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. 2011 Dec 13;108(50):20213-8.
doi: 10.1073/pnas.1108497108. Epub 2011 Nov 28.

Palmitoylation influences the function and pharmacology of sodium channels

Frank Bosmans  1 , Mirela Milescu, Kenton J Swartz
Affiliations

Palmitoylation influences the function and pharmacology of sodium channels

Frank Bosmans et al. Proc Natl Acad Sci U S A. .

Abstract

Palmitoylation is a common lipid modification known to regulate the functional properties of various proteins and is a vital step in the biosynthesis of voltage-activated sodium (Nav) channels. We discovered a mutation in an intracellular loop of rNav1.2a (G1079C), which results in a higher apparent affinity for externally applied PaurTx3 and ProTx-II, two voltage sensor toxins isolated from tarantula venom. To explore whether palmitoylation of the introduced cysteine underlies this observation, we compared channel susceptibility to a range of animal toxins in the absence and presence of 2-Br-palmitate, a palmitate analog that prevents palmitate incorporation into proteins, and found that palmitoylation contributes to the increased affinity of PaurTx3 and ProTx-II for G1079C. Further investigations with 2-Br-palmitate revealed that palmitoylation can regulate the gating and pharmacology of wild-type (wt) rNav1.2a. To identify rNav1.2a palmitoylation sites contributing to these phenomena, we substituted three endogenous cysteines predicted to be palmitoylated and found that the gating behavior of this triple cysteine mutant is similar to wt rNav1.2a treated with 2-Br-palmitate. As with chemically depalmitoylated rNav1.2a channels, this mutant also exhibits an increased susceptibility for PaurTx3. Additional mutagenesis experiments showed that palmitoylation of one cysteine in particular (C1182) primarily influences PaurTx3 sensitivity and may enhance the inactivation process of wt rNav1.2a. Overall, our results demonstrate that lipid modifications are capable of altering the gating and pharmacological properties of rNav1.2a.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Gating characteristics and sensitivity of wt rNav1.2a and G1079C to PaurTx3. (A) Effect of 10 nM PaurTx3 on wt rNav1.2a and the mutant channel. Sodium currents are elicited by a depolarization to 0 mV before (black) and after (red) addition of PaurTx3 from a holding potential of −90 mV. (B) Apparent affinity for PaurTx3 interacting with wt rNav1.2a (black) and the mutant channel (red). Concentration dependence for toxin inhibition plotted as fraction unbound (Fu) is shown. Lines represent a fit with the Hill equation; n = 3–5 for each toxin concentration and error bars represent SEM. (C) Normalized conductance–voltage and steady-state inactivation relationships of the wt rNav1.2a channel (black) and the G1079C mutation (red). (D) Recovery from fast inactivation of the wt channel (black) and mutant (red) determined by a double pulse protocol to 0 mV with a varying time between pulses (0–50 ms). Values are reported in Tables S1 and S2. n = 12, and error bars represent SEM.
Fig. 2.
Fig. 2.
Effects of palmitoylation on the pharmacology and function of wt rNav1.2a and G1079C. (A) Apparent affinity for PaurTx3 interacting with wt rNav1.2a (black) and the G1079C mutant (green) after channel depalmitoylation. Concentration dependence for toxin inhibition plotted as Fu measured at negative voltages is shown. Data are compared with that shown in Fig. 1B, which is represented in this figure by a dotted line and symbols. Solid lines represent a fit with the Hill equation. n = 3–5 for each toxin concentration, and error bars represent SEM. (B) Apparent affinity for ProTx-II interacting with wt rNav1.2a (black) and the G1079C mutant (green) after channel depalmitoylation. Data are compared with rNav1.2a (gray) and G1079C (red) before addition of 2-Br-palmitate. (C) Comparison of Nav channel fast inactivation before (black) and after (green) channel depalmitoylation for rNav1.2a (Left) and G1079C (Right). Nav channels were depolarized to −20 mV from a holding potential of −90 mV. (D and E) Side-by-side comparison of the effects of 2-Br-palmitate on the gating properties of rNav1.2a (Left) and G1079C (Right). D shows deduced conductance–voltage and steady-state inactivation relationships before (black) and after (green) depalmitoylation. E shows recovery from fast inactivation before (black) and after (green) depalmitoylation determined by a double pulse protocol to 0 mV with a varying time between pulses (0–50 ms). n = 3, and error bars represent SEM. Values are reported in Tables S1 and S2.
Fig. 3.
Fig. 3.
Gating characteristics and PaurTx3 sensitivity of rNav1.2AAA. (A) Comparison of Nav channel fast inactivation for wt rNav1.2a (black) and rNav1.2AAA before (blue) or after (green) depalmitoylation with 2-Br-palmitate. Nav channels were depolarized to −20 mV from a holding potential of −90 mV. (B and C) Comparison of the gating properties of rNav1.2a (black) and rNav1.2AAA before (blue) or after (green) depalmitoylation. B shows deduced conductance–voltage and steady-state inactivation relationships; C shows recovery from fast inactivation as determined by a double-pulse protocol to −20 mV with a varying time between pulses (0–50 ms). n = 3, and error bars represent SEM. (D) Apparent affinity of PaurTx3 for rNav1.2a (black) and rNav1.2AAA before (blue), or after (green) depalmitoylation with 2-Br-palmitate. Concentration dependence for toxin inhibition plotted as Fu measured at negative voltages is shown. Solid lines represent a fit with the Hill equation. n = 3–5 for each toxin concentration, and error bars represent SEM. Values are reported in Tables S3 and S4.
Fig. 4.
Fig. 4.
Gating characteristics and PaurTx3 sensitivity of C1182A. (A and B) Comparison of the gating properties of rNav1.2a (black) and C1182A before (blue) or after (green) depalmitoylation with 2-Br-palmitate. A shows deduced conductance–voltage and steady-state inactivation relationships; B shows recovery from fast inactivation as determined by a double-pulse protocol to −20 mV with a varying time between pulses (0–50 ms). n = 3–5, and error bars represent SEM. (C) Apparent affinity of PaurTx3 for rNav1.2a (black) and C1182A before (blue) or after (green) depalmitoylation. (D) Apparent affinity of PaurTx3 for rNav1.2a (black), C650A (blue, open circles), and C1053A (blue, filled circles). n = 3–4 for each toxin concentration, and error bars represent SEM. Values are reported in Tables S3 and S4.
Fig. 5.
Fig. 5.
Effects of cholesterol on PaurTx3 interaction with wt rNav1.2a and G1079C. (A) Effects of cholesterol depletion on the gating properties of rNav1.2a; deduced conductance–voltage and steady-state inactivation relationships (Left) and recovery from fast inactivation (Right) before (black) and after (red) 5 mM methyl-β-cyclodextrin are shown. n = 3, and error bars represent SEM. (B and C) Apparent affinity of PaurTx3 interacting with rNav1.2a (B) and G1079C (C) before (black) and after (red) membrane cholesterol depletion. Solid line represents a fit with the Hill equation, and error bars represent SEM.
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