Rémy Sauvé

External ATP triggers a biphasic activation process of a calcium-dependent K+ channel in cultured bovine aortic endothelial cells.

We have used the patch-clamp method in order to investigate the single-channel events underlying the effect of external ATP on the potassium permeability of bovine aortic endothelial cells (BAE). The results obtained from cell-attached and inside-out experiments led first to conclude that BAE cells possess an inward rectifying potassium channel activated by internal calcium at micromolar concentrations. The channel conductance for inward currents was estimated at 40 pS in symmetrical 200 mM KCl and the open-channel probability was found to be voltage insensitive within the membrane voltage range −50 to −100 mV. Based on results obtained in the cell-attached configuration, it could next be established that external ATP and ADP at micromolar concentrations could trigger, via the stimulation of P2 purinergic receptors, a time variable activation process of the observed calcium-dependent potassium channel. This activation process was found to occur in a biphasic manner with an initial phase independent of the presence of calcium in the cell bathing medium. The second phase which could be blocked by calcium channel blockers such as Co2+ or La3+ required, however, the presence of external calcium and could be abolished by depolarizing the cells using high K+ external solutions. Another important aspect related to this phenomenon was the observation that removing ATP from the external medium during the second phase led to a complete abolition of the associated calcium-dependent potassium channel activation process. It is suggested from these results that the action of ATP on the potassium permeability of BAE cells is related to a second messenger mediated release of calcium from internal calcium stores coupled to an ATP-dependent calcium influx abolished at depolarizing voltages.

Effect of anisotonic media on volume, ion and amino-acid content and membrane potential of kidney cells (MDCK) in culture

SummaryEffects of anisotonic media on a monolayer of confluent kidney cells in culture (MDCK) were studied by measuring: cell thickness and cross-section changes, ion and amino-acid content and membrane potential. The volume was also determined with cells in suspension. When cells in a monolayer were incubated in hypotonic media, the lateral and the apical membranes were rapidly stretched. Afterwards the lateral membranes returned to their initial state while the apical membranes remained stretched. This partial regulatory volume decrease (RVD) was verified with cells in suspension. RVD was accompanied by a loss of K+, Cl− and amino acids, but there was no loss of inorganic phosphate. Also a transient hyperpolarization of the membrane potential was observed, suggesting an increase of the K+ conductance during RVD. Upon restoring the isotonic medium, a regulatory volume increase (RVI) was observed accompanied by a rapid Na+ and Cl− increase and followed by a slow recovery of the initial K+ and Na+ content while amino acids remained at their reduced content. A transient depolarization of the membrane potential was measured during this RVI, suggesting that Na+ and Cl− conductance could have increased. In hypertonic media, only a small and slow RVI was observed accompanied by an increase in K+ and Cl− content but without any change of membrane potential. Quinine partly inhibited RVD in hypotonic media with cells in a monolayer while inhibiting RVD completely with cells in suspension. Incubation during four hours in a Ca2+ free medium had no effect on RVD. Furosemide and amiloride had no effect on RVD and RVI. Volume regulation, RVD or RVI, was not affected by replacing Cl− by nitrate. When cells in a monolayer were incubated in a hypotonic K2SO4 medium, no RVD was observed. From these results, it seems that MDCK cells in a confluent monolayer regulate their volume by activating specific ion and amino-acid transport pathways. Selective K+ and Na+ conductances are activated during RVD and RVI, while the activated anion conductance has a low selectivity. The controlling mechanism might not be the free intracellular Ca2+ concentration.

