Voltage and concentration dependence of Ca(2+) permeability in recombinant glutamate receptor subtypes

Abstract

The channels associated with glutamate receptor (GluR) subtypes, namely N-methyl-D-aspartate receptors (NMDARs), and Ca(2+)-permeable alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptors (AMPARs) and kainate receptors (KARs), are to varying degrees permeable to Ca(2+). To compare the mechanism of Ca(2+) influx, we measured Ca(2+) permeability relative to that of Na(+) (P(Ca)/P(Na)) using fractional Ca(2+) currents (P(f)) and reversal potential measurements over a wide voltage and Ca(2+) concentration range in recombinant NMDAR NR1-NR2A, AMPAR GluR-A(Q) and KAR GluR-6(Q) channels. For NR1-NR2A channels, P(Ca)/P(Na) derived from P(f) measurements was voltage independent but showed a weak concentration dependence. A stronger concentration dependence was found when P(Ca)/P(Na) was derived from changes in reversal potentials on going from a Na(+) reference solution to a solution with Ca(2+) as the only permeant ion ('biionic' condition). In contrast, P(Ca)/P(Na) was concentration independent when derived from changes in reversal potentials on going from a Na(+) reference solution to the same solution with added Ca(2+) ('high monovalent' condition). For GluR-A(Q) channels, P(Ca)/P(Na) derived from all three approaches was concentration independent, and for the reversal potential-based approaches were of comparable magnitude. Their most distinctive property was that P(Ca)/P(Na) derived from P(f) measurements was strongly voltage dependent. For GluR-6(Q) channels, P(Ca)/P(Na) derived from P(f) measurements was weakly voltage dependent. On the other hand, P(Ca)/P(Na) derived from all three approaches was the most strongly concentration dependent of any GluR subtype and, except for low Ca(2+) concentrations, the values were of comparable magnitude. Thus, the three Ca(2+)-permeable GluR subtypes showed unique patterns of Ca(2+) permeability, indicating that distinct biophysical and molecular events underlie Ca(2+) influx in each subtype.

Figure 4. Ca²⁺ permeability in GluR subtypes under biionic conditions

Average P_Ca/P_Na (○) or P_Ca/P_Cs (•) derived from ΔE_rev for individual Ca²⁺ concentrations using the Lewis equation (n > 4). ΔE_rev was measured on replacing Na⁺ (or Cs⁺) in a reference solution with 0.5, 1.8, 10 or 110 mm Ca²⁺ (see Wollmuth & Sakmann, 1998). The lines through the points have no theoretical meaning. The weak NMDG⁺ permeability in non-NMDAR channels (see Methods) strongly alters the magnitude of P_Ca/P_Na, especially in 0.5 mm Ca²⁺, and these values should be viewed cautiously. Nevertheless, assuming a P_NMDG/P_Na value of between 0 and 0.02 for GluR-6(Q) does not alter the overall pattern of concentration dependence (the values shown were derived using P_NMDG/P_Na = 0.01). P_Ca/P_Cs was not measured in 0.5 mm Ca²⁺.

Figure 5. Ca²⁺ permeability in GluR subtypes under high monovalent conditions

A, mean ΔE_rev on going from the Na⁺-based reference solution to the same solution but with added Ca²⁺ (0.3–50 mm Ca²⁺; n > 4). The continuous lines are fits of eqn (3), yielding P_Ca/P_Na values of 4.35 for NR1-NR2A, 1.74 for GluR-A(Q) and 0.32 for GluR-6(Q). B, average P_Ca/P_Na derived from ΔE_rev (shown in A) for individual Ca²⁺ concentrations using eqn (2). The continuous lines are from the fits in A. The dashed lines are the average of the individually derived P_Ca/P_Na values (3.6 for NR1-NR2A and 1.55 for GluR-A(Q)).

Figure 1. Fractional Ca²⁺ currents (P_f) in GluR subtypes

Simultaneous measurement of whole-cell currents (I, top) and fluorescence intensity with 380 nm excitation (F₃₈₀, bottom) evoked by glutamate applications (filled bars) in HEK 293 cells expressing NMDAR NR1-NR2A (left panel), AMPAR GluR-A(Q) (middle panel) or KAR GluR-6(Q) (right panel) channels. The potential (V) was −60 mV to the reversal potential (see Methods). In the current records (upper panels), the dashed lines represent zero current, and the shaded regions correspond to the current integral (Q_T), which was approximately the same for each of the example records. The F₃₈₀ plot is expressed in bead units (BU). ΔF₃₈₀ was derived as the difference between the F₃₈₀ amplitude at the indicated time (arrow) and the baseline F₃₈₀ signal (continuous line), extrapolated from a linear fit to the F₃₈₀ amplitudes prior to glutamate application.

