experiments. A corresponding effect was seen in isotonic Ringer containing GdCl3. In either osmotic situation Gd3+-induced cell swelling was abolished by monoclonal mouse anti-human type-1 porin antibodies. Agonist and antibody effects were dose dependent. Finally, videocamera-monitored control experiments with adherent HeLa cells verified the direct effect of the agonist on cell swelling in hypo- or isotonic situations and its prevention by the antibodies. We conclude that GdCl3 opens plasmalemma-integrated porin channels, allowing ions to following their gradients, resulting in cell swelling. Since respiratory epithelium expresses porin channels in the apical membrane, the use of gadolinium to activate ORCC may represent a new therapeutic approach in CF. [ABSTRACT FROM AUTHOR]

An alternatively spliced isoform of the P2X1 receptor (P2X1a) was cloned from rat mesenteric artery. The spliced isoform does not have the 27 amino acids that are in the middle of the putative extracellular loop domain of the P2X1 original subunit (P2X1). Reverse transcriptase polymerase chain reaction (RT-PCR) revealed co-localization of P2X1a mRNA and P2X1 mRNA in vascular and other smooth muscle tissues and heart, but not in the spinal cord. In HEK293 cells transfected with P2X1 cDNA, ATP (1 µM) evoked an inward current which strongly desensitized, and an intense signal for GFP (green fluorescent protein) fused with P2X1 was detected at the membrane. Neither of these results was obtained in HEK293 cells expressing P2X1a alone. The fluorescent GFP signal was detected at the membrane when GFP-fused P2X1a was co-expressed with P2X1, and no significant difference in the ATP-activated current was noted between cells expressing P2X1 and those co-expressing P2X1 and P2X1a. These results indicate that the 27-amino-acid sequence (175–201) is important for protein trafficking to the membrane and for the formation of a functional P2X1 receptor. Our results also show that P2X1a is transported to the membrane when P2X1a is co-expressed with P2X1, although the co-expression of P2X1a does not modify the channel's current properties. [ABSTRACT FROM AUTHOR]

Genistein, an inhibitor of protein tyrosine kinase (PTK), enhanced the activation of the cardiac isoform of the protein kinase A (PKA)-regulated cystic fibrosis transmembrane conductance regulator (CFTR) Cl– conductance in guinea-pig ventricular cells. We examined the mechanism(s) underlying this excitatory action of genistein by using patch-clamp techniques. The CFTR Cl– conductance, activated by isoproterenol (ISO, 10 nM; [Cl–] 153 mM extracellular, 21 mM intracellular; 36 °C), was enhanced by 20 µM genistein. Daidzein, a structural analogue of genistein with little inhibitory action on PTK, also enhanced CFTR Cl– currents. After maximal activation of the Cl– conductance by a cocktail of adenosine 3’,5’-cyclic monophosphate, 3-isobutyl-1-methylxanthine and okadaic acid or vanadate plus forskolin in the pipette, genistein was no longer stimulatory but was rather slightly inhibitory at 100 µM. Direct exposure of myocytes to higher concentrations of genistein (50–100 µM) elicited outwardly rectifying currents with a reversal potential of –47 mV in the absence of ISO. In the presence of 50 µM H-89, a PKA inhibitor, genistein had no effect. Vanadate in the pipette at a concentration (100 µM) inhibiting phosphotyrosine phosphatases alone did not prevent the action of genistein. In contrast, no conductance was activated by tyrphostins B42 or 51 or lavendustin A, other PTK inhibitors. Genistein’s stimulation of cardiac CFTR Cl– conductance appears to be independent of the PTK pathway and to be due to its direct interaction with CFTR Cl– channels. [ABSTRACT FROM AUTHOR]