A general solution to the time interval omission problem applied to single channel analysis

To obtain the open or closed time interval distributions of patch clamp signals, several workers have used a half-amplitude minimum time interval criterion. Within this framework, no transition between states of different conductance levels is considered to have taken place if it leads to a time interval smaller than a certain critical value. This procedure modifies substantially the open or closed time interval distribution of the random signal to be analyzed, since time intervals well above the time resolution of the recording system may be interrupted by short gaps that may or may not satisfy the minimum time interval criterion. We present here a general theoretical framework by means of which the effect of time interval omission on time interval distributions can be taken into account. Based on the mathematical formalism provided by the Kolmogorov forward equation, special matrix operators are first defined. The general solution to the time omission problem in its integral form is then derived. In view of the poor computational feasibility of the resulting solution, a first-order approximation is also presented. This approximation consists essentially in neglecting the contribution of the undetected gaps to the total length of the resulting time interval. The exact and approximate solutions are then applied to two special kinetic schemes commonly found in single-channel studies, namely the O-C and C-O-C models. The applicability of the proposed formalism to the time interval distribution problem of a damped random signal is finally discussed.

EFFECTS OF THIOL-MODIFYING AGENTS ON A K(CA2+) CHANNEL OF INTERMEDIATE CONDUCTANCE IN BOVINE AORTIC ENDOTHELIAL CELLS

Abstract. Ca2+-activated K+ channels (K(Ca2+)) constitute key regulators of the endothelial cell electrophysiological response to InsP3-mobilizing agonists. Inside-out and outside-out patch clamp experiments were thus undertaken to determine if the gating properties of a voltage-insensitive K(Ca2+) channel of intermediate conductance present in bovine aortic endothelial (BAE) cells could be modified by specific sulfhydryl (SH) oxidative and/or reducing reagents. The results obtained first indicate that cytosolic application of hydrophilic oxidative reagents such as 5,5′-dithio-bis(2-nitrobenzoic acid) (DTNB) (0.2 to 5 mm) or [(O-carboxyphenyl)thio]ethyl mercury sodium salt (thimerosal) (0.5 to 5 mm) reduces gradually the K(Ca2+) channel activity with no modification of the channel unitary conductance. The inhibitory action of DTNB (1 to 5 mm) or thimerosal (1 to 5 mm) was not reserved following withdrawal of the oxidative agents, but channel activity could partly be restored by the addition of the SH group reducing agents dithiothreitol (DTT) (5 mm) or reduced glutathione (GSH) (5 mm) in 53% and 50% of the inside-out experiments performed with DTNB and thimerosal respectively. Similar results were obtained using H2O2 at concentrations ranging from 500 μm to 10 mm as oxidative reagent. In contrast, the lipid soluble oxidative agent 4,4′-dithiodipyridine (4-PDS) (1 mm) appeared in inside-out experiments less potent than DTNB and thimerosal at inhibiting the K(Ca2+) channel activity, suggesting that the critical SH groups involved in channel gating are localized at the inner face of the cell membrane. This conclusion was further substantiated by a series of outside-out patch clamp experiments which showed that DTNB (5 mm) and thimerosal (5 mm) were unable to inhibit the K(Ca2+) channel activity when applied to the external surface of the excised membrane. Finally, no significant changes of the gating properties of the K(Ca2+) channel were observed in inside-out experiments where the SH group reducing agents DTT and GSH were applied immediately following membrane excision. However, the application of either GSH or DTT was found to partly restore channel activity in experiments where the K(Ca2+) channels showed significant rundown.

Single-channel analysis of the potassium permeability in HeLa cancer cells: evidence for a calcium-activated potassium channel of small unitary conductance.

SummaryCell-attached and inside-out patch-clamp experiments (O.P. Hamill et al.,Pfluegers Arch.391: 85–100, 1981) were undertaken in order to characterize the molecular mechanisms responsible for the calcium-dependent potassium permeability observed in HeLa cancer cells. Our result essentially indicate that the HeLa cell external membrane contains potassium channels whose activity can be triggered within an internal calcium concentration range of 0.1 to 1 μm. This particular channel was found to behave as an inward rectifier in symmetrical 200mm KCl with a conductance of 50 and 10 pS at large negative and large positive membrane potentials, respectively.I/V curves were also measured in 10, 20, 75, 200 and 300mm KCl and the data interpreted in terms of a one-site-two-barrier model. The channel activity appeared to be nearly voltage independent within the voltage range −100 to +100mV, an increase ofPo, the open channel probability, being observed at large negative potentials only. In addition, the results obtained from inside-out experiments on the relationship betweenPo and the cytoplasmic freecalcium concentration have led to conclude that four calcium ions are probably required in order to open the channel. In this regard it was found that an increase of the internal free-calcium level affects more the number of channel openings per second than the actual channel mean lifetime. Finally, it is concluded following a time interval distribution analysis, that this particular channel has at least three closed states and two open states.

