The spatio-temporal profile of intraneuronal free [Ca2+] is highly regulated because it controls vital cellular functions, such as excitability, plasticity and gene expression (Kennedy, 1989; Ghosh & Greenberg, 1995), under physiological conditions and chronic disease (Gibbons, Brorson, Bleakman, Chard & Miller, 1993). Ligand-gated ion channels, voltage-gated Ca2+ channels (VGCCs) and Ca2+ extruding pumps regulate the net Ca2+ flux across the cell membrane, while intracellular Ca2+ stores and buffers control the dynamics of intracellular free [Ca2+] (Miller, 1991).
A negative feedback mechanism between Ca2+ entry and intracellular [Ca2+] is provided by the Ca2+-dependent inactivation of Ca2+ currents (ICa) (Brehm & Eckert, 1978). Basal levels of inactivation have been shown to increase with elevated intracellular [Ca2+] (Branchaw, Banks & Jackson, 1997) and inactivation rates of ICa are sensitive to exogenous Ca2+ buffers added intracellularly (Chad, 1989). The precise mechanism whereby Ca2+ entry mediates the inactivation of ICa is unknown. For L-type channels (α1C) inactivation requires an intact C-terminus of the protein. Binding of Ca2+ to an EF-hand like region in this terminus (de Leon et al. 1995), or to another short sequence nearby (Zhou, Olcese, Qin, Noceti, Birnbaumer & Stefani, 1997) is thought to reduce the probability of channel opening. Therefore, a disease-related change in the endogenous Ca2+ buffering or in the affinity of the Ca2+ binding site responsible for inactivation would be expected to control the gain of the negative feedback.
An increase in this feedback on Ca2+ entry through high-voltage activated (HVA) ICa is associated with the loss of the Ca2+ binding protein calbindin D-28k (CB) in rat dentate gyrus granule cells (DGGCs) during kindling, an animal model of epilepsy (Köhr, Lambert & Mody, 1991). It was hypothesized that CB normally acts as a local Ca2+ buffer which competes for entering Ca2+ ions with the binding site responsible for inactivation. A recent histochemical study (Maglóczky, Halász, Vajda, Czirják & Freund, 1997) has demonstrated the selective loss of CB from granule cells in the human hippocampus in temporal lobe epilepsy (TLE) patients. Therefore, we wanted to find out whether a similar functional relationship might exist between intraneuronal Ca2+ buffering and ICa inactivation in human TLE granule cells following the loss of CB. Our experiments in human granule cells acutely dissociated from TLE patients reveal a strongly inactivating Ca2+ conductance with its inactivation sensitive to intraneuronal Ca2+ buffering.
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Voltage-clamped inward currents (Fig. 1A) were elicited by stepping the potential from a holding potential of -60 mV in the whole-cell mode using recording solutions designed to isolate the Ca2+ conductance. Cadmium (50 μm, n= 3 cells, data not shown) blocked the currents, identifying them as Ca2+ currents. The peak ICavs.V plot was bell shaped and peaked at +10 mV (-308 ± 22 pA), which is characteristic of well-clamped calcium currents. Since a single Boltzmann function yielded a good fit (χ2= 0.00083) to the steady-state activation curve (Fig. 1B), we conclude that the predominant conductance activated by voltage steps from -60 mV is of the high-threshold type (HVA).
Figure 1. Human TLE granule cells possess a HVA calcium conductance which inactivates in a Ca2+-dependent fashion
A, whole-cell voltage-clamped Ca2+ currents were elicited from a -60 mV holding potential by steps of 160 ms duration delivered every 8 s. The currents (○) were fitted with mono-exponential decay functions (continuous lines). See Methods for details. For the maximal ICa: τ= 44 ms, a= -265 pA, c= -160 pA. B, the normalized peak conductance g is plotted as a function of the membrane potential step and fitted with a single Boltzmann function (dashed line). The voltage at half-maximal activation (V1/2) is -6.5 ± 0.5 mV. C, the peak Ca2+ current amplitude and the decay time constant τ for the whole-cell recordings (n= 8) are plotted as a function of step voltages. Note the correlation between current amplitudes and decay time constants.
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To find out whether the currents inactivated in a Ca2+-dependent manner, ICa traces were fitted with a mono-exponential decay function (Fig. 1A), and the resulting decay time constants (τ) were plotted as a function of the step potential (Fig. 1C). The τ had a U-shaped dependency on membrane voltage, indicating that calcium entry was correlated with current inactivation, which is a characteristic of the Ca2+-dependent inactivation process of Ca2+ currents (Brehm & Eckert, 1978).
