Oxygen-sensing persistent sodium channels in rat hippocampus

Abstract

1. Persistent sodium channel activity was recorded before and during hypoxia from cell-attached and inside-out patches obtained from cultured hippocampal neurons at a pipette potential (Vp) of +30 mV. Average mean current (IU) of these channels was very low under normoxic conditions and was similar in cell-attached and excised inside-out patches (-0.018 +/- 0.010 and -0.025 +/- 0.008 pA, respectively, n = 24). 2. Hypoxia increased the activity of persistent sodium channels in 10 cell-attached patches (IU increased from -0. 026 +/- 0.016 pA in control to -0.156 +/- 0.034 pA during hypoxia, n = 4, P = 0.013). The increased persistent sodium channel activity was most prominent at a VP between +70 and +30 mV (membrane potential, Vm = -70 to -30 mV) and could be blocked by lidocaine, TTX or R56865 (n = 5). Sodium cyanide (NaCN, 5 mM; 0.5-5 min) increased persistent sodium channel activity in cell-attached patches (n = 3) in a similar manner. 3. Hypoxia also increased sodium channel activity in inside-out patches from hippocampal neurons. Within 2-4 min of exposure to hypoxia, I had increased 9-fold to -0. 18 +/- 0.04 pA (n = 21, P = 0.001). Sodium channel activity increased further with longer exposures to hypoxia. 4. The hypoxia-induced sodium channel activity in inside-out patches could be inhibited by exposure to 10-100 microM lidocaine applied via the bath solution (I = -0.03 +/- 0.01 pA, n = 8) or by perfusion of the pipette tip with 1 microM TTX (I = -0.01 +/- 0.01 pA, n = 3). 5. The reducing agent dithiothreitol (DTT, 2-5 mM) rapidly abolished the increase in sodium channel activity caused by hypoxia in excised patches (I = -0.01 +/- 0.01 pA, n = 4). Similarly, reduced glutathione (GSH, 5-20 mM) also reversed the hypoxia-induced increase in sodium channel activity (IU = -0.02 +/- 0.02 pA, n = 5). 6. These results suggest that persistent sodium channels in neurons can sense O2 levels in excised patches of plasma membrane. Hypoxia triggers an increase in sodium channel activity. The redox reaction involved in increasing the sodium channel activity probably occurs in an auxiliary regulatory protein, co-localized in the plasma membrane.

Figure 1. Effect of bubbling the bath solution with 100% N₂ on O₂ tension

The graph shows the level of O₂ recorded at various times after commencing the bubbling of the perfusing solution with 100% N₂. O₂ tension reached levels of less than 30% of normal air O₂ within 3 min (see Methods). This time corresponds well with the initial increase in persistent Na⁺ channel activity.

Figure 2. Effect of hypoxia on Na⁺ channel activity in a cell-attached hippocampal patch

Representative traces recorded from a cell-attached hippocampal patch at various potentials (-V_P) stepped from a holding potential (V_h) of +70 mV (pre-pulse to V_P =+120 mV) in control (A), hypoxia (B) and after addition of 100 μm lidocaine to the hypoxic perfusing solution (C). All traces start 100 ms after the beginning of the 400 ms test pulse, hence they only show persistent Na⁺ channel activity. D, the relationship between single channel current amplitude (I) and potential. The current amplitude is the mean of several measurements at each potential (n = 3-15) obtained from the same patch shown in A–C during normoxia (○) and hypoxia (•), and ±1 s.e.m. is shown where greater than the size of the symbol. The slope conductance was 9.7 pS and the extrapolated reversal potential (V_P = -12 mV) would suggest that these channels are also permeable to K⁺, as reported previously (Hammarström & Gage, 1999a). As persistent Na⁺ channel activity was very rare in control conditions, no reliable current-voltage realtionship could be established during normoxia. When Na⁺ channel activity was observed in control solution it did not appear to change in amplitude or reversal potential with hypoxia (see ○).

