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Predicting a double mutant in the twilight zone of low homology modeling for the skeletal muscle voltage-gated sodium channel subunit beta-1 (Nav1.4 β1).

Scior T, Paiz-Candia B, Islas ÁA, Sánchez-Solano A, Millan-Perez Peña L, Mancilla-Simbro C, Salinas-Stefanon EM - Comput Struct Biotechnol J (2015)

Bottom Line: Despite the distant phylogenic relationships, we found a 3D-template to identify two adjacent amino acids leading to the long-awaited loss of function (inactivation) of Nav1.4 channels.Exhaustive and unbiased sampling of "all β proteins" (Ig-like, Ig) resulted in a plethora of 3D templates which were compared to the target secondary structure prediction.The location of TANA was made possible thanks to another "all β protein" structure in complex with an irreversible bound protein as well as a reversible protein-protein interface (our "Rosetta Stone" effect).

View Article: PubMed Central - PubMed

Affiliation: Facultad de Ciencias Químicas, Universidad Autónoma de Puebla, Puebla, Mexico.

ABSTRACT
The molecular structure modeling of the β1 subunit of the skeletal muscle voltage-gated sodium channel (Nav1.4) was carried out in the twilight zone of very low homology. Structural significance can per se be confounded with random sequence similarities. Hence, we combined (i) not automated computational modeling of weakly homologous 3D templates, some with interfaces to analogous structures to the pore-bearing Nav1.4 α subunit with (ii) site-directed mutagenesis (SDM), as well as (iii) electrophysiological experiments to study the structure and function of the β1 subunit. Despite the distant phylogenic relationships, we found a 3D-template to identify two adjacent amino acids leading to the long-awaited loss of function (inactivation) of Nav1.4 channels. This mutant type (T109A, N110A, herein called TANA) was expressed and tested on cells of hamster ovary (CHO). The present electrophysiological results showed that the double alanine substitution TANA disrupted channel inactivation as if the β1 subunit would not be in complex with the α subunit. Exhaustive and unbiased sampling of "all β proteins" (Ig-like, Ig) resulted in a plethora of 3D templates which were compared to the target secondary structure prediction. The location of TANA was made possible thanks to another "all β protein" structure in complex with an irreversible bound protein as well as a reversible protein-protein interface (our "Rosetta Stone" effect). This finding coincides with our electrophysiological data (disrupted β1-like voltage dependence) and it is safe to utter that the Nav1.4 α/β1 interface is likely to be of reversible nature.

No MeSH data available.


Related in: MedlinePlus

Representative current traces of the Nav1.4 sodium channel. Its α subunit was co-transfected with wild type (WT) β1 subunit (blue) or the double mutation TANA (red). Leftmost panel (black), the electrophysiological effects of a ß1 subunit mutation on the I-V relationship of Nav 1.4. Sodium currents were generated by step depolarizations from a holding potential of − 100 mV in 10 mV increments from − 100 mV to + 50 mV (30 ms duration) in several mammalian CHO cells transfected with either WT α together with WT ß1 subunits as control or WT α subunit with TANA (red traces). Calibrations as shown, n = 6. For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.
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f0050: Representative current traces of the Nav1.4 sodium channel. Its α subunit was co-transfected with wild type (WT) β1 subunit (blue) or the double mutation TANA (red). Leftmost panel (black), the electrophysiological effects of a ß1 subunit mutation on the I-V relationship of Nav 1.4. Sodium currents were generated by step depolarizations from a holding potential of − 100 mV in 10 mV increments from − 100 mV to + 50 mV (30 ms duration) in several mammalian CHO cells transfected with either WT α together with WT ß1 subunits as control or WT α subunit with TANA (red traces). Calibrations as shown, n = 6. For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.

Mentions: Voltage-gated Na+ channels formed by α and β subunits, characteristically display gating kinetics on millisecond time scales, ensuring rapid electrical communication between cells [2]. Navβ1 subunits interact non-covalently with pore-forming α subunits in the extracellular space, which accelerates gating kinetics, and modifies voltage dependence [2,4,8,87,88]. In this part of our study we observed that TANA disrupted inactivation, delayed recovery from inactivation and disrupted β1-like voltage-dependence (Fig. 10). TANA had neither an effect on the current–voltage (I–V) curve nor in the total amplitude of the current demonstrating that TANA did not change the peak voltage of activation (cf. leftmost inlay chart in Fig. 10). The observed loss of function can be classified as a general loss of function type because TANA had a double effect on: (i) kinetics of recovery from inactivation and (ii) high frequency stimulation. The general loss was about 80% by looking at the maximum difference between wild type (WT) and mutant type (MT) TANA (Fig. 11).