In order to avoid imposing an exogenous Ca2+ buffering capacity on the neurones, in control whole-cell recordings, we omitted any exogenous Ca2+ buffers from the pipette solutions (Köhr & Mody, 1991). To test whether the Ca2+-dependent ICa inactivation recorded in the buffer-free whole-cell mode corresponds to unperturbed endogenous Ca2+ buffering conditions, we also recorded calcium currents in the perforated patch mode (Fig. 2A), which provides electrical but not diffusional access to the cell. Based on the calculated steady-state voltage error, the shape of the rising phase of ICa, the steady-state activation curve (Fig. 2B), and the I-V plot (Fig. 2C) the quality of the voltage clamp appears to have been adequate for the experiments in spite of the high Rs in such recordings. As shown in Fig. 2C, the inactivation time constant τ and the peak ICa amplitude were not significantly different between the perforated and whole-cell recordings (perforated: for Vstep=+10 mV, Ipeak= -273 ± 57 pA, τ= 40 ± 1.4 ms, n= 4; whole-cell: for Vstep=+10 mV, Ipeak= -308 ± 22 pA, τ= 42.4 ± 4.5 ms, n= 8).
Figure 2. Inactivation of HVA ICa in perforated patch recordings that preserve the endogenous Ca2+ buffering
A, the perforated patch method was used to elicit Ca2+ currents which were analysed in the same way as the whole-cell currents. The fit to the maximal ICa yielded the following values: τ= 49 ms, a = -234 pA, c = -114 pA. B, the normalized peak conductance g is plotted as a function of the step potential and fitted with a single Boltzmann function (dashed line). The V½ is -4.3 ± 0.4 mV, not significantly different from the V½ obtained for the whole-cell recordings. C, the peak Ca2+ current amplitude and the decay time constant (τ) for perforated patch recordings (n= 4) are plotted as functions of step voltages. Note the similarity to the I-V and τvs.V relationships obtained for the whole-cell recordings (Fig. 1C).
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Having shown that the inactivation process is intact in buffer-free whole-cell recording, we further probed the Ca2+ dependence of ICa inactivation in this configuration by perfusing the cells with equimolar Ba2+ substituted for Ca2+ (n= 6) or added 5 mm BAPTA, a rapid Ca2+ chelator, to the pipette solution (n= 5). The normalized current traces in Fig. 3A show that both conditions effectively reduced the ICa inactivation. BAPTA increased the decay time constant associated with the maximal ICa to 66 ± 4 ms and Ba2+ increased it to 85 ± 17 ms. Both values were significantly larger than the control τ. Furthermore, under these recording conditions, τ no longer displayed the pronounced U-shaped dependency on membrane voltage (Fig. 3B). The addition of BAPTA to the internal solution also affected the ICa amplitudes, which were significantly larger (Ipeak= 429 ± 45 pA) than in control recordings. The peak I-V curve for Ba2+ currents (Fig. 3B) reflects the characteristic left shift in IBa activation and increased channel permeability for Ba2+ over Ca2+ (McDonald, Pelzer, Trautwein & Pelzer, 1994).
Figure 3. Inactivation of Ca2+ currents is Ca2+ dependent and sensitive to intracellular Ca2+ buffering
A, the whole-cell currents were recorded under different ionic and intracellular buffering conditions, and are shown here normalized. One trace (control current) was recorded with 5 mm Ca2+ in the extracellular solution and no Ca2+ buffer in the internal solution, the second was obtained with 5 mm Ca2+ outside and 5 mm BAPTA inside the pipette, and the third was recorded after equimolar replacement of Ca2+ with Ba2+ in the extracellular solution and no Ca2+ buffer inside. B,I-V plot for the Ba2+ (▪) and Ca2+ with BAPTA inside (○) currents and the voltage dependence of the associated decay time constants τ. Note that the τ values are elevated for the Ba2+ and BAPTA cases compared with the Ca2+ case and have lost the characteristic U-shape. C, the time course of inactivation of HVA Ca2+ currents is similar in granule cells obtained from human TLE and experimental animal model of TLE (kindling). Representative traces of normalized whole-cell Ca2+ currents recorded from epileptic human, control and kindled rat granule cells are superimposed. The recording conditions are the same as those described in Fig. 1A.
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Without access to human control granule cells which have been shown to contain CB (Maglóczky et al. 1997), we compared the ICa recorded in human TLE neurones to Ca2+ currents recorded in DGGCs acutely dissociated using the same protocols from hippocampal slices prepared from kindled and control rats. Representative HVA Ca2+ current traces recorded under identical whole-cell voltage-clamp conditions from epileptic human, control and kindled (epileptic) rat DGGCs are shown superimposed in Fig. 3C. The time course of ICa inactivation is comparable between kindled rat and epileptic human and between control rat and 5 mm BAPTA-loaded epileptic human dentate gyrus granule cells.
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We have described a significant Ca2+-dependent component contributing to the inactivation of HVA calcium currents in human granule cells obtained from TLE patients. Moreover, we have compared the time course of current inactivation in human epileptic, kindled and control rat DGGCs.
