Brenes O et al. (2021), Functional and Structural Characterization of C...

XB-ART-57891

Cells 2021 Feb 11;102:. doi: 10.3390/cells10020374.

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Functional and Structural Characterization of ClC-1 and Nav1.4 Channels Resulting from CLCN1 and SCN4A Mutations Identified Alone and Coexisting in Myotonic Patients.

Brenes O , Barbieri R , Vásquez M , Vindas-Smith R , Roig J , Romero A , Valle GD , Bermúdez-Guzmán L , Bertelli S , Pusch M , Morales F .

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Non-dystrophic myotonias have been linked to loss-of-function mutations in the ClC-1 chloride channel or gain-of-function mutations in the Nav1.4 sodium channel. Here, we describe a family with members diagnosed with Thomsen's disease. One novel mutation (p.W322*) in CLCN1 and one undescribed mutation (p.R1463H) in SCN4A are segregating in this family. The CLCN1-p.W322* was also found in an unrelated family, in compound heterozygosity with the known CLCN1-p.G355R mutation. One reported mutation, SCN4A-p.T1313M, was found in a third family. Both CLCN1 mutations exhibited loss-of-function: CLCN1-p.W322* probably leads to a non-viable truncated protein; for CLCN1-p.G355R, we predict structural damage, triggering important steric clashes. The SCN4A-p.R1463H produced a positive shift in the steady-state inactivation increasing window currents and a faster recovery from inactivation. These gain-of-function effects are probably due to a disruption of interaction R1463-D1356, which destabilizes the voltage sensor domain (VSD) IV and increases the flexibility of the S4-S5 linker. Finally, modelling suggested that the p.T1313M induces a strong decrease in protein flexibility on the III-IV linker. This study demonstrates that CLCN1-p.W322* and SCN4A-p.R1463H mutations can act alone or in combination as inducers of myotonia. Their co-segregation highlights the necessity for carrying out deep genetic analysis to provide accurate genetic counseling and management of patients.

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Genes referenced: clcn1 nav1 scn4a
GO keywords: chloride channel activity

???displayArticle.disOnts??? myotonia congenita [+]

