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. 1997 Jun 24;94(13):7012-7.
doi: 10.1073/pnas.94.13.7012.

Extracellular calcium sensed by a novel cation channel in hippocampal neurons

Z Xiong  1 , W Lu, J F MacDonald
Affiliations

Extracellular calcium sensed by a novel cation channel in hippocampal neurons

Z Xiong et al. Proc Natl Acad Sci U S A. .

Abstract

Extracellular concentrations of Ca2+ change rapidly and transiently in the brain during excitatory synaptic activity. To test whether such changes in Ca2+ can play a signaling role we examined the effects of rapidly lowering Ca2+ on the excitability of acutely isolated CA1 and cultured hippocampal neurons. Reducing Ca2+ excited and depolarized neurons by activating a previously undescribed nonselective cation channel. This channel had a single-channel conductance of 36 pS, and its frequency of opening was inversely proportional to the concentration of Ca2+. The inhibition of gating of this channel was sensitive to ionic strength but independent of membrane potential. The ability of this channel to sense Ca2+ provides a novel mechanism whereby neurons can respond to alterations in the extracellular concentration of this key signaling ion.

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Figures

Figure 1
Figure 1
The effect of lowering Ca2+ on the excitability of hippocampal neurons. (A) The membrane potential of a cultured neuron was recorded using the whole-cell current-clamp configuration (KCl instead of CsF in the recording pipette). Decreasing Ca2+ from 1.5 to 0.5 mM for a period of 2 s depolarized and strongly excited the cell. (B) In the presence of 0.5 μM tetrodotoxin and 1.5 mM Mg2+, reductions of Ca2+ from 1.3 to 1.2, 1.0, 0.5, 0.2, and 0 mM respectively, evoked graded depolarizations. (C) In a series of six recordings, the amplitude of the depolarization was recorded and values plotted versus the concentration of Ca2+. The control solution contained 1.3 mM Ca2+, and all of the solutions contained 1.5 mM Mg2+. (D) The normalized dose–response curve for the depolarization induced by lowering Ca2+. The depolarization was normalized to that induced by the solution containing no added Ca2+. The nominal concentration of Ca2+ in this solution was estimated to be about 20 μM (24). The logistic equation in the form R = Rmax/[1 + (D/EC50)nH] was used to fit the points. R represents the normalized depolarization at any given Ca2+ concentration, Rmax is the response with no added Ca2+, the EC50 value is the concentration of Ca2+ that produced 50% of the maximal response, and nH is the estimated Hill coefficient (EC50 = 0.39 ± 0.11 mM; nH = 1.4 ± 0.3; n = 6).
Figure 2
Figure 2
Lowering Ca2+ induces a nonspecific cation current in cultured and isolated hippocampal neurons. (A) A step reduction in the concentration of Ca2+ evoked a substantial inward current in a cultured hippocampal neuron. This inward current was associated with an increase in membrane conductance as assessed by repeating a depolarizing voltage step. (B) In a group of 15 neurons the current–voltage relationships (I-V) were determined for the response to a change from 2 to 0 mM Ca2+ while holding at values from −60 to +20 mM. The currents demonstrated a near-linear I-V curve and reversed at +0.3 ± 1.8 mV. (C) Superimposed responses to NMDA (100 μM, 3 μM glycine) with and without a simultaneous decrease in Ca2+ (to 200 μM) are shown for two cultured neurons (a and b). The smallest current in each case is the superimposed response to a decrease in Ca2+ alone. A response of a HEK293 cell to a decrease in Ca2+ (2 mM to 200 μM) alone (NMDA was not applied) is shown before and following a partial block with Gd3+ (2 μM) (c). These currents ranged from 50 to 1500 pA in different HEK cells.
Figure 3
Figure 3