Two previous studies have examined calcium currents in acutely dissociated human neurones obtained from TLE patients (Sayer, Brown, Schwindt & Crill, 1993). Unlike Beck, Steffens, Heinemann & Elger (1997B), we did not detect a T-type ICa component. This lack may be due to minor differences in the dissociation procedure which may differentially preserve a T-type conductance possibly located on truncated dendrites. Alternatively, the holding potentials employed in our study may have inactivated a large proportion of the T channels. This latter possibility is, however, not supported by our recordings (n= 2) in which we failed to observe a T-type conductance in spite of a -90 mV holding potential. Given the scarcity of human dissociated neurones, we did not use specific pharmacological tools to separate the underlying Ca2+ conductances. While in guinea-pig DGGCs at least four different ICa components (T, L, N and P) were demonstrated (Eliot & Johnston, 1994), under our experimental conditions the predominant source of Ca2+ influx into dissociated human granule cells is a HVA ICa composed of up to three Ca2+ conductances. Our recordings also differ from those of Beck et al. (1997b) in the substantially smaller peak IBa densities (60 ± 6 pA pF−1versus the 144.3 ± 24.3 pA pF−1 in their report). This difference may stem from their use of 10 mm intracellular EGTA, thereby possibly lowering resting [Ca2+] and consequently basal Ca2+-dependent inactivation (see below). However, the absolute peak ICa amplitudes in the presence of intracellular BAPTA in our recordings are quite similar to the currents measured by Sayer et al. (1993) in neocortical neurones.
Previous studies of Ca2+ currents in human neurones have not addressed the Ca2+ dependence of ICa inactivation. We specifically examined this issue in light of the anatomical study of Maglóczky et al. (1997) describing the loss of CB, a putative intraneuronal Ca2+ buffer (Chard et al. 1995; Li, Decavel & Hatton, 1995; Airaksinen, Eilers, Garaschuk, Thoenen, Konnerth & Meyer, 1997). The inactivation of Ca2+ currents observed in our recordings fulfils all criteria established for the Ca2+ dependency of ICa inactivation (Chad, 1989): (1) the presence of a U-shaped relationship between inactivation time constants and membrane voltages, (2) the correlation between the amount of Ca2+ entry and τ, (3) the suppression of inactivation by enhancing internal Ca2+ buffering, and (4) the divalent ion specificity of inactivation (Ca2+ > Ba2+).
In addition to slowing the decay of ICa, the intracellular presence of Ca2+-free BAPTA is expected to reduce resting free [Ca2+]. This would tend to diminish the level of basal ICa inactivation by resting [Ca2+] and should lead to an increase in the peak ICa. Indeed, intracellular BAPTA significantly increased peak ICa amplitudes. Since this effect of BAPTA was not observed in kindled rat neurones (Köhr & Mody, 1991), it might reflect a difference in the endogenous steady-state Ca2+ buffering between human TLE and kindled rat DGGCs.
Our findings are of interest in the context of TLE, in which CB is selectively lost from the DGGCs (Maglóczky et al. 1997). As shown by our study, a functional consequence of this loss may be an increased negative feedback on Ca2+ entry through HVA channels. A similar outcome was postulated for the kindled rat model, where CB is also selectively lost from the DGGCs (Baimbridge, 1992) and ICa inactivation rates are enhanced (Köhr & Mody, 1991). The comparable time courses of ICa inactivation (Fig. 3C) and the restored inactivation by exogenous internal Ca2+ buffering in human TLE and kindled rat neurones (Köhr & Mody, 1991)underscore the similarities between cellular alterations in human TLE and kindled DGGCs.
Whereas CB is not expected to affect the resting level of free [Ca2+] in normal human DGGCs, it might critically restrict the dynamic free [Ca2+] profile at the point of Ca2+ entry since it is present in a high concentration (as much as a few hundred micromolar), has four or six functional EF-hand Ca2+ binding domains (Chard, Bleakman, Christakos, Fullmer & Miller, 1993), and is associated with membranes (Winsky & Kuznicki, 1995). Thus, CB may effectively compete for entering Ca2+ ions with the EF-hand Ca2+ binding site on the channel (de Leon et al. 1995) or with other Ca2+ binding sites with similar affinities (Zhou et al. 1997) thought to be responsible for inactivation.
According to our studies, an epilepsy-associated loss of CB may be responsible for the enhanced negative feedback on ICa. This process may represent a neuroprotective mechanism aimed at reducing potentially cytotoxic Ca2+ loads following prolonged trains of action potentials known to occur during seizures. Furthermore, a decrease in endogenous Ca2+ buffering could promote the activation of a Ca2+-dependent K+ conductance in epileptic DGGCs (Beck, Clusmann, Kral, Schramm, Heinemann & Elger, 1997a) thereby invoking another mechanism to protect the neurones from hyperexcitability.