Cell-attached measurements of the firing threshold of rat hippocampal neurones.

1. The cell-attached configuration of the patch-clamp technique was used to assess resting membrane potential and firing threshold of CA1 pyramidal cells and interneurones of rat hippocampal slices. 2. Resting potential was inferred from the reversal potential of voltage-gated K+ currents with symmetrical intracellular and pipette K+ concentrations. Its mean value was -74 +/- 9 mV for silent interneurones (mean +/- s.d.; n = 17) and -84 +/- 7 mV for silent pyramidal cells (n = 8). Spontaneous action currents occurred in thirteen out of thirty-two interneurones and two out of ten pyramidal cells. In active cells, membrane potential values fluctuated by up to 20 mV, due in part to the large hyperpolarizations that followed an action current. 3. Membrane potential values determined from K+ current reversal were 13 +/- 6 mV more hyperpolarized than those measured in whole-cell recordings from the same neurones (n = 8), probably due to a Donnan equilibrium potential between pipette and cytoplasm. 4. Firing threshold of silent cells was determined by elevating external K+ until action currents were generated, while membrane potential was monitored from the cell-attached K+ current reversal. Spike threshold was attained at -49 +/- 8 mV for interneurones (n = 17) and at -60 +/- 8 mV for pyramidal cells (n = 8). Increasing external Ca2+ from 2 to 4 mM shifted the neuronal voltage threshold by +5 mV, without affecting resting potential. 5. For comparison with these values, we examined how the rate of membrane polarization influenced firing threshold in whole-cell records. Ramp current injections, of duration 15-1500 ms, revealed that current threshold followed a classical strength-duration relationship. In contrast voltage threshold, determined from current injection or by elevating extracellular K+, varied little with the rate of membrane polarization. 6. The state of activation and inactivation of Na+ and K+ currents might contribute to the stability of the voltage threshold. Cell-attached records showed that 79 +/- 10 % of Na+ channels and 64 +/- 10 % of K+ channels were available for activation at resting potential in silent cells (n = 8). As cells were depolarized to threshold, Na+ current availability was reduced to 23 +/- 10 %, and K+ current availability to 31 +/- 12 %. 7. The speed of transition into the inactivated states also appears to contribute to the invariance of threshold for all but the fastest depolarizations. At potentials close to threshold, the rate of inactivation of Na+ and K+ followed a double exponential time course, such that Na+ currents were 62 % inactivated and K+ currents were 63 % inactivated within 15 ms.

Noninvasive measurements of the membrane potential and GABAergic action in hippocampal interneurons.

Neurotransmitters affect the membrane potential (Vm) of target cells by modulating the activity of receptor-linked ion channels. The direction and amplitude of the resulting transmembrane current depend on the resting level of Vm and the gradient across the membrane of permeant ion species. Vm, in addition, governs the activation state of voltage-gated channels. Knowledge of the exact level of Vm is therefore crucial to evaluate the nature of the neurotransmitter effect. However, the traditional methods to measure Vm, with microelectrodes or the whole-cell current-clamp technique, have the drawback that the recording pipette is in contact with the cytoplasm, and dialysis with the pipette solution alters the ionic composition of the interior of the cell. Here we describe a novel technique to determine the Vm of an intact cell from the reversal potential of K+ currents through a cell-attached patch. Applying the method to interneurons in hippocampal brain slices yielded more negative values for Vm than subsequent whole-cell current-clamp measurements from the same cell, presumably reflecting the development of a Donnan potential between cytoplasm and pipette solution in the whole-cell mode. Cell-attached Vm measurements were used to study GABAergic actions in intact CA1 interneurons. In 1- to 3-week-old rats, bath-applied GABA inhibited these cells by stabilizing Vm at a level depending on contributions from both GABAA and GABAB components. In contrast, in 1- to 4-d-old animals, only GABAA receptors were activated resulting in a depolarizing GABA response.

