A gain-of-function sodium channel β2-subunit mutation in painful diabetic neuropathy

Abstract

Diabetes mellitus is a global challenge with many diverse health sequelae, of which diabetic peripheral neuropathy is one of the most common. A substantial number of patients with diabetic peripheral neuropathy develop chronic pain, but the genetic and epigenetic factors that predispose diabetic peripheral neuropathy patients to develop neuropathic pain are poorly understood. Recent targeted genetic studies have identified mutations in α-subunits of voltage-gated sodium channels (Na_vs) in patients with painful diabetic peripheral neuropathy. Mutations in proteins that regulate trafficking or functional properties of Na_vs could expand the spectrum of patients with Na_v-related peripheral neuropathies. The auxiliary sodium channel β-subunits (β1-4) have been reported to increase current density, alter inactivation kinetics, and modulate subcellular localization of Na_v. Mutations in β-subunits have been associated with several diseases, including epilepsy, cancer, and diseases of the cardiac conducting system. However, mutations in β-subunits have never been shown previously to contribute to neuropathic pain. We report here a patient with painful diabetic peripheral neuropathy and negative genetic screening for mutations in SCN9A, SCN10A, and SCN11A-genes encoding sodium channel α-subunit that have been previously linked to the development of neuropathic pain. Genetic analysis revealed an aspartic acid to asparagine mutation, D109N, in the β2-subunit. Functional analysis using current-clamp revealed that the β2-D109N rendered dorsal root ganglion neurons hyperexcitable, especially in response to repetitive stimulation. Underlying the hyperexcitability induced by the β2-subunit mutation, as evidenced by voltage-clamp analysis, we found a depolarizing shift in the voltage dependence of Na_v1.7 fast inactivation and reduced use-dependent inhibition of the Na_v1.7 channel.

Keywords: Diabetic neuropathy; neuropathic pain; sodium channel beta-subunits; voltage-gated sodium channels.

Figure 1.

Structure of the β2-subunit extracellular domain illustrates the location of the D109N mutation with respect to Cys. (a) PyMol model of the human β2-subunit with the D109 residue (red) shown near the C55 residue (cyan) responsible for disulfide bridge formation to the Na_v1.2 channel. (b) Surface maps illustrate a common surface between the D109 residue (red) and the C55 residue (cyan).

Figure 2.

The β2 D109N variant does not alter passive membrane properties or action potential characteristics of DRG neurons. (a) Sample action potential waveforms representative of collected current-clamp recordings from either wild-type (n = 17) or D109N expressing DRG neurons (n = 21). (b) Comparison of total action potential amplitude for DRG neurons expressing either wild-type (113.28 ± 2.48 mV) or D109N variant (112.58 ± 2.85 mV, p = 0.85) β2-subunits. (c) Comparison of maximum slope during rising phase of action potential between DRG neurons expressing either wild-type (97.06 ± 10.21 mV/ms) or D109N variant (84.71 ± 10.22, p = 0.39). (d) Comparison of resting membrane potential of DRG neurons overexpressing either wild-type (−56.8 ± 1.8 mV) or D109N variant (−53.7 ± 1.7 mV, p = 0.12) subunit. (e) Comparison of current injection threshold for action potential spiking between DRG neurons expressing either wild-type (278.23 ± 60.40 pA) or D109N variant (213.57 ± 45.29 pA, p = 0.39) β2-subunit.

Figure 3.

β2 D109N confers hyperexcitability to DRG neurons. (a) Comparison of repetitive action potential firing between DRG neurons expressing wild-type (n = 17) and D109N (n = 21) β2-subunits across a range of 500 ms current injections from 25 to 500 pA. Overexpression of β2-subunits with the D109N mutation permits continuous spiking of rat DRG neurons even in the presence of high-current stimuli, whereas DRG expressing wild-type β2-subunits adapt and cease firing. (b) The D109N mutation in the β2-subunit does not increase the proportion of cells that fire repetitively. Approximately 52% of neurons recorded in either condition fired multiple action potentials. (c and d) Sample traces at approximately threshold, 5× threshold, and 10× threshold for DRG cells expressing wild-type β2 (c, black) and mutant β2-subunits (d, red).

Figure 4.

Voltage-clamp analysis of D109N variant. (a) Comparison of use-dependent inhibition of total sodium current after 20 Hz stimulation in DRG neurons expressing either wild-type (I/I_max at 1.5 s = 59.1%, n = 12) or D109N (I/I_max at 1.5 s = 67.9%, n = 14) β2-subunit. (b) Comparison of use-dependent inhibition of TTX-R sodium channels in DRG neurons expressing either wild-type (n = 8) or D109N variant (n = 9) showing no difference at 20 Hz stimulation. (c) Comparison of voltage dependence of activation in wild type (−20.1 ± 3.6 mV, n = 9) and D109N (−17.5 ± 4.3 mV, n = 9, p = 0.65). (d) Comparison of voltage dependence of fast inactivation in DRG neurons expressing either wild-type (−77.4 ± 2.7 mV, n = 8) or D109N variant (−67.8 ± 2.0 mV, n = 7).

Figure 5.

Voltage-clamp analysis of Na_v1.7 in the presence of the β2 D109N-subunit. (a) Comparison of voltage dependence of fast inactivation of Na_v1.7 in the presence of the D109N variant (−76.0 ± 2.2 mV, n = 8) and the wild-type β2 (−82.9 ± 2.3 mV, n = 9, p = 0.043). (b) The presence of the D109N variant (n = 6) results in a 9% reduction in use-dependent inhibition of Na_v1.7 compared to wild-type β2 (n = 6) after 1.5 s of 20 Hz stimulation.