Fig. 6.

Constitutive overexpression of S68E mutant inhibits Na⁺/Ca²⁺ exchanger current (I_NaCa). Pipette solution contained (in mM) 100 Cs⁺ glutamate, 7.25 NaCl, 1 MgCl₂, 20 HEPES, 2.5 Na₂ATP, 10 EGTA, and 6 CaCl₂ (pH 7.2). Free Ca²⁺ in the pipette solution was 205 nM, measured fluorimetrically with fura-2. External solution contained (in mM) 130 NaCl, 5 CsCl, 1.2 MgSO₄, 1.2 NaH₂PO₄, 5 CaCl₂, 10 HEPES, 10 Na⁺ HEPES, and 10 glucose (pH 7.4). Verapamil (1 μM) was used to block L-type Ca²⁺ current (I_Ca). Our measurement conditions were biased toward measuring outward (3 Na⁺ out: 1 Ca²⁺ in) I_NaCa. A: after holding the myocyte at the calculated reversal potential (−73 mV) of I_NaCa for 5 min (to minimize fluxes through NCX1 and thus allowed [Na⁺]_i and [Ca²⁺]_i to equilibrate with those in pipette solution), I_NaCa (30^°C) was measured in WT and conS68E myocytes using a descending (from +100 to −120 mV; 500 mV/s) and ascending (from −120 to +100 mV; 500 mV/s) voltage ramp, first in the absence and then in the presence of 1 mM NiCl₂. B: raw currents measured in a WT myocyte. I_NaCa was defined as the difference current measured in the absence and presence of Ni⁺ during the descending voltage ramp. Note that with the exception of small contamination of the ascending ramp by the cardiac Na⁺ current, there were little to no differences in currents measured between the descending and ascending voltage ramps. This suggests that [Ca²⁺]_i and [Na⁺]_i sensed by NCX1 did not appreciably change by NCX1 fluxes during the brief (880 ms) voltage ramp. I_NaCa was divided by C_m before comparisons. C: current-voltage relationships of I_NaCa (means ± SE) from WT (▴; n = 6) and conS68E (◊; n = 12) myocytes are shown. The reversal potential of I_NaCa was ∼−60 mV, close to the theoretical reversal potential of −73 mV. Error bars are not shown if they fall within the boundaries of the symbol. I_NaCa data are fitted with 3^rd order polynomial equation.