Elevation of intracellular Ca2+ in the physiologically relevant range does not inhibit voltage-gated K+ channels in human T lymphocytes.

1. Human T lymphocytes express both voltage-gated (K(V)) and Ca2+-activated (K(Ca)) potassium channels. The K(Ca) channel is activated by elevations of intracellular Ca2+ ([Ca2+]i) at concentrations attained during physiological Ca2+ signalling. Whether or not the K(V) channel is affected by [Ca2+]i is a matter of controversy. Here, the interaction between the K+ channels of lymphocytes and [Ca2+]i was studied using cell-attached and whole-cell patch-clamp recordings, while [Ca2+]i of the same cell was monitored simultaneously by fura-2 imaging or from the activity of the K(Ca) channels. 2. The K+ channels in cell-attached patches were measured using a high K+ pipette solution. The K(V) conductance was quantified as the integral of the inward current during voltage ramp stimulation, yielding a measure independent of the cell membrane potential. Whereas the open probability of the K(Ca) channel showed an absolute dependence on [Ca2+]i, the K(V) channel was little affected by [Ca2+]i. The K(V) conductance is not reduced by elevations of [Ca2+]i in the range 0-8 muM. On the contrary, a modest but consistent increase in the K(V) current component in cell-attached currents was observed when [Ca2+]i was elevated. 3. The absence of inhibition of the K(V) current by [Ca2+]i was also apparent from whole-cell measurements with pipette solutions buffered to 1 microM free Ca2+: following break-in to whole-cell configuration, depolarizing voltage ramps were applied at regular intervals to activate the K(V) current while the K(Ca) current was measured from the slope of the ramp current below the activation of the voltage-gated current. During the gradual activation of the K(Ca) current, as the cell interior was perfused with the pipette solution, the K(V) current remained constant in amplitude. 4. In the initial period following break-in to whole-cell configuration, a gradual increase in the rate of K(V) current inactivation was generally observed. However, the time course of this change in kinetics was much slower than the perfusion of the cell interior, as judged from the activation of the K(Ca) conductance, ruling out a direct effect of Ca2+, within the physiological range, on K(V) channel kinetics.

A charybdotoxin-insensitive conductance in human T lymphocytes: T cell membrane potential is set by distinct K+ channels.

1. Changes in the membrane potential (Vm) of human T lymphocytes upon K+ channel block were inferred from alterations in K+ current reversal potential in cell-attached patches. It was found that a high concentration of charybdotoxin (100 nM, CTX), which blocks both voltage-gated (K(V)) and Ca(2+)-activated (K(Ca)) potassium channels in these cells, depolarizes Vm of lymphocytes only partially. Subsequent whole-cell measurements of the same cells showed that 39 +/- 25% of the voltage-gated current remains in the presence of CTX. 2. The CTX-resistant current reverses at potentials between -80 and -90 mV, indicating that it is K+ selective. The current is activated at more depolarized potentials compared with the unblocked IK(V) current with a threshold between -40 and -20 mV and a half-maximal activation at +50 mV. Inactivation during prolonged depolarization is slow. Steady-state inactivation is half-maximal at -45 mV and complete at potentials > -20 mV. The CTX-resistant IK(V) is completely blocked by nifedipine and is not sensitive to dendrotoxin. 3. The effect of nifedipine on the Vm of lymphocytes varies between cells depending on the contribution of the nifedipine-sensitive current to whole-cell IK(V). Combined application of CTX and nifedipine completely depolarizes Vm. 4. The extent to which T cell receptor-evoked Ca2+ signals of resting cells are inhibited by K+ channel blockers correlates with the magnitude of the depolarization induced by the drugs. Complete suppression of the response is achieved only by combined block of the CTX-sensitive and -insensitive IK(V). The enhanced Ca2+ response of activated cells, which express increased numbers of K(Ca) channels, is in addition subject to modulation by blockers which prevent the hyperpolarization during the Ca2+ rise mediated by these channels.

