Figure 3.

H₂S inhibits Na⁺/Ca²⁺ exchanger current (I_NaCa): reversal by MB. 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 I_Ca,L. Our measurement conditions were biased towards 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 Na⁺/Ca²⁺ exchanger and thus allowed [Na⁺]_i and [Ca²⁺]_i to equilibrate with those in pipette solution), I_NaCa (30°C) was measured in myocytes using a descending (from +100 to −120 mV; 500 mV/s) – 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 I_NaCa measured between the descending and ascending voltage ramps. This suggests that [Ca²⁺]_i and [Na⁺]_i sensed by Na⁺/Ca²⁺ exchanger did not appreciably change by ion fluxes during the brief (880 ms) voltage ramp. I_NaCa was divided by C_m prior to comparisons. (C). At time 0, either saline or NaHS (100 μM) was added followed by MB (20 μg/ml) or saline at 3 min before I_NaCa was measured at 7 min. Current-voltage relationships of I_NaCa (means ± SE) from control (▲; n=6), MB (●; n=3), NaHS (■; n=5) and NaHS + MB (◆; n=5) 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.