Figure 7. Effect of 5 mm NaCN on Na⁺ channel activity in a cell-attached hippocampal patch

Representative Na⁺ channel traces recorded at various potentials (-V_P, all potentials corrected for a liquid junction potential of 18.8 mV) from a V_h of +70 mV (pre-pulse to +120 mV) in control (A), in the presence of 5 mm NaCN (B) and after addition of lidocaine to the NaCN-containing perfusing solution (C). All traces start 100 ms after the beginning of the 400 ms test pulse, and hence only show the persistent Na⁺ channel activity. D, current-voltage relationship established from the same cell-attached patch (n = 3-23 measurements at each potential) as in A–C, showing ±1 s.e.m. where greater than the size of the symbol. No channel activity was recorded around 0 ± 10 mV either in control (○) or in the presence of NaCN (•). The extrapolated reversal potential for this patch was +20 mV (-V_P), and the slope conductance was 10.1 pS. This reversal potential is less negative than the calculated reversal potential of +42 mV. This would suggest that these channels, like those activated by hypoxia, are also permeable to K⁺. Very few channel openings were recorded across the full voltage range in control, when no NaCN was present. The current amplitudes recorded during control, normoxic conditions (○) suggest that there was no change in current amplitude caused by NaCN.

Figure 3. Effect of hypoxia on inside-out patches excised from hippocampal neurons

Representative traces from a hippocampal patch held at a V_P of +30 mV in control (A) and after 30 s of hypoxia (non-continuous traces in B). Dotted lines show the closed channel current level. C–E, all-points histograms calculated from full 0.5-3 min recordings sampled at 10 kHz obtained from another hippocampal patch held at a V_P of +30 mV (P, probability). C, control; D, 0-30 s hypoxia (inset shows open peak more clearly); E, 1-3 min of hypoxia. F, increase in mean current during hypoxia measured in control patches (n = 21, ▪), and after 0-4 min (n = 21) and 4-8 min (n = 8) of hypoxia (). Histograms show mean data and the vertical bars ±1 s.e.m.

Figure 4. Effect of TTX on I_Na,P induced by hypoxia

A–E, effect of pipette perfusion with TTX in one inside-out hippocampal patch. Representative traces recorded at a V_P of +30 mV during control (A), after 5 min of hypoxia (B), after 10 min of hypoxia (C), after 1 μm TTX has reached the tip of the pipette and the membrane patch abolishing activity (D), and 12 min later (continuous hypoxia) when it can be assumed that TTX has diffused away and channel activity has resumed (E). The mean current (I′) from a full 0.5-4 min recording is shown below each set of traces. Dashed lines indicate closed current levels. F, mean data from excised patches exposed to extracellular TTX in the continued presence of hypoxia. Histograms show I′ (pA) and the vertical bars ±1 s.e.m. during control (), hypoxia (□) and exposure to TTX (▪).

Figure 5. Effect of lidocaine on I_Na,P induced by hypoxia

A–F, effect of lidocaine in two inside-out hippocampal patches. Three representative traces (V_P =+30 mV) during control (A), after 5-6 min of hypoxia (B) and after 2.5 min exposure to 100 μm lidocaine added to the hypoxic perfusing solution (C). The mean current (I′) from a full 0.5-4 min recording is shown below each set of traces. Dashed lines indicate closed current levels. D–F, all-points histograms obtained from 4 min recordings sampled at 10 kHz from another inside-out patch held at a V_P of +30 mV during control (D), 4 min of hypoxia (E) and after 2 min the the presence of 100 μm lidocaine (F). G, cumulative data for the effect of lidocaine on hypoxia-elicited activity. Histograms show I′ (pA) and the vertical bars ±1 s.e.m. during control (), hypoxia (□) and exposure to lidocaine (▪, n = 8).

Figure 6. Reducing agents inhibit the effect of hypoxia

A–C, effect of DTT in one inside-out hippocampal patch. Three representative traces (V_P =+30 mV) are shown during control (A), after ≈12 min of hypoxia (B) and within 2 min of exposure to 5 mm DTT in the perfusing solution (C). The mean current (I’) from a full 0.5-4 min recording is shown below each set of traces. Dashed lines indicate the closed current level. All-points histograms obtained from 4 min recordings sampled at 10 kHz from the same inside-out patch held at a V_P of +30 mV during control, after 23 min of hypoxia and within 2 min of adding 5 mm DTT to the perfusing solution accompany each set of traces. D and E, mean effect of reducing agents on hypoxia-elicited activity. Histograms show I′ and the vertical bars ±1 s.e.m. during control (), hypoxia (hypo, □) and exposure to drugs (D, DTT, n = 4; E, GSH, n = 5; ▪).