Enhancement of calcium signaling and proliferation responses in activated human T lymphocytes. Inhibitory effects of K+ channel block by charybdotoxin depend on the T cell activation state.

T cell receptor (TCR) stimulation, leading to T cell activation and ultimately to cell proliferation and differentiation, evokes elevations of [Ca2+]i with a high variability between individual T lymphocytes. We have used Ca(2+)-imaging of Fura-2 loaded cells to study the origin of the variation in Ca2+ signals and its consequences for the final cellular response. We found that, compared to resting cells, the percentage of responding cells and the average amplitude of the Ca2+ signal upon TCR re-stimulation by PHA increases in the first 5 days of T cell activation and declines thereafter, with more pronounced [Ca2+]i oscillations in later stages. In parallel, an enhancement of T cell proliferation is observed. Stronger stimulation of the TCR/CD3 complex by co-crosslinking CD3 with CD4/CD8 molecules evokes oscillating Ca2+ responses irrespective of the activation state, indicating that the basic capacity for Ca2+ signaling is essentially the same in resting and activated cells. Nevertheless, also the amplitude of the CD3+CD4/8 response shows a transient additional increase during the first days of T cell activation. Experiments with the K+ channel blocker charybdotoxin (CTX) indicate that [Ca2+]i oscillations depend critically on K+ channel functioning, but suppression of these oscillations by CTX does not significantly affect the average amplitude of the Ca2+ signal nor PHA-induced proliferation. However, when applied during the first 4-5 days of activation, CTX reduces in addition the average level of the TCR evoked Ca2+ response and inhibits subsequent proliferation.

Voltage-gated and Ca(2+)-activated K+ channels in intact human T lymphocytes. Noninvasive measurements of membrane currents, membrane potential, and intracellular calcium.

Voltage-gated n-type K(V) and Ca(2+)-activated K+ [K(Ca)] channels were studied in cell-attached patches of activated human T lymphocytes. The single-channel conductance of the K(V) channel near the resting membrane potential (Vm) was 10 pS with low K+ solution in the pipette, and 33 pS with high K+ solution in the pipette. With high K+ pipette solution, the channel showed inward rectification at positive potentials. K(V) channels in cell-attached patches of T lymphocytes inactivated more slowly than K(V) channels in the whole-cell configuration. In intact cells, steady state inactivation at the resting membrane potential was incomplete, and the threshold for activation was close to Vm. This indicates that the K(V) channel is active in the physiological Vm range. An accurate, quantitative measure for Vm was obtained from the reversal potential of the K(V) current evoked by ramp stimulation in cell-attached patches, with high K+ solution in the pipette. This method yielded an average resting Vm for activated human T lymphocytes of -59 mV. Fluctuations in Vm were detected from changes in the reversal potential. Ionomycin activates K(Ca) channels and hyperpolarizes Vm to the Nernst potential for K+. Elevating intracellular Ca2+ concentration ([Ca2+]i) by ionomycin opened a 33-50-pS channel, identified kinetically as the CTX-sensitive IK-type K(Ca) channel. The Ca2+ sensitivity of the K(Ca) channel in intact cells was determined by measuring [Ca2+]i and the activity of single K(Ca) channels simultaneously. The threshold for activation was between 100 and 200 nM; half-maximal activation occurred at 450 nM. At concentrations > 1 microM, channel activity decreased. Stimulation of the T-cell receptor/CD3 complex using the mitogenic lectin, PHA, increased [Ca2+]i, and increased channel activity and current amplitude resulting from membrane hyperpolarization.

Intracellular Ca2+ oscillations and membrane potential fluctuations in intact human T lymphocytes: role of K+ channels in Ca2+ signaling.