The cytosolic concentration of chloride correlates directly with renin secretion from renal juxtaglomerular granular (JG) cells. In the present study, the mechanism by which chloride stimulates renin release was investigated in a preparation of permeabilized rat glomeruli with attached JG cells. An isosmotic increase in the concentration of chloride by 129 mM stimulated renin release 16- to 20-fold. Substitution of K+ by the impermeant cation N-methyl- d-glucamine (NMDG) abolished this response, while substitution with Na+ caused marginal inhibition. Substitution with Cs+ had no effect. Addition of sucrose, which permeates the secretory granules poorly, also abolished the stimulation of renin secretion by KCl. The response to KCl was not affected by K+-channel antagonists or by agonists of K+ channels. Chloride channel blockers were also without effect on the secretory response to KCl. When the ATP concentration was lowered from 1 to 0.1 mM renin release was stimulated, while an increase in the ATP concentration from 1 to 5 mM had no effect. Blockers of ATP-sensitive (KATP) channels did not modify the response to chloride. The present data suggest that chloride stimulates renin release after entry of KCl into the renin secretory granules which results in swelling and release of renin. [ABSTRACT FROM AUTHOR]

Connexin 46 (cx46), when expressed in Xenopus oocytes, not only forms typical gap junction channels between paired cells but also forms open gap junction hemichannels in the plasma membrane of single cells. The gap junction hemichannels share properties with complete gap junction channels in terms of permeability and gating. Here we characterize the gate that closes hemichannels in response to increased calcium concentration with whole-cell and single-channel records. The channels close within a narrow range of extracellular calcium concentrations (1–2 mM) which includes the calcium concentration prevailing in the primary site of cx46 expression, the lens. The effect of calcium on the channels is determined by voltage. A cysteine mutant of cx46, cx46L35C, was used to determine the localization of the gate. Experimental evidence suggests that position 35 is pore lining. The localization protocol tests the accessibility of position 35 for thiol reagents applied extra- or intracellularly to the channel closed by calcium. Channel closure by calcium excluded the thiol reagent from the outside but not from the inside. Consequently, the gate results in a regional closure of the pore and it is located extracellular to the position 35 of cx46. The present data also suggest that the cx46 gap junction hemichannel may exert a physiological function in the lens. Considering the association of calcium with cataract formation, it is feasible that misregulation of cx46 gap junction hemichannels could be a cause for cataract. [ABSTRACT FROM AUTHOR]

The patch-clamp technique was used to characterise the ion channels in cells located in the mid region of mouse jejunal crypts. Six different channels were seen. A large outwardly rectified K+ channel (BK) (conductance, g at 0 mV = 92 ± 6 pS), which was highly selective for K+ [ PK + (1) > PRb + (0.6) >> PCs +) in the on-cell (O/C) configuration and appeared in 66% of the patches. In inside-out (I/O) patches, this channel had a linear current/voltage ( I/ V) relationship ( g = 132 ± 3 pS), PNa + (0.07) > PLi channel (IK) which was present in 49% of O/C patches, had a linear I/ V ( g = 38 ± 3 pS), ran-down in O/C patches, and was not seen in I/O patches. A number of smaller channels (SC) with conductances ranging from 5 to 20 pS were seen in 16% of O/C patches. Also present in the basolateral membrane were a Cl+ channel (ICOR) and a nonselective cation channel (NSCC). These channels were only seen in I/O patches. ICOR had an outwardly rectified conductance ( g at 0 mV = 36 ± 2 pS), its Po) in the on-cell (O/C) configuration and appeared in 66% of the patches. In inside-out (I/O) patches, this channel had a linear current/voltage ( I/ V) relationship ( g = 132 ± 3 pS), Po was voltage dependent and it was blocked by cytoplasmic Ba2+ (5 mmol/l). An intermediate K+ channel (IK) which was present in 49% of O/C patches, had a linear I/ V ( g = 38 ± 3 pS), ran-down in O/C patches, and was not seen in I/O patches. A number of smaller channels (SC) with conductances ranging from 5 to 20 pS were seen in 16% of O/C patches. Also present in the basolateral membrane were a Cl– channel (ICOR) and a nonselective cation channel (NSCC). These channels were only seen in I/O patches. ICOR had an outwardly rectified conductance ( g at 0 mV = 36 ± 2 pS), its Po was independent of voltage and unaffected by variations in cytoplasmic Ca2+ channel and thus is most likely located in the apical membrane. [ABSTRACT FROM AUTHOR]o increased with depolarisation and elevation of cytoplasmic [Ca2+] (≥ 10 μmol/l), but was reduced by cytoplasmic ATP. None of the basolateral channels described here were activated by cAMP-dependent secretagogues, although a Cl– conductance was activated. This cAMP-dependent Cl– conductance was distinct from the basolateral Cl– channel and thus is most likely located in the apical membrane. [ABSTRACT FROM AUTHOR]

