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PLoS One
2013 Dec 09;812:e81995. doi: 10.1371/journal.pone.0081995.
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Identification of Navβ1 residues involved in the modulation of the sodium channel Nav1.4.
Islas AA
,
Sánchez-Solano A
,
Scior T
,
Millan-PerezPeña L
,
Salinas-Stefanon EM
.
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Voltage-gated sodium channels (VGSCs) are heteromeric protein complexes that initiate action potentials in excitable cells. The voltage-gated sodium channel accessory subunit, Navβ1, allosterically modulates the α subunit pore structure upon binding. To date, the molecular determinants of the interface remain unknown. We made use of sequence, knowledge and structure-based methods to identify residues critical to the association of the α and β1 Nav1.4 subunits. The Navβ1 point mutant C43A disrupted the modulation of voltage dependence of activation and inactivation and delayed the peak current decay, the recovery from inactivation, and induced a use-dependent decay upon depolarisation at 1 Hz. The Navβ1 mutant R89A selectively delayed channel inactivation and recovery from inactivation and had no effect on voltage dependence or repetitive depolarisations. Navβ1 mutants Y32A and G33M selectively modified the half voltage of inactivation without altering the kinetics. Despite low sequence identity, highly conserved structural elements were identified. Our models were consistent with published data and may help relate pathologies associated with VGSCs to the Navβ1 subunit.
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24358138
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Figure 2. Representative current traces of Nav1.4 channel in the absence and presence of β1 and point mutants.Sodium currents were generated by step depolarisations from a holding potential of −100 mV in 10 mV increments from −100 mV to +50 mV (30 ms duration).
Figure 3. Effects of β1 mutants on Nav1.4 channel voltage-dependence of activation and inactivation.(A) Normalized conductance plotted as a function of the voltage potential of Nav1.4 channels expressed alone and with β1 and each mutant (inset). (B) Steady-state inactivation curves of the same groups. Data were expressed as mean ± SEM (n = 5–9). Data were fitted with a Boltzman equation (protocols described in methods).
Figure 4. Effects of β1 and mutants on Nav1.4 channel recovery from inactivation.(A) The current amplitude at the second pulse was normalized to the current amplitude at the pre-pulse and plotted as a function of the variable time interval (Δt), currents were evoked according to the protocol in the inset. (B) Representative traces of currents evoked by a depolarizing step of −20 mV (peak current activation). The trace in bold was obtained from WT channels (Nav1.4+β1) and the dashed trace represented the Nav1.4 α channel alone. (C) Effects of β1 and mutants on Nav1.4 after repetitive depolarisations (30 ms duration) from a holding potential of −100 mV to −20 mV at a frequency of 1 Hz. Data were expressed as mean ± SE (n = 4 to 9 cells).
Figure 5. Effects of β1 single residue substitutions on the electrostatic surface potential (ESP).(A) ESP of the extracellular domain of WT β1 (left) and the R89A mutant (right). The point mutation increased the protein’s negative charge by the loss of a salt bridge between Arg89 and Glu87. The acidic carboxylic side chain was now exposed and reactive. (B) ESP of WT (left) and the C43A mutant (right). This substitution prevented the formation of an intermolecular disulphide bond between C43 and C21, increasing entropy. Calculations were made under the AMBER-Gasteiger force field in Chimera 1.3.5 after energy minimization under AMMP in VEGA ZZ 2.3.2.
Figure 1. Atomic homology model of the extracellular domain of the Navβ1 subunit.(A) The functionally relevant residues identified are shown in magenta. Cysteine 43 was predicted to form a disulphide bond with cysteine 21 and arginine 89 may form a salt bridge with glutamate 87. The buried disulphide bridge between C40 and C121 is also shown. (B) The model of β1 (cyan) was superimposed over the template structure, myelin protein P0 (gray), and 3 Ig-like proteins including NTRK2_HUMAN, TRGC2_HUMAN, and a camelid VHH antibody (PDB codes: 1neu_A, 1wwb_X, 1hxm_B, 1kxq_H). Despite their divergent functions, the general topologies of the crystal structures were conserved. The conserved basic residues at the position equivalent to Arg89 are displayed as atom sticks (colour code: β1 model in cyan, 1wwb_X in marine blue, 1hxm_B in magenta, and 1kxq_H in yellow).
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