In intact human T lymphocytes, voltage-gated K+ [K(V)] channels and Ca(2+)-activated K+ [K(Ca)] channels have been recorded using the patch clamp technique in the cell-attached configuration. The reversal potential of the voltage-gated current with high K+ solution in the pipette gives a measure for the cell membrane potential (VM). The open probability of the K(Ca) channels gives a measure for intracellular Ca2+ concentration ([Ca2+]i). By simultaneous recording of both types of K+ channels, the interaction of VM and [Ca2+]i in T lymphocytes was investigated. It was demonstrated that VM fluctuates under resting conditions in a 20 mV range around an average value of -60 mV. In response to T cell receptor stimulation by PHA, rises in [Ca2+]i occur, which vary between cells from transient or sustained elevations to Ca2+ oscillations, in parallel with amplification of the hyperpolarizing deflections of VM. The correlation between VM and [Ca2+]i suggests that Ca2+ oscillations are modulated by positive feedback between Ca2+ influx, [Ca2+]i and VM mediated by K(Ca) channels and by intrinsic VM fluctuations caused by negative feedback between VM and the K(V) channel. Differences in the ratio between K(Ca) and K(V) channel numbers can account for the variability in Ca2+ responses between cells. The results predict periodic K(V) channel activity at rest and alternating K(V) and K(Ca) channel activity during Ca2+ signaling, which was consistent with subsequent observations.

Characterization of Ca(2+)-activated K+ channels in excised patches of human T lymphocytes.

Ca(2+)-activated K+ [K(Ca)] channels were studied in excised patches of resting and activated human peripheral blood T lymphocytes. The K(Ca) channel had a single-channel conductance of 50 +/- 6 pS in symmetrical high-K+ solutions in the potential range of -100 to -10 mV and was inwardly rectifying at more depolarized potentials. The channel was sensitive to block by charybdotoxin (10 nM) and insensitive to apamin (3 nM). Half-maximum activation occurred at an internal free Ca2+ concentration of 360 +/- 110 nM. The concentration-effect curve had a slope factor of 0.83 +/- 0.12, suggesting a 1:1 interaction of Ca2+ ions with the channel. Ca2+ affects the open time probability of the K(Ca) channels, mainly by modulating the frequency of channel opening. The open probability did not show voltage dependence. The kinetics of the channel could be described assuming one open state and two closed states. The time constant of the exponential describing the open time distribution amounted to 2.8 +/- 1.2 ms, whereas the closed time distribution could be described with two exponentials with time constants of 0.2 +/- 0.05 ms and 8.0 +/- 2.1 ms, respectively. Resting T lymphocytes expressed a low number of channels but the density of channels increased dramatically during chronic phytohaemagglutinin stimulation.

Veratridine blocks voltage-gated potassium current in human T lymphocytes and in mouse neuroblastoma cells.

(i) Effects of veratridine on ionic conductances of human peripheral blood T lymphocytes have been investigated using the whole-cell patch-clamp technique. (ii) Veratridine reduces the net outward current evoked by membrane depolarizations. The reduction originates from block of a 4-aminopyridine-sensitive, voltage-gated K+ current. (iii) Human T lymphocytes do not appear to express voltage-gated Na+ channels, since inward currents are observed neither in control nor in veratridine- and bretylium-exposed lymphocytes. (iv) The effect of veratridine consists of an increase in the rate of decay of the voltage-gated K+ current and a reduction of the peak current amplitude. Both effects depend on veratridine concentration. Half-maximum block occurs at 97 microM and the time constant of decay is reduced by 50% at 54 microM of veratridine. (v) Possible mechanisms of veratridine action are discussed. The increased rate of K+ current decay is most likely due to open channel block. The decrease of current amplitude may involve an additional mechanism. (vi) In cultured mouse neuroblastoma N1E-115 cells, veratridine blocks a component of voltage-gated K+ current, in addition to its effect on voltage-gated Na+ current. This result shows that the novel effect of veratridine is not confined to lymphocytes.