Neuropeptide Y (NPY) is probably the most abundant neuropeptide, with a plethora of central as well as peripheral effects, including its proabsorptive action in the gastro-intestinal tract. The effects of NPY on electrical parameters related to three different pathways stimulating ion secretion were investigated using the human intestinal cell line HT29cl.19A. Transepithelial potential and resistance were measured with the preparation maintained in a horizontal Ussing chamber, allowing simultaneous measurement of the membrane potential and determination of the fractional resistance of the apical cell membrane. It was found that application of NPY, after the adenylyl-cyclase-activating drug forskolin, resulted in complete inhibition of forskolin-induced effects within approximately 20 min. The secretion stimulated by adenosine appeared to be insensitive to NPY. The acetylcholine analogue carbachol stimulates ion secretion by increasing intracellular free calcium concentrations ([Ca2+]i) which activates the basolateral potassium (K+) conductance. NPY caused 50% inhibition of the effect of carbachol. Measurements of [Ca2+]i showed that NPY inhibited the carbachol-induced rise in [Ca2+]i, which correlates with the reduced activation of basolateral K+ channels. From this study we conclude that NPY inhibits cAMP-stimulated as well as Ca2+-stimulated secretion via a reduction in the apical Cl– and basolateral K+ conductance. This double effect makes NPY an effective proabsorptive peptide. [ABSTRACT FROM AUTHOR]

The aim of this study was to examine the question of whether activation of wt-CFTR (wild-type cystic fibrosis transmembrane conductance regulator) by cAMP and the opening of a Cl–conductance is paralleled by exocytosis and corresponding increases in membrane capacitance. To this end three types of Chinese hamster ovary (CHO) cells were examined: a control group of CHO cells; a group of CHO cells stably expressing wt-CFTR at high levels (also called BQ2-CHO); and a group of CHO cells stably expressing the frequent mutation ΔF508-CFTR. Whole-cell patch-clamp studies were performed to measure the membrane voltage ( Vm), the membrane conductance ( Gm) and the membrane capacitance ( Cm). Cm was assessed by a two-frequency lock-in amplifier method. Forskolin (Fsk, 0.1 μmol/l) and isobutylmethylxanthine (IBMX, 0.1 mmol/l) were used to increase cytosolic cAMP. It is shown that Fsk and IBMX had no effect on Vm and Gm in control CHO and ΔF508-CFTR-CHO cells. Fsk and IBMX depolarized wt-CFTR-expressing CHO cells significantly (from –40 ± 1.5 to –32 ± 1.6 mV, n = 41) and enhanced Gm strongly from 5.0 ± 0.9 to 36 ± 3.9 nS ( n = 65). The conductance increase was mostly for Cl–, because under stimulated conditions a reduction in bath Cl–concentration depolarized these cells further and significantly from –26 ± 1.8 to –10 ± 1.2 mV ( n = 16). This conductance had the characteristic wt-CFTR selectivity of Br– > Cl– > I–( n = 16). Despite this large increase in the Fsk- and IBMX-induced conductance Cm was not altered significantly (15.5 versus 15.7 pF, n = 50). These data indicate that stable overexpression of wt-CFTR but not of ΔF508-CFTR in CHO cells induces a cAMP-activated Cl–conductance. The activation of this large conductance obviously proceeds with little if any exocytosis. [ABSTRACT FROM AUTHOR]