Single-Channel Characterization of the Pharmacological Properties of the K(Ca2+) Channel of Intermediate Conductance in Bovine Aortic Endothelial Cells

Abstract. The pharmacological profile of a voltage-independent Ca2+-activated potassium channel of intermediate conductance (IK(Ca2+)) present in bovine aortic endothelial cells (BAEC) was investigated in a series of inside-out and outside-out patch-clamp experiments. Channel inhibition was observed in response to external application of ChTX with a half inhibition concentration of 3.3 ± 0.3 nm (n= 4). This channel was insensitive to IbTX, but channel block was detected following external application of MgTX and StK leading to the rank order toxin potency ChTX > StK > MgTX >>IbTX. A reduction of the channel unitary current amplitude was also measured in the presence of external TEA, with half reduction occurring at 23 ± 3 mm TEA (n= 3). The effect of TEA was voltage insensitive, an indication that TEA may bind to a site located on external side of the pore region of this channel. Similarly, the addition of d-TC to the external medium caused a reduction of the channel unitary current amplitude with half reduction at 4.4 ± 0.3 mm (n= 4). In contrast, application of d-TC to the bathing medium in inside-out experiments led to the appearance of long silent periods, typical of a slow blocking process. Finally, the IK(Ca2+) in BAEC was found to be inhibited by NS1619, an activator of the Ca2+-activated potassium channel of large conductance (Maxi K(Ca2+)), with a half inhibition value of 11 ± 0.8 μm (n= 4). These results provide evidence for a pharmacological profile distinct from that reported for the Maxi K(Ca2+) channel, with some features attributed to the voltage-gated KV1.2 potassium channel.

The Role of the GX9GX3G Motif in the Gating of High Voltage-activated Ca2+ Channels

The putative hinge point revealed by the crystal structure of the MthK potassium channel is a glycine residue that is conserved in many ion channels. In high voltage-activated (HVA) CaV channels, the mid-S6 glycine residue is only present in IS6 and IIS6, corresponding to G422 and G770 in CaV1.2. Two additional glycine residues are found in the distal portion of IS6 (Gly432 and Gly436 in CaV1.2) to form a triglycine motif unique to HVA CaV channels. Lethal arrhythmias are associated with mutations of glycine residues in the human L-type Ca2+ channel. Hence, we undertook a mutational analysis to investigate the role of S6 glycine residues in channel gating. In CaV1.2, α-helix-breaking proline mutants (G422P and G432P) as well as the double G422A/G432A channel did not produce functional channels. The macroscopic inactivation kinetics were significantly decreased with CaV1.2 wild type > G770A > G422A ≅ G436A >> G432A (from the fastest to the slowest). Mutations at position Gly432 produced mostly nonfunctional mutants. Macroscopic inactivation kinetics were markedly reduced by mutations of Gly436 to Ala, Pro, Tyr, Glu, Arg, His, Lys, or Asp residues with stronger effects obtained with charged and polar residues. Mutations within the distal GX3G residues blunted Ca2+-dependent inactivation kinetics and prevented the increased voltage-dependent inactivation kinetics brought by positively charged residues in the I-II linker. In CaV2.3, mutation of the distal glycine Gly352 impacted significantly on the inactivation gating. Altogether, these data highlight the role of the GX3G motif in the voltage-dependent activation and inactivation gating of HVA CaV channels with the distal glycine residue being mostly involved in the inactivation gating.

