Stoichiometry of Na+-Ca2+ exchange in inside-out patches excised from guinea-pig ventricular myocytes

Abstract

1. The stoichiometry (nx) of cardiac Na+-Ca2+ exchange was examined by measuring the reversal potential of the Na+-Ca2+ exchange current (INa-Ca) in large inside-out patches, 'macro patches', excised from intact guinea-pig ventricular cells. 2. Cytoplasmic application of Na+ (Na+i) or Ca2+ (Ca2+i) induced INa-Ca which showed properties similar to INa-Ca in the giant membrane patch. The outward INa-Ca was depressed by an exchanger inhibitory peptide, XIP. 3. The reversal potential of the XIP-sensitive current indicated that nx was approximately 4 (3.6-4.2) at 9-40 mM Na+i, and nx tended to increase as Na+i was increased. Proteolysis by trypsin did not significantly affect the stoichiometry. Similar results were obtained from the reversal potential of INa-Ca that was induced by application of both Na+i and Ca2+i. 4. At 0.1 microM Ca2+i, nx was approximately 4 (3.7-4. 4) at 6-25 mM Na+i and tended to increase as Na+i was increased. When Ca2+i was changed from 0.1 to 1 and 1000 microM at constant 50 mM Na+i, the value was approximately 4 (3.6-4.4). 5. When the extracellular Na+ (Na+o) and Ca2+ (Ca2+o) concentrations were varied in the presence of 25 or 9 mM Na+i and 1 microM Ca2+i, nx was almost constant ( approximately 4) over the range 0.3-20 mM Ca2+o and 10-145 mM Na+o. 6. These results indicated that the stoichiometry of Na+-Ca2+ exchange is different from generally accepted 3Na+:1Ca2+, and suggested that the stoichiometry is either 4Na+:1Ca2+ or variable depending on Na+i and Ca2+i.

Figure 1. I_Na-Ca in the ‘macro patch’

A, 10 μM Ca²⁺_i-induced inward I_Na-Ca. Na⁺₀= 100 mM, Ca²⁺_o= 0 mM, and Na⁺_i= 0 mM. Ca²⁺_i was increased from 0 to 10 μM during the period indicated. Right panel is the I–V relation of I_Na-Ca (‘b – a’), which was obtained as a difference current in the presence and absence of 10 μM Ca²⁺_i. B, 100 mM Na⁺_i-induced outward I_Na-Ca. Na⁺₀= 0 mM and Ca²⁺_o= 5 mM. Na⁺_i (100 mM) was applied in the presence and absence of 1 μM Ca²⁺_i. Then the cytoplasmic side of the patch was treated by trypsin for 20 s. The I–V relations in the presence (‘b – a’) and absence (‘d – c’) of 1 μM Ca²⁺_i, and I–V relation after trypsin treatment (‘f – e’) were superimposed. Note that 1 μM Ca²⁺_i did not affect background current (‘a – c’). C, inhibition of I_Na-Ca by XIP. The patch was pretreated with trypsin. XIP (0.2 μM) reversibly inhibited 100 mM Na⁺_i-induced I_Na-Ca. Note that the effect of XIP on the background current was small (‘a – c’). Data in A, B and C were obtained from different patches.

Figure 2. XIP-sensitive I_Na-Ca

A, XIP-sensitive current. Na⁺₀= 145 mM, Ca²⁺_o= 2 mM, Na⁺_i= 9 mM and Ca²⁺_i= 1 μM. XIP (0.2 μM) suppressed inward I_Na-Ca at the holding potential (left panel) in the trypsin-treated patch. The XIP-sensitive current (‘b – a’) is shown in the right panel. Note that the background current did not significantly change (‘c – a’). B, I–V relation of the XIP-sensitive current at different Na⁺_i. XIP (0.2 μM)-sensitive currents at 40, 25, 15 and 9 mM Na⁺_i are shown. Dotted curves are fitting functions and arrows indicate reversal potentials. C, effect of XIP on the background current. XIP (0.2 μM)-sensitive current in the presence of 100 mM Na⁺_i and 0 μM Ca²⁺_i is shown (Na⁺₀= 145 mM, Ca²⁺_o= 2 mM, trypsin-untreated patch).

Figure 3. Reversal potentials and stoichiometry of the XIP-sensitive current

A, Na⁺_i concentration-reversal potential relationship. Dotted lines indicate theoretical E_Na-Ca of 4Na⁺:1Ca²⁺ and 3Na⁺:1Ca²⁺ exchange, respectively, and the continuous line is the fitted function (n_x= 4.1). Data were obtained from trypsin-treated patches (^, 7 patches) and non-trypsin-treated patches (•, 6 patches). B, Na⁺_i concentration- stoichiometry relationship. n_x was calculated from the experimental data in A using eqn (2). Na⁺₀= 145 mM, Ca²⁺_o= 2 mM and Ca²⁺_i= 1 μM. The number of values at each Na⁺_i concentration was 3–7.