Figure 2. Voltage dependence of P_f measurements in GluR subtypes

A, mean P_f values measured in GluR subtypes over a wide voltage range (n > 5). Voltages are relative to V_rev. The lines are predicted P_f values, using eqn (1), with P_Ca/P_Na derived from the P_f measurement either at −60 mV (continuous line), or at −20 mV (dashed line; see Table 1). B, average P_Ca/P_Na derived from P_f values measured at different membrane potentials (only P_f values greater than 2% were included in this plot). The continuous lines are fits of the relationship P_Ca/P_Na(V) = P_Ca/P_Na(V_rev) × exp(VzδF/RT), where P_Ca/P_Na(V_rev) is the estimated P_Ca/P_Na at the reversal potential and zδ is the voltage dependence of the process. For non-NMDAR channels, P_Ca/P_Na was fitted only at potentials positive to −60 mV where a clear voltage dependence existed. P_Ca/P_Na(V_rev) and zδ were: 1.50, 0.41 for GluR-A(Q) (○); 1.40, 0.40 for GluR-B(Q) (⊙, lower); 2.10, 0.32 for GluR-B(N) (⊙, upper); and 0.63, 0.18, for GluR-6(Q).

Figure 3. Concentration dependence of P_f measurements in GluR subtypes

A, mean P_f values, at −60 mV to the reversal potential, in cells expressing NR1-NR2A, GluR-A(Q) or GluR-6(Q) channels (n > 4). The continuous lines through the points are fitted Hill equations (P_f,max/(1 + (K_0.5/[Ca²⁺])^nH)) where P_f,max is the maximal P_f, K_0.5 the half-maximal response, and n_H the Hill coefficient. For all fits, the Hill coefficient was around 1. The fits yielded P_f,max and K_0.5 of approximately: 90%, 10 mm for NR1-NR2A; 100%, 57 mm for GluR-A(Q); and 34%, 28 mm for GluR-6(Q). Measurements at −20 mV were not made for all concentrations (see B) so comparable fits could not be made. B, average P_Ca/P_Na derived using eqn (1) from P_f values measured at −60 mV (□; shown in A) or at −20 mV (⋄). The continuous lines are the average of the P_Ca/P_Na values measured around physiological concentrations (1 and 1.8 mm Ca²⁺) and were, at −60 and −20 mV: 3.1, 3.1 for NR1-NR2A; 0.68, 1.10 for GluR-A(Q); and 0.49, 0.57 for GluR-6(Q).

Figure 6. P_f measurements in low extracellular Na⁺

A, mean P_f values measured in low extracellular Na⁺. Cells were bathed in a solution containing 1.8 mm Ca²⁺, 20 mm Na⁺ and 120 mm NMDG⁺. Values were measured at −60 mV. The number of cells recorded was, from left to right, 4, 3, 3. B, comparison of P_Ca/P_Na values derived from P_f measurements at −60 mV either in 143.5 mm Na⁺ or in 20 mm Na⁺.

Figure 7. Divalent cation permeability in GluR subtypes

Mean permeability ratios, P_D/P_Cs, for different divalent ions in the GluR subtypes (n > 3). The divalent ions tested included Ca²⁺ (○), Ba²⁺ (•), Sr²⁺ (□), Mg²⁺ (▪) and Co²⁺ (▵). For GluR-6(Q), only Ca²⁺ and Mg²⁺ could be measured at 10 mm because of the very negative reversal potential and the small current size in the test solution.

Figure 8. Comparison of P_Ca/P_Na derived from P_f measurements and from changes in reversal potentials

Comparison of P_Ca/P_Na derived from P_f measurements at −60 mV (□) or at −20 mV (⋄) (from Fig. 3) to P_Ca/P_Na derived from changes in reversal potentials using either the biionic (left panel, ○; from Fig. 4) or the high monovalent (right panel, ▵; from Fig. 5) approach. The dashed lines in all panels show the average P_Ca/P_Na derived from P_f measurements in 1 and 1.8 mm Ca²⁺ either at −60 or at −20 mV. A, NMDAR NR1-NR2A. Dashed line, 3.1. The continuous line in the left panel has no theoretical meaning, whereas that in the right panel is the average P_Ca/P_Na (3.60). B, AMPAR GluR-A(Q). Dashed lines, 0.68 (−60 mV) or 1.10 (−20 mV). The continuous lines are the average of the individually derived P_Ca/P_Na (1.73, left panel and 1.55, right panel). The ‘×’ in the left panel represents the estimated P_Ca/P_Na at 0 mV (1.50) from Fig. 2B. C, KAR GluR-6(Q). Dashed lines, 0.49 (−60 mV). The line at −20 mV is not shown. The continuous lines have no theoretical meaning. P_Ca/P_Na in 0.5 mm Ca²⁺ under the biionic conditions is not shown in order to compare more readily the values at higher Ca²⁺ concentrations.