Analysis of a novel double-barreled anion channel from rat liver rough endoplasmic reticulum

The presence of anionic channels in stripped rough endoplasmic reticulum membranes isolated from rat hepatocytes was investigated by fusing microsomes from these membranes to a planar lipid bilayer. Several types of anion-selective channels were observed including a voltage-gated Cl- channel, the activity of which appeared in bursts characterized by transitions among three distinct conductance levels of 0 pS (0 level), 160 pS (O1 level), and 320 pS (O2 level), respectively, in 450 mM (cis) 50 mM (trans) KCl conditions. A chi 2 analysis on current records where interburst silent periods were omitted showed that the relative probability of current levels 0 (baseline), O1, and O2 followed a binomial statistic. However, measurements of the conditional probabilities W(level 0 at tau/level O2 at 0) and W(level O2 at tau/level 0 at 0) provided clear evidence of direct transitions between the current levels 0 and O2 without any detectable transitions to the intermediate level O1. It was concluded on the basis of these results that the observed channel was controlled by at least two distinct gating processes, namely 1) a voltage-dependent activation mechanism in which the entire system behaves as two independent monomeric channels of 160 pS with each channel characterized by a simple Open-Closed kinetic, and 2) a slow voltage-dependent process that accounts for both the appearance of silent periods between bursts of channel activity and the transitions between the current levels 0 and O2. Finally, an analysis of the relative probability for the system to be in levels 0, O1, and O2 showed that our results are more compatible with a model in which all the states resulting from the superposition of the two independent monomeric channels have access at different rates to a common inactivated state than with a model where a simple Open-Closed main gate either occludes or exposes simultaneously two independent 160-pS monomers.

Oscillatory activation of calcium-dependent potassium channels in HeLa cells induced by histamine H1 receptor stimulation: A single-channel study

SummaryWe have used the patch-clamp method (O.P. Hamill et al.,Pfluegers Arch.,391:85–100, 1981) in order to investigate the activation pattern of a calcium-dependent potassium channel following H1 receptor stimulation in HeLa cells. Our results essentially indicate that the stimulation of H1 receptors by exogenous histamine at concentrations greater than 1 μm induces an oscillatory activation pattern of calcium-dependent potassium channels characterized by the occurrence of channel current bursts separated by long silent periods. It was also found that the occurrence of these bursts could be directly correlated with transmembrane potential oscillations, the latter being the resulting effect of the calcium-dependent potassium channel synchronous openings. In addition, the cyclic activation of the calciumdependent potassium channels could be initiated by the addition of histamine to a calcium-free external medium, indicating that the stimulation of the H1 receptors in HeLa cells is mainly related to the release of calcium from internal stores. Finally, the membrane-permeable cyclic AMP analog dibutyryl cyclic AMP was found to be ineffective in initiating single-channel events such as those triggered by exogenous histamine. It is proposed that the oscillatory activation of the calcium-dependent potassium channels in HeLa cells results from a repetitive transient increase in cytosolic free calcium concentration consequent to the H1 receptor stimulation.

Single channel K+ currents from HeLa cells

SummaryThe extracellular patch-clamp technique was used in order to investigate the presence of ionic channels in HeLa cells, a well-known cultured cell type obtained from an epidermoid carcinoma of the cervix. Under Gigohm-seal conditions, discrete current jumps could be observed with patch electrodes containing KCl. These channels were found to be mainly permeable to K+ and showed multiple levels of conductance. From single-channelI–V curve measurements, a strong rectification effect, characterized by a large inward and no detectable outward current, was observed. For negative membrane potentials (0 to −90 mV), the measured current-voltage relationship was found to be mostly linear, corresponding to a single-channel conductance of 40 pS. An analysis of some selected time records has revealed in addition that the probability of the channel to be in the open state was a function of the KCl concentration in the patch pipette.