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	Figure 1. Pedigrees of the three families segregating CLCN1 and SCN4A mutations. (Family 1) Clinical information for this family has been published already, including detailed information on which individuals were clinically evaluated and that resulted affected [13]. Coded (NDM1-15) family members were studied clinically and/or genetically. Circles represent females and squares represent males. Individuals marked with a + (upper right of the symbol) indicates that those individuals had been previously clinically evaluated [13]. Clinical and genetic information is indicated below each symbol. EMG+ indicates positive for myotonia in the electromyographic test. Genotype for each individual tested for the CLCN1 and SCN4A mutations is also indicated below the symbol. For CLCN1, genotypes GA/AA indicate that the individual carries the W322* mutation, while genotype GG indicates that the individual is W322-free. For SCN4A, genotype GA indicates that the individual carries the R1463H mutation, while genotype GG indicates that the individual is R1463H-free. The figure also shows the code to determine which patients carry both or just one of the mutations. Clinical and genetic information for the uncoded family members is unavailable. (Family 2) Individual NDM16 resulted carrier of two CLCN1 mutations. Coded (NDM16) family member was studied clinically and/or genetically. Filled symbol indicates that the individual is symptomatic. Genotype for NDM16 for both mutations is indicated below the symbol. Genotype GA/GA indicates that the NDM16 is carrier of W322 and G355R mutations, respectively. Clinical and genetic information for the uncoded family members is unavailable. (Family 3) In this family, two individuals are affected (filled symbols). The genotype for NDM17 for the SCN4A mutation is indicated below the symbol. Genotype GT indicates that the NDM17 is carrier of T1313M mutations. Clinical and genetic information for the uncoded family members is unavailable.
	Figure 2. G355R mutant channels characterization. (A) Sample traces of macroscopic currents recorded in oocytes injected with the vehicle (water) or expressing wild-type (WT), homozygous (G355R) or heterozygous (WT/G355R) channels. (B) I-V relationship of the instantaneous current observed in the oocytes, the maximal currents from Boltzmann fitting are showed in the bottom panel. (C) Right panel shows the pore path prediction in MOLEonline for ClC-1; left panel shows the dimeric channel reconstruction with both pores highlighted. (D) Amino acid interactions on WT (left panel) and predicted structural changes resulting from the G355R mutation (right panel); dotted lines represent amino acid bonds in or between alpha-helix structures. (E) Change of vibrational entropy upon mutation, where blue represents a rigidification of the structure and red a gain in flexibility. Values indicates mean Â± s.e.m. * p < 0.05, *** p < 0.001 in G355R with respect to WT.
	Figure 3. W322* mutant channels currents. (A) Sample traces of macroscopic currents recorded in oocytes injected with water or expressing WT, homozygous (W322) or heterozygous (WT/W322) channels. (B) I-V relationship of the instantaneous current observed in the oocytes, the maximal currents from Boltzmann fitting are showed in the bottom panel. (C) Sample recordings of the macroscopic currents recorded in oocytes injected with WT or with both mutants (G355R and W322) in a 1:1 ratio and I-V relationship of the instantaneous current observed in these oocytes. Values indicates mean Â± s.e.m. ** p < 0.001 in W322* or G355R/W322* with respect to WT.
	Figure 4. R1463H mutant channels currents. (A) Sample traces of macroscopic currents recorded in oocytes injected with the WT or with the mutant channel R1463H at âˆ’20 mV. (B) I-V relationship of the instantaneous current observed in the oocytes, maximal current and the voltage of half activation (V1/2) obtained by Boltzmann equation fitting are shown as inserts. (C) Sample traces at âˆ’20 mV for the steady-state inactivation after voltage steps from âˆ’60 to âˆ’30 mV. (D) Normalized I-V relationship for steady-state activation (circles) and inactivation (squares), the overlapping gray region of the curves represents the window current of the WT (dark gray) and R1463H (light gray). (E) Left panel shows sample traces of the currents developed during the protocol of recovery from inactivation in oocytes injected with the WT or with the R1463H channel, central panel shows the time course of the recovery of inactive channels, and the time constant (tau) of the recovery is shown in the right panel. Value indicates mean Â± s.e.m. * p < 0.05, p < 0.01, * p < 0.001 in R1463H with respect to WT.
	Figure 5. Structural changes predicted for mutations R1463H and T1313M. (A) Right panel shows the pore path prediction for Nav1.4 channel, the pore length was 85.6 Ã… with a radius <9 Ã…, and pore lining residues are negative as expected for a cation channel; left panel shows the channel reconstruction with pore highlighted. (B) Amino acid interactions on WT (left panel) and structural changes derived from R1463H mutation (right panel), where dotted lines represent amino acid bonds in or between structures. (C) Changes of vibrational entropy upon mutation, where blue represents a rigidification and red a gain in flexibility of the structure. (D) Amino acid interactions on WT (left panel) and structural changes predicted for T1313M (right panel). (E) Changes of vibrational entropy upon mutation, where blue represents a rigidification and red a gain in flexibility of the structure.

