Protein kinase C co-expression and the effects of halothane on rat skeletal muscle sodium channels

Abstract

1. Voltage-gated Na channels, which are potential targets for general anaesthetics, are substrates for PKC, which phosphorylates a conserved site in the channel inactivation gate. We investigated the idea that PKC modulates the effect of volatile anaesthetics on Na channels via phosphorylation of this inactivation gate site. 2. Na currents through rat skeletal muscle Na channel alpha-subunits expressed in Xenopus oocytes were measured by two-microelectrode voltage clamp in the presence of the volatile anaesthetic agent halothane (2-bromo-2-chloro-1,1,1-trifluroethane). PKC activity was modulated by co-expression of a constitutively active PKC alpha-isozyme. 3. Halothane (0.4 mM) had no effect on Na currents. With co-expression of PKC, however, halothane dose-dependently enhanced the rate of Na current decay and caused a small, but statistically significant reduction in Na current amplitude. 4. The enhancement of Na current decay was absent in a Na channel mutant in which the inactivation gate phosphorylation site was disabled. Effects of halothane on amplitude were independent of this mutation. 5. Co-expression of a PKC alpha-isozyme permits an effect of halothane to hasten current decay and reduce current amplitude, at least in part through interaction with the inactivation gate phosphorylation site. We speculate that the interaction between halothane and Na channels is direct, and facilitated by PKC activity and by phosphorylation of a site in the channel inactivation gate.

Figure 1

Effect of repeated depolarizations, 30 s apart, on currents through oocyte-expressed rat skeletal muscle Na channels. The original current records (A) reveal a steady decrement in amplitude and decay kinetics. The individual current traces were fitted to a single exponential, and the decay time constant (τ) was a linear function of time (B, squares). A regression line had a slope of −0.09 msec min⁻¹, (r=0.980, P<0.001). This linear decay was subtracted from the raw data to yield an unchanging corrected τ over the 22 min course of the experiment (circles). The drift of amplitude was also linear (C), and a regression line had a slope of 0.1 μA min⁻¹ (r=0.998, P<0.001). When this was subtracted from the raw data, corrected amplitude was unchanging.

Figure 2

The effect of halothane on oocytes expressing rat skeletal muscle Na channels alone (left panels) and with PKC co-expression (right panels). (A and B) show currents recorded from oocytes expressing Na channels α-subunits alone; (C and D) show data from oocytes co-expressing the β-subunit. Sample recordings taken under control conditions (i), after superfusion with halothane for 10 min (ii), and after 10 min wash (in oocytes expressing Na channels alone) (iii), are shown superimposed. Note that currents recorded from the oocyte co-expressing the β-subunit were larger and decayed much more quickly than the currents recorded from the oocyte expressing Na channels alone.

Figure 3

Subtraction of the linear drift in decay τ (A and B) and amplitude (C and D) for the examples shown in Figure 2A and B. (A and C) show data from the oocyte expressing Na channels alone; (B and D) show data from the oocyte co-expressing PKC. Halothane was applied between minutes 2 and 12 as shown by the bars. Raw data are displayed as squares; corrected data as circles. The drift of τ and amplitude in this example were 0.05 msec min⁻¹ and 0.3 μA min⁻¹ in oocytes expressing Na channels alone, and 0.05 msec min⁻¹ and 0.1 μA min⁻¹ in oocytes co-expressing PKC.

Figure 4

Summary data for the effects of 0.4 and 1.4 mM halothane on Na current decay τ and amplitude. Open circles represent oocytes expressing Na channels alone; filled circles represent oocytes co-expressing PKC. Data have been corrected for linear drift. In oocytes expressing Na channels alone, the mean drift in decay τ was 0.07 msec min⁻¹ in 0.4 mM halothane and 0.06 msec min⁻¹ in 1.4 mM halothane. The mean drift in amplitude was 1.8% min⁻¹ in 0.4 mM halothane compared with 2.3% min⁻¹ in 1.4 mM halothane (P=NS, t-test). In oocytes co-expressing PKC, the mean drifts in decay τ were 0.06 and 0.09 msec min⁻¹, and the mean drifts in amplitude were 1.5% and 1.8% min⁻¹ in 0.4 and 1.4 mM halothane (P=NS, t-test). ^*P<0.05 (t-test) for the comparison between oocytes expressing Na channels alone and oocytes co-expressing PKC. ‡P<0.05 (t-test) for the comparison between halothane 0.4 and 1.4 mM. Data were derived from 10–21 oocytes, three to six frogs.

Figure 5

Box plots of the effect of PKC co-expression on the amplitude of currents through wild-type and phos(−) Na channels. Currents were normalized so that the mean current amplitude in oocytes expressing wild-type or phos(−) channels alone in each batch was 1.0. In the box plot symbol, the line represents the median, the boxes enclose 50% of the data and the hatches enclose 80% of the data. Data were derived from 30–64 oocytes, four to six frogs. ^*P<0.001 (rank sum test).

Figure 6

(A and B) The effect of halothane (0.4 mM, 10 min) on the proportion of Na channels available in oocytes expressing Na channels alone (A), and in oocytes co-expressing PKC (B). The lines are least squares fits of the data to a Boltzmann function. The slope factor and midpoints were −11.0 and −17 mV in oocytes expressing Na channels alone, and −11.4 and −18 mV in oocytes expressing PKC. Halothane had no significant effect on either parameter (t-test). The data points are mean (s.d.) from five oocytes, two frogs. (C and D) The effect of halothane (0.4 mM, 10 min) on the current-voltage relationship in oocytes expressing Na channels alone (C), and in oocytes co-expressing PKC (D). The data points are mean (s.d.) from five oocytes, two frogs. The current amplitudes were normalized to the pre-halothane value to emphasize effects on the potential dependence of amplitude. Open circles represent control data; filled circles were obtained after exposure to halothane.

Figure 7

Summary data for the effects of halothane on Na current decay rate and amplitude in phos(−) Na channels. Data were derived from 10–15 oocytes, two to four frogs. Experimental details and symbols as in Figure 4. In oocytes expressing Na channels alone, the mean drift in decay τ was 0.07 and 0.09±0.01 msec min⁻¹ in 0.4 and 1.4 mM halothane. The mean drifts in amplitude were 1.2% min⁻¹ compared with 1.1% min⁻¹ (P=NS, t-test). In oocytes co-expressing PKC the mean drifts in decay τ were 0.05 and 0.1 msec min⁻¹, and the mean drifts in amplitude were 1.8 and 2.5% min⁻¹ in 0.4 and 1.4 mM halothane (P=NS, t-test).