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PLoS One
2013 Jan 01;87:e67551. doi: 10.1371/journal.pone.0067551.
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Differential effects of TipE and a TipE-homologous protein on modulation of gating properties of sodium channels from Drosophila melanogaster.
Wang L
,
Nomura Y
,
Du Y
,
Dong K
.
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β subunits of mammalian sodium channels play important roles in modulating the expression and gating of mammalian sodium channels. However, there are no orthologs of β subunits in insects. Instead, an unrelated protein, TipE in Drosophila melanogaster and its orthologs in other insects, is thought to be a sodium channel auxiliary subunit. In addition, there are four TipE-homologous genes (TEH1-4) in D. melanogaster and three to four orthologs in other insect species. TipE and TEH1-3 have been shown to enhance the peak current of various insect sodium channels expressed in Xenopus oocytes. However, limited information is available on how these proteins modulate the gating of sodium channels, particularly sodium channel variants generated by alternative splicing and RNA editing. In this study, we compared the effects of TEH1 and TipE on the function of three Drosophila sodium channel splice variants, DmNav9-1, DmNav22, and DmNav26, in Xenopus oocytes. Both TipE and TEH1 enhanced the amplitude of sodium current and accelerated current decay of all three sodium channels tested. Strikingly, TEH1 caused hyperpolarizing shifts in the voltage-dependence of activation, fast inactivation and slow inactivation of all three variants. In contrast, TipE did not alter these gating properties except for a hyperpolarizing shift in the voltage-dependence of fast inactivation of DmNav26. Further analysis of the gating kinetics of DmNav9-1 revealed that TEH1 accelerated the entry of sodium channels into the fast inactivated state and slowed the recovery from both fast- and slow-inactivated states, thereby, enhancing both fast and slow inactivation. These results highlight the differential effects of TipE and TEH1 on the gating of insect sodium channels and suggest that TEH1 may play a broader role than TipE in regulating sodium channel function and neuronal excitability in vivo.
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Figure 2. Enhancement of sodium current decay by co-expression of TipE or TEH1 with DmNav channel variants.Sodium current decay in DmNav9-1, DmNav22, or DmNav26 co-expressed with TipE (A, B, C, respectively) or TEH1 (D, E, and F, respectively). The decay of sodium current was fitted by a single exponential to generate time constants of current decay (τdecay). Each data point represents mean ± SEM for 12-20 oocytes. * indicates a significant difference compared to that of DmNav channel only (p<0.05).
Figure 3. Effects of co-expression of TipE or TEH1 on the voltage-dependence of activation and fast inactivation of DmNa v9-1 channels.(A) Voltage-dependences of activation. (B) Voltage-dependence of fast inactivation. Data were fitted with a two-state Boltzmann equation and fitting parameters are shown in Table 1. Data points are shown as mean ± SEM. Recording protocols are indicated and the details of the protocols and data analysis are described in the Materials and Methods.
Figure 4. Sensitivity of DmNa v9-1 to deltamethrin is modulated by co-expression of TEH1.(A) A representative tail current induced by 1 µM deltamethrin. (B) Percentage of channel modification by deltamethrin (multiple-pulse test). Tail currents were elicited by a 66.7-Hz train of 100 5-ms depolarization from -120 to 0 mV. (C) Co-expression of TEH1 significantly reduced the stability of DmNav9-1 peak sodium current under repeated conditioning depolarizations. Sodium currents were recorded during 20-ms step depolarizations from -120 mV to -10 mV after 0-100 conditioning pulses (5-ms pulses from -120 mV to 0 mV at 66.7 Hz). (D) Percentage of channel modification by deltamethrin (single-pulse test). Tail currents were elicited by a 500 ms depolarization from -120 mV to 0 mV. All data are shown as mean ± SEM for 9-15 oocytes. * indicates significant difference compared to the DmNav9-1 channel using one-way ANOVA with Scheffe’s post hoc analysis (p<0.05).
Figure 5. Effects of co-expression of TipE or TEH1 on entry into or recovery from fast inactivation of DmNav9-1 channels.(A) Time course of development of fast inactivation with a pre-pulse of -45 mV. (B) τ values of development of fast inactivation under different pre-pulse voltages. τ values were determined by fitting time course of the development of fast inactivation with a single exponential decay (n ≥ 12). (C) Recovery from fast inactivation with a repolarizing voltage of -70 mV. (D) τ values of recovery from fast inactivation at different repolarizing voltages. τ values were calculated by fitting recovery from fast inactivation data from different repolarizing voltages by a single exponential function (n≥ 10). Recording protocols are indicated and the details of the protocols and data analysis are described in the Materials and Methods.
Figure 6. Co-expression of TEH1 inhibits recovery from slow inactivation of DmNav9-1 channels.(A) Voltage-dependence of slow inactivation. (B) Time course of development of slow-inactivation. Development of slow inactivation was fitted by an exponential decay and the parameters are summarized in Table S1. (C) Time course of recovery from slow-inactivation. Recovery from slow inactivation was fitted by a double exponential function and the parameters are summarized in Table 2. Recording protocols are indicated and the details of the protocols and data analysis are described in the Materials and Methods.
Figure 1. Modulatory effects of TipE and TEH1 on peak sodium currents of DmNav9-1, DmNav22, or DmNav26 channels.(A) Representative traces of peak sodium currents from oocytes expressing DmNav9-1, DmNav9-1+TipE, and DmNav9-1+TEH1 sodium channels. Note that the sodium current of the DmNav9-1 channel possesses a non-inactivating component, known as persistent current (10% of the maximal transient peak current). Both TipE and TEH1 enhanced the persistent current. However, the persistent current remained to be about 10% of the maximal transient peak current. (B) Both TipE and TEH1 significantly increased peak sodium currents of all three Para sodium variants tested: DmNav9-1, DmNav26, and DmNav22, but there was no significant difference between the effects of TipE and TEH1. Sodium currents were recorded 48 hours after cRNA injection. Sodium currents were recorded by a step depolarization to from -80 to 65 mV in 5mV increments with a holding potential of -120 mV. Data are presented as means ± SEM for 12-15 oocytes. * indicates a significant difference compared to peak of DmNav channel only using one-way ANOVA with Scheffe’s post hoc analysis (p<0.05).
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