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	Figure 1. Pedigrees of the three families segregating CLCN1 and SCN4A mutations. (Family 1) Clinical information for this family has been published already, including detailed information on which individuals were clinically evaluated and that resulted affected [13]. Coded (NDM1-15) family members were studied clinically and/or genetically. Circles represent females and squares represent males. Individuals marked with a + (upper right of the symbol) indicates that those individuals had been previously clinically evaluated [13]. Clinical and genetic information is indicated below each symbol. EMG+ indicates positive for myotonia in the electromyographic test. Genotype for each individual tested for the CLCN1 and SCN4A mutations is also indicated below the symbol. For CLCN1, genotypes GA/AA indicate that the individual carries the W322* mutation, while genotype GG indicates that the individual is W322-free. For SCN4A, genotype GA indicates that the individual carries the R1463H mutation, while genotype GG indicates that the individual is R1463H-free. The figure also shows the code to determine which patients carry both or just one of the mutations. Clinical and genetic information for the uncoded family members is unavailable. (Family 2) Individual NDM16 resulted carrier of two CLCN1 mutations. Coded (NDM16) family member was studied clinically and/or genetically. Filled symbol indicates that the individual is symptomatic. Genotype for NDM16 for both mutations is indicated below the symbol. Genotype GA/GA indicates that the NDM16 is carrier of W322 and G355R mutations, respectively. Clinical and genetic information for the uncoded family members is unavailable. (Family 3) In this family, two individuals are affected (filled symbols). The genotype for NDM17 for the SCN4A mutation is indicated below the symbol. Genotype GT indicates that the NDM17 is carrier of T1313M mutations. Clinical and genetic information for the uncoded family members is unavailable.
	Figure 2. G355R mutant channels characterization. (A) Sample traces of macroscopic currents recorded in oocytes injected with the vehicle (water) or expressing wild-type (WT), homozygous (G355R) or heterozygous (WT/G355R) channels. (B) I-V relationship of the instantaneous current observed in the oocytes, the maximal currents from Boltzmann fitting are showed in the bottom panel. (C) Right panel shows the pore path prediction in MOLEonline for ClC-1; left panel shows the dimeric channel reconstruction with both pores highlighted. (D) Amino acid interactions on WT (left panel) and predicted structural changes resulting from the G355R mutation (right panel); dotted lines represent amino acid bonds in or between alpha-helix structures. (E) Change of vibrational entropy upon mutation, where blue represents a rigidification of the structure and red a gain in flexibility. Values indicates mean Â± s.e.m. * p < 0.05, *** p < 0.001 in G355R with respect to WT.
	Figure 3. W322* mutant channels currents. (A) Sample traces of macroscopic currents recorded in oocytes injected with water or expressing WT, homozygous (W322) or heterozygous (WT/W322) channels. (B) I-V relationship of the instantaneous current observed in the oocytes, the maximal currents from Boltzmann fitting are showed in the bottom panel. (C) Sample recordings of the macroscopic currents recorded in oocytes injected with WT or with both mutants (G355R and W322) in a 1:1 ratio and I-V relationship of the instantaneous current observed in these oocytes. Values indicates mean Â± s.e.m. ** p < 0.001 in W322* or G355R/W322* with respect to WT.
	Figure 4. R1463H mutant channels currents. (A) Sample traces of macroscopic currents recorded in oocytes injected with the WT or with the mutant channel R1463H at âˆ’20 mV. (B) I-V relationship of the instantaneous current observed in the oocytes, maximal current and the voltage of half activation (V1/2) obtained by Boltzmann equation fitting are shown as inserts. (C) Sample traces at âˆ’20 mV for the steady-state inactivation after voltage steps from âˆ’60 to âˆ’30 mV. (D) Normalized I-V relationship for steady-state activation (circles) and inactivation (squares), the overlapping gray region of the curves represents the window current of the WT (dark gray) and R1463H (light gray). (E) Left panel shows sample traces of the currents developed during the protocol of recovery from inactivation in oocytes injected with the WT or with the R1463H channel, central panel shows the time course of the recovery of inactive channels, and the time constant (tau) of the recovery is shown in the right panel. Value indicates mean Â± s.e.m. * p < 0.05, p < 0.01, * p < 0.001 in R1463H with respect to WT.
	Figure 5. Structural changes predicted for mutations R1463H and T1313M. (A) Right panel shows the pore path prediction for Nav1.4 channel, the pore length was 85.6 Ã… with a radius <9 Ã…, and pore lining residues are negative as expected for a cation channel; left panel shows the channel reconstruction with pore highlighted. (B) Amino acid interactions on WT (left panel) and structural changes derived from R1463H mutation (right panel), where dotted lines represent amino acid bonds in or between structures. (C) Changes of vibrational entropy upon mutation, where blue represents a rigidification and red a gain in flexibility of the structure. (D) Amino acid interactions on WT (left panel) and structural changes predicted for T1313M (right panel). (E) Changes of vibrational entropy upon mutation, where blue represents a rigidification and red a gain in flexibility of the structure.