Figure 4. Na⁺_i,Ca²⁺_i-induced I_Na-Ca

A, I_Na-Ca induced by application of Na⁺_i and Ca²⁺_i. Na⁺₀= 145 mM, Ca²⁺_o= 2 mM. The patch was pretreated with trypsin. An application of 12 mM Na⁺_i and 1 μM Ca²⁺_i induced an inward I_Na-Ca at the holding potential (left panel). The I–V relation of the current (‘b – a’) is shown in the right panel. Note that background current did not change significantly after the activation of I_Na-Ca (‘c – a’). B, the I–V relation at different Na⁺_i concentrations. The Na⁺_i concentration was changed from 12 to 50 mM in a patch with the same protocol as A. C, 100 mM Na⁺_i-induced current in the absence of both Na⁺₀ and Ca²⁺_o in the pipette solution. Na⁺₀ was replaced by NMDG.

Figure 5. Reversal potentials and stoichiometry of the Na⁺_i,Ca²⁺_i-induced I_Na-Ca

A, Na⁺_i concentration-reversal potential relationship. Dotted lines indicate theoretical E_Na-Ca of 4Na⁺:1Ca²⁺ and 3Na⁺:1Ca²⁺ exchange, respectively, and the continuous line is the fitted function (n_x= 4.3). Data were from trypsin-treated patches (□, 9 patches) and non-trypsin-treated patches (▪, 9 patches). B, Na⁺_i concentration-stoichiometry relationship. n_x was calculated from the data in A using eqn (2).

Figure 6. Ca²⁺_i dependence of the reversal potential

All patches were pretreated with trypsin. Data were obtained from the 0.2 or 0.8 μM XIP-sensitive current (^) and from the Na⁺_i,Ca²⁺_i-induced current (□). The two dotted lines have the same meanings as in Fig. 5. A, Na⁺_i concentration-reversal potential (upper panel) and -stoichiometry (lower panel) relationships at 0.1 μM Ca²⁺_i. Na⁺₀= 145 mM and Ca²⁺_o= 2 mM. Data were from 6 patches. The continuous line is the fitted function (n_x= 4.3). B, Ca²⁺_i concentration-reversal potential (upper panel) and -stoichiometry (lower panel) relationships. Na⁺₀= 145 mM, Ca²⁺_o= 2 mM and Na⁺_i= 50 mM. Data were from 7 patches. The continuous line is the fitted function (n_x= 4.6).

Figure 7. Na⁺₀ and Ca²⁺_o concentration dependencies of the reversal potential

A, Ca²⁺_o concentration-reversal potential (upper panel) and -stoichiometry (lower panel) relationships. Na⁺₀= 145 mM, Na⁺_i= 25 mM and Ca²⁺_i= 1 μM. The fitted n_x is 4.2 (continuous line). Data were from 17 patches. B, Na⁺₀ concentration-reversal potential (upper panel) and -stoichiometry (lower panel) relationships. Ca²⁺_o= 2 mM, Na⁺_i= 9 mM and Ca²⁺_i= 1 μM. The fitted n_x is 4.3 (continuous line). Data were from 24 patches. All the data were obtained from the 0.2 μM XIP-sensitive current (^) and from the Na⁺_i,Ca²⁺_i-induced current (□) in trypsin-treated patches. In each case the two dotted lines have the same meanings as in Fig. 5.

Figure 8. Reversal potential of I_Na-Ca in the giant membrane patch from cardiac bleb

A, Na⁺_i concentration-reversal potential relation. Na⁺₀= 145 mM, Ca²⁺_o= 2 mM and Ca²⁺_i= 1 μM. Data were from the 0.2 μM XIP-sensitive current (^) and from the Na⁺_i,Ca²⁺_i-induced current (□). B, Na⁺_i concentration- stoichiometry relation. In A and B the two dotted lines have the same meanings as in Fig. 5. The number of values at each point was 3–5.

Figure 9. Simulation of ion flux in the inside-out patch

A, activation of outward I_Na-Ca. The pipette solution contains 0 mM Na⁺₀ and 5 mM Ca²⁺_o. The patch was pretreated with trypsin. Na⁺_i (50 mM)-induced I_Na-Ca and simulated I_Na-Ca with the patch distance of 4, 6, 8 and 10 μm (smooth curves from left to right) are superimposed. B, suppression of outward I_Na-Ca by Ca²⁺_i. Ca²⁺ (2 mM: 10 mM EGTA + 12 mM CaCl₂) inhibited the 50 mM Na⁺-induced I_Na-Ca. Simulated I_Na-Ca with the patch distance of 4, 6, 8 and 10 μm (smooth curves from left to right) are also superimposed. C, open-tip response. Current response to bath solution change from the control Tyrode solution to 20 % diluted Tyrode solution was recorded after disruption of the patch membrane. Data from A, B and C were from the same pipette. D, simulation of I_Na-Ca, and Na⁺ and Ca²⁺ concentrations in a compartment just under the membrane. Na⁺ (50 mM) and Ca²⁺ (1 μM) were added at the bulk cytoplasmic space at time 0. I_Na-Ca had an I_max of 10 pA (upper panel). Na⁺ simulation in the middle panel had I_max values of 10 pA, 10 nA and 10 μA, and the results are superimposed (a, b and c). Ca²⁺ simulation in the bottom panel had I_max values of 10 pA (a), 10 nA (b) and 10 μA (c).