The inward current evoked by decreasing Ca2+ is carried by Na+ and K+ but not by Cl−. (A) Example traces showing the responses of cultured hippocampal neurons to a decrease in Ca2+. Ca2+ was reduced from 2.0 to 0 mM in four different extracellular solutions: (i) No Na+ or K+, replacement with 150 mM NMDG; (ii) in the presence of 140 NaCl and 5.4 mM KCl; (iii) in 20 mM NaCl; and (iv) in 20 mM KCl. In the absence of monovalent cations, step reductions of Ca2+ evoked little or no inward current, demonstrating that the response was not due to some kind of nonspecific breakdown of the membrane. However, when the usual extracellular solution (140 NaCl, 5.4 KCl) was used, an inward current with an amplitude of about 500 pA was recorded from the same cell. A smaller current was recorded when the extracellular solution contained only 20 mM Na+ or only K+. The holding potential was −60 mV. (B) A step reduction in the concentration of Ca2+ was used to evoke responses in the presence of a solution containing 150 mM NaCl and 5.4 mM KCl or a second solution with half the concentration of Na+ (75 mM) and K+ (2.7 mM). In each case the holding potential of the cell was varied between −60 and +20 mV. (C) Such data (n = 3) was then used to construct I-V curves. When the concentration of monovalent ions was reduced by one-half, the reversal potential shifted toward more hyperpolarized values (see text). (D) Substitution of all of the extracellular Cl− with gluconate had little influence on the responses to lowered Ca2+ and did not alter the I-V curve (n = 3) for this response (E).
Figure 4
Figure 4
Divalent and polyvalent cations block the inward currents induced by lowering Ca2+. (A) Dose-dependent block of inward currents. The inward current was induced by a reduction of Ca2+ from 2 to 0 mM at a holding potential of −60 mV. Different concentrations of cations were added to the low Ca2+ solution, and the normalized inhibition of this current was plotted against the concentration of a given cation (B). The dose-inhibition curves were fitted using the logistic equation. The doses that produced 50% block of the current were: Gd3+ (◊), 0.0014 ± 0.00049 mM (n = 5); neomycin (⧫), 0.0043 ± 0.0011 mM (n = 6); Ca2+ (○), 0.15 ± 0.04 mM (n = 7); Ba2+ (•), 0.32 ± 0.10 mM (n = 4); Mg2+ (▵), 0.34 ± 0.14 mM (n = 5); and Cd2+ (▴), 0.37 ± 0.05 mM (n = 4). Higher concentrations of neomycin activated an additional inward current, thus apparently limiting the inhibition of the nonspecific cation current. (C) The apparent affinity of divalent cations was increased when the ionic strength in the extracellular solution was reduced. The open symbols represent Ca2+ (○) and Mg2+ (▵) dose-inhibition curves in the presence of solutions of normal ionic strength (140 mM NaCl, 5.4 mM KCl), and the filled symbols represent Ca2+ (•) and Mg2+ (▴) in the presence of solution of reduced ionic strength (35 mM NaCl, 1.4 mM KCl, supplemented with sucrose). The recording pipettes contained either CsCl (or CsF) and 2 mM TEA.
Figure 5
Figure 5
The nonselective cation current demonstrates little or no voltage dependence. (A) An example recording from a cultured hippocampal neuron (holding, −40 mV). In (a) the response to a series of voltage steps from −120 to +80 mV in the presence of Ca2+ (2 mM) is shown. The same series was then repeated following a decrease of Ca2+ to nominally free values (b), and the difference responses (b-a) were calculated (c). The dotted line marks the initial holding current. No current relaxations were observed, and the I-V curve (d) was linear. Identical results were observed in another four cells. The reversal potential was slightly more positive than 0 mV because of the pipette solution that had been diluted (120 mM CsF). (B) Steady-state currents in response to two different changes in Ca2+ concentration (0 and 0.5 mM) were also examined. The I-V curves (n = 3) for each were approximately linear over the range tested, and both reversed at 0 mV, demonstrating that Ca2+ did not cause an obvious voltage-dependent block.
Figure 6
Figure 6
Single-channel recordings in outside-out patches taken from cultured hippocampal neurons. (A) An example record showing the activation of single-channel openings in response to a step reduction in the concentration (2 to 0 mM) of Ca2+. The holding potential was −60 mV. (B) I-V relationship for single-channel currents. Representative trances show the activation of these currents at holding potentials ranging from −60 to 0 mV. The I-V curve was constructed from the results of recordings from eight such patches. The channel showed a linear I-V relationship with a slope conductance of 36 ± 2.6 pS (n = 8). (C) Effect of Gd3+ on single-channel currents. Gd3+ (0.002 mM) significantly decreased the channel-open probability (control Po = 0.21 ± 0.13; Gd3+ Po = 0.10 ± 0.07, n = 5, P < 0.05) without having any effect on single-channel amplitude.
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