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Channels (Austin)
2021 Dec 01;151:79-93. doi: 10.1080/19336950.2020.1860399.
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Functional modulation of the human voltage-gated sodium channel NaV1.8 by auxiliary β subunits.
Nevin ST
,
Lawrence N
,
Nicke A
,
Lewis RJ
,
Adams DJ
.
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The voltage-gated sodium channel Nav1.8 mediates the tetrodotoxin-resistant (TTX-R) Na+ current in nociceptive primary sensory neurons, which has an important role in the transmission of painful stimuli. Here, we describe the functional modulation of the human Nav1.8 α-subunit in Xenopus oocytes by auxiliary β subunits. We found that the β3 subunit down-regulated the maximal Na+ current amplitude and decelerated recovery from inactivation of hNav1.8, whereas the β1 and β2 subunits had no such effects. The specific regulation of Nav1.8 by the β3 subunit constitutes a potential novel regulatory mechanism of the TTX-R Na+ current in primary sensory neurons with potential implications in chronic pain states. In particular, neuropathic pain states are characterized by a down-regulation of Nav1.8 accompanied by increased expression of the β3 subunit. Our results suggest that these two phenomena may be correlated, and that increased levels of the β3 subunit may directly contribute to the down-regulation of Nav1.8. To determine which domain of the β3 subunit is responsible for the specific regulation of hNav1.8, we created chimeras of the β1 and β3 subunits and co-expressed them with the hNav1.8 α-subunit in Xenopus oocytes. The intracellular domain of the β3 subunit was shown to be responsible for the down-regulation of maximal Nav1.8 current amplitudes. In contrast, the extracellular domain mediated the effect of the β3 subunit on hNav1.8 recovery kinetics.
Figure 1. Expression of human Nav1.8 and rat Nav1.2 in Xenopus oocytes. (a) When expressed in Xenopus oocytes, hNav1.8 mediates an inward Na+ current with slow activation and inactivation kinetics that is unaffected by 1 μM tetrodotoxin (TTX). (b) In contrast, rat Nav1.2 mediates a Na+ current exhibiting fast activation and inactivation kinetics that is completely abolished by the application of 1 μM TTX. Oocytes were held at – 70 mV and depolarized to voltages between – 50 and +40 mV in 10 mV increments. External solutions containing TTX (1 µM) were applied through the perfusion system
Figure 2. Biochemical analysis of the synthesis and plasma membrane transport of the sodium channel β3 subunit in Xenopus laevis oocytes. Oocytes injected with cRNA encoding the His-tagged β3 subunit or non-injected controls (c) were metabolically labeled with [35S]-methionine (left panel) or surface-iodinated with [125I]-sulfo-SHPP (right panel). His-tagged protein was purified via Ni2+-NTA-agarose, treated with endoglycosidases (concentrations given in IUB milliunits/ml sample) as indicated, and separated on a 10% SDS-PAGE gel. Black and white triangles indicate complex glycosylated and completely deglycosylated protein, respectively. Numbers 1–4 indicate the Endo H-sensitive core-glycosylated and partly deglycosylated forms of the protein
Figure 3. Modulation of hNav1.8 by auxiliary β subunits. (a) Effects of β subunits on Na+ current amplitude. I represents maximal Na+ current amplitude of oocytes expressing hNav1.8 (2 ng cRNA/oocyte) alone or in combination with β1, β2 or β3 (5 ng cRNA/oocyte). I(average control) represents the average maximal Na+ current amplitude of oocytes expressing only hNav1.8. Maximal Na+ current amplitude was determined by step depolarizations to voltages between – 50 and +50 mV (5 mV increments) from a holding potential of – 70 mV. The voltages at which maximal Na+ current amplitude was obtained was +5 mV for hNav1.8 + β1 and +10 mV for the other combinations (including Nav1.8 in the absence of β subunits). Curves show the Na+ conductance (G) obtained at different voltages relative to the maximal conductance (Gmax). Conductance curves were fitted with single exponential functions for the hNav1.8 α subunit alone and in the presence of the various β subunits. (c) Voltage-dependence of inactivation. I represents the Na+ current elicited by a depolarizing pulse to the voltage generating maximal Na+ current amplitude immediately after long (1 s) pre-pulses to different voltages. I–120 represents the Na+ current amplitude elicited by an identical depolarizing pulse generated after a long pre-pulse to – 120 mV, where inactivation is minimal. I/I−120 represents the fraction of maximal Na+ current available after steady-state inactivation at each voltage. (d) Inactivation kinetics. Superimposed traces normalized to the same value are shown for Na+ currents mediated by hNav1.8 in the absence and presence of the β1, β2 and β3 subunit (5 ng cRNA/oocyte). Oocytes were held at – 70 mV and depolarized to the voltage that elicited maximal Na+ current amplitude. (e) Recovery from inactivation. The fraction of Na+ current recovering from steady-state inactivation after different periods of time (2.5 ms – 1 s) was determined for hNav1.8 (2.5 ng cRNA/oocyte) expressed alone or together with the β1, β2 or β3 subunit (5 ng cRNA/oocyte). Na+ current was first inactivated by a 1 s pulse to 0 mV. After a variable recovery period ranging from 2.5 ms – 1 s, a depolarizing pulse to elicit maximal Na+ current amplitude was applied. The Na+ current amplitude after different recovery times (I) was compared to the Na+ current amplitude elicited by an identical control pulse that was not preceded by inactivation (Imax). The recovered fraction of Na+ current (I/Imax) was plotted against recovery time and fitted with double exponential functions
Figure 4. Effects of the α:β3 ratio on β3-mediated modulation of the hNav1.8 current amplitude and recovery from inactivation. (a) Effects of the α:β3 ratio on current amplitude. Maximal Na+ current amplitude was recorded from oocytes injected with cRNA for hNav1.8 (2.5 ng/oocyte) alone or together with 0.5, 1, or 5 ng of cRNA encoding the β3-subunit. I represents maximal Na+ current amplitude in the various groups while I(average control) represents the average maximal Na+ current amplitude of control (oocytes expressing only hNav1.8). N = 30–41 oocytes/group. (b) Effects of the α:β3 ratio on the repriming kinetics of hNav1.8. Recovery from inactivation was determined as described for hNav1.8 alone. The Na+ current amplitude after different recovery times (i) was compared to the Na+ current amplitude generated by an identical control pulse (Imax). The repriming curves were fitted with double exponential functions (N ≥ 10 oocytes/group)
Figure 5. Comparison of modulation of hNav1.8 by the rat and human β3 subunits. (a). Effects on Na+ current amplitude. Maximal Na+ current amplitude was determined for oocytes expressing hNav1.8 alone or in combination with the rat or human β3-subunit. I represents the maximal Na+ current amplitude of oocytes expressing hNav1.8 alone or in combination with the β3 subunit. I(average control) represents the average maximal Na+ current amplitude of oocytes expressing only hNav1.8. ****significantly different from control, p ≤ 0.0001. (b) Comparison of the modulation of recovery from inactivation of hNav1.8 by the rat and human β3 subunits (N = 15–23 oocytes/group)
Figure 6. Effects of β subunits chimeras on maximal current amplitude and recovery from inactivation of hNav1.8. (a). Schematic showing the structure of the wild-type β1 and β3 subunits and the constructed chimeras (β1: white, β3: black). (b) Effects on Na+ current amplitude. Maximal Na+ current amplitude was determined for oocytes expressing hNav1.8 alone or in combination with the rat β3 chimera subunits. I represents maximal Na+ current amplitude of oocytes expressing hNav1.8 alone or in combination with the β3 chimera subunits
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