Predicting a double mutant in the twilight zone of low homology modeling for the skeletal muscle voltage-gated sodium channel subunit beta-1 (Nav1.4 β1).

Scior T, Paiz-Candia B, Islas ÁA, Sánchez-Solano A, Millan-Perez Peña L, Mancilla-Simbro C, Salinas-Stefanon EM - Comput Struct Biotechnol J (2015)

Representative current traces of the Nav1.4 sodium channel. Its α subunit was co-transfected with wild type (WT) β1 subunit (blue) or the double mutation TANA (red). Leftmost panel (black), the electrophysiological effects of a ß1 subunit mutation on the I-V relationship of Nav 1.4. Sodium currents were generated by step depolarizations from a holding potential of − 100 mV in 10 mV increments from − 100 mV to + 50 mV (30 ms duration) in several mammalian CHO cells transfected with either WT α together with WT ß1 subunits as control or WT α subunit with TANA (red traces). Calibrations as shown, n = 6. For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.
© Copyright Policy - CC BY
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4402383&req=5

f0050: Representative current traces of the Nav1.4 sodium channel. Its α subunit was co-transfected with wild type (WT) β1 subunit (blue) or the double mutation TANA (red). Leftmost panel (black), the electrophysiological effects of a ß1 subunit mutation on the I-V relationship of Nav 1.4. Sodium currents were generated by step depolarizations from a holding potential of − 100 mV in 10 mV increments from − 100 mV to + 50 mV (30 ms duration) in several mammalian CHO cells transfected with either WT α together with WT ß1 subunits as control or WT α subunit with TANA (red traces). Calibrations as shown, n = 6. For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.
Mentions: Voltage-gated Na+ channels formed by α and β subunits, characteristically display gating kinetics on millisecond time scales, ensuring rapid electrical communication between cells [2]. Navβ1 subunits interact non-covalently with pore-forming α subunits in the extracellular space, which accelerates gating kinetics, and modifies voltage dependence [2,4,8,87,88]. In this part of our study we observed that TANA disrupted inactivation, delayed recovery from inactivation and disrupted β1-like voltage-dependence (Fig. 10). TANA had neither an effect on the current–voltage (I–V) curve nor in the total amplitude of the current demonstrating that TANA did not change the peak voltage of activation (cf. leftmost inlay chart in Fig. 10). The observed loss of function can be classified as a general loss of function type because TANA had a double effect on: (i) kinetics of recovery from inactivation and (ii) high frequency stimulation. The general loss was about 80% by looking at the maximum difference between wild type (WT) and mutant type (MT) TANA (Fig. 11).

Bottom Line: Despite the distant phylogenic relationships, we found a 3D-template to identify two adjacent amino acids leading to the long-awaited loss of function (inactivation) of Nav1.4 channels.Exhaustive and unbiased sampling of "all β proteins" (Ig-like, Ig) resulted in a plethora of 3D templates which were compared to the target secondary structure prediction.The location of TANA was made possible thanks to another "all β protein" structure in complex with an irreversible bound protein as well as a reversible protein-protein interface (our "Rosetta Stone" effect).

View Article: PubMed Central - PubMed

Affiliation: Facultad de Ciencias Químicas, Universidad Autónoma de Puebla, Puebla, Mexico.

ABSTRACT
The molecular structure modeling of the β1 subunit of the skeletal muscle voltage-gated sodium channel (Nav1.4) was carried out in the twilight zone of very low homology. Structural significance can per se be confounded with random sequence similarities. Hence, we combined (i) not automated computational modeling of weakly homologous 3D templates, some with interfaces to analogous structures to the pore-bearing Nav1.4 α subunit with (ii) site-directed mutagenesis (SDM), as well as (iii) electrophysiological experiments to study the structure and function of the β1 subunit. Despite the distant phylogenic relationships, we found a 3D-template to identify two adjacent amino acids leading to the long-awaited loss of function (inactivation) of Nav1.4 channels. This mutant type (T109A, N110A, herein called TANA) was expressed and tested on cells of hamster ovary (CHO). The present electrophysiological results showed that the double alanine substitution TANA disrupted channel inactivation as if the β1 subunit would not be in complex with the α subunit. Exhaustive and unbiased sampling of "all β proteins" (Ig-like, Ig) resulted in a plethora of 3D templates which were compared to the target secondary structure prediction. The location of TANA was made possible thanks to another "all β protein" structure in complex with an irreversible bound protein as well as a reversible protein-protein interface (our "Rosetta Stone" effect). This finding coincides with our electrophysiological data (disrupted β1-like voltage dependence) and it is safe to utter that the Nav1.4 α/β1 interface is likely to be of reversible nature.

No MeSH data available.


Related in: MedlinePlus