Pancreatic acini secrete Na+, Cl–and H2O in response to secretagogues such as acetylcholine. Cl–channels in the luminal membrane are a prerequisite for this secretion. The properties of the corresponding conductance have previously been examined using whole-cell recordings. The present study attempts to examine the properties of the single channels in cell-attached and cell-free excised patches from the luminal membrane. To this end the pipettes were filled with an N-methyl- D-glucamine (NMDG+) chloride/gluconate solution. The voltage-clamp range was chosen to be pipette positive (cell negative, –60 to –130 mV) in order to increase the driving force for outward Cl–currents. Under resting conditions cell attached luminal patches had very few single-channel currents (12 out of 45 experiments). Their incidence was sharply increased by carbachol (CCH, 1 μmol/l) in 41 out of 45 experiments. The single-channel conductance of these channels was 1.97 ± 0.05 pS. The properties of these channels in excised patches were examined further: their single-channel conductance was 2.2 ± 0.07 pS ( n = 59) and their conductance selectivity was I– > Br– > Cl– >> gluconate. None of the typical Cl–channel blockers (DIDS, NPPB, glibenclamide 100 μmol/l) blocked these channels. It is concluded that the luminal membrane of the rat pancreatic acinus possesses Cl–channels with very low conductance which are activated by carbachol. [ABSTRACT FROM AUTHOR]

Ca2+-dependent conductances have been hypothesized to play a role in the bursting pattern of electrical activity of insulin-secreting β cells in response to high plasma glucose. A Maxi K+ channel has received the most attention, while a low-conductance Ca2+-activated K+ current has also been identified. We used an increasingly popular β cell model system, the βTC-3 cell line, and the perforated-patch technique to describe the properties of a novel Ca2+-dependent Cl–current [ ICl(Ca)] in insulin-secreting pancreatic β cells. The reported ICl(Ca) could be activated under physiological Ca2+ concentrations and is the first of its kind to be described in pancreatic insulin-secreting cells. We found that long depolarizing steps above –20 mV elicited an outward current which showed slow inward relaxation upon repolarization to negative membrane potentials. Both the outward currents and the inward tails showed dependence on Ca2+ influx: their current/voltage ( I/ V) relations followed that of the ”L-like” Ca2+ current ( ICa) present in these cells; they were blocked completely by the removal of external Ca2+ or application of Cd2+ at concentrations sufficient for complete block of ICa; and their magnitude increased with the depolarizing step duration. Moreover, the inward tail decayed monoexponentially with a time constant which at voltages negative to activation of ICa showed a weak linear voltage dependence, while at voltages positive to activation of ICa it followed the voltage dependence of ICa. This Ca2+-dependent current reversed at –21.5 mV and when the external Cl–concentration was reduced from 159 mM to 62 mM the reversal potential shifted by ≈+20 mV as predicted by the Nernst relation for a Cl–-selective current. Cl–channel blockers such as DIDS (100 μM) and niflumic acid (100 μM) blocked this current. We concluded that this current was a Ca2+-dependent Cl–current [ ICl(Ca)]. From substitution of the external Cl–with various monovalent anions and from the reversal potentials we obtained the following permeability sequence for ICl(Ca): I–>NO3 –>Br–>Cl–>Acetate–. [ABSTRACT FROM AUTHOR]

During the development of mammalian muscle the γ-subunit of the nicotinic acetylcholine receptor (AChR) is replaced by the ɛ-subunit to produce well-defined alterations in the conductance and gating of the channel. To gain a better unterstanding of the functional role of the γ and ɛ-subunits, we have studied the properties of an AChR channel lacking these subunits. The AChR expressed in Xenopus oocytes injected with the bovine α-, β and δ-subunit-specific mRNAs (referred to as αβδ-AChR) is unusual in that its channel opens spontaneously at a high frequency in the absence of agonist. From a comparison of the αβδ-AChR with complete receptors containing either the γ or ɛ-subunit, we conclude that the γ and ɛ-subunits influence most channel properties, including agonist binding, and are especially important for stabilizing the closed state of the unliganded receptor channel. The αβδ-AChR can form when a complete set of four subunit-specific mRNAs is injected. The ease with which it is assembled raises the possibility that the αβδ- AChR contributes to some of the variations in receptor properties that occur during development. [ABSTRACT FROM AUTHOR]

The ability to form gigaohm seals is essential for patch-clamp studies. Cells with otherwise useful properties must be abandoned for electrophysiological studies if seal formation is not possible. We have found that by inhibiting the growth of extracellular matrix with ß-D-xyloside, the success of forming gigaohm seals increased from near 0% to near 100%. Treated cells remained viable and appeared morphologically similar to untreated cells. Prototype treatment protocols are given for the renin secreting cell line As4.1. [ABSTRACT FROM AUTHOR]