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Shetuan Zhang - One of the best experts on this subject based on the ideXlab platform.
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Kv1.5 Channels are regulated by PKC-mediated endocytic degradation.
The Journal of biological chemistry, 2021Co-Authors: Tingzhong Wang, Jun Guo, Tonghua Yang, Mark Szendrey, Shetuan ZhangAbstract:The voltage-gated Potassium Channel Kv1.5 plays important roles in the repolarization of atrial action potentials and regulation of the vascular tone. While the modulation of Kv1.5 function has been well studied, less is known about how the protein levels of Kv1.5 on the cell membrane are regulated. Here, through electrophysiological and biochemical analyses of Kv1.5 Channels heterologously expressed in HEK293 cells and neonatal rat ventricular myocytes, as well as native Kv1.5 in human induced pluripotent stem cell (iPSC)-derived atrial cardiomyocytes, we found that activation of protein kinase C (PKC) with phorbol 12-myristate 13-acetate (PMA, 10 nM) diminished Kv1.5 current (IKv1.5) and protein levels of Kv1.5 in the plasma membrane. Mechanistically, PKC activation led to monoubiquitination and degradation of the mature Kv1.5 proteins. Overexpression of Vps24, a protein that sorts transmembrane proteins into lysosomes via the multivesicular body (MVB) pathway, accelerated, whereas the lysosome inhibitor bafilomycin A1 completely prevented PKC-mediated Kv1.5 degradation. Kv1.5, but not Kv1.1, Kv1.2, Kv1.3, or Kv1.4, was uniquely sensitive to PMA treatment. Sequence alignments suggested that residues within the N terminus of Kv1.5 are essential for PKC-mediated Kv1.5 reduction. Using N-terminal truncation as well as site-directed mutagenesis, we identified that Thr15 is the target site for PKC that mediates endocytic degradation of Kv1.5 Channels. These findings indicate that alteration of protein levels in the plasma membrane represents an important regulatory mechanism of Kv1.5 Channel function under PKC activation conditions.
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mechanical stretch increases Kv1.5 current through an interaction between the s1 s2 linker and n terminus of the Channel
Journal of Biological Chemistry, 2020Co-Authors: Alexandria O Milton, Tingzhong Wang, Jun Guo, Shetuan ZhangAbstract:The voltage-gated Potassium Channel Kv1.5 plays important roles in atrial repolarization and regulation of vascular tone. In the present study, we investigated the effects of mechanical stretch on Kv1.5 Channels. We induced mechanical stretch by centrifuging or culturing Kv1.5-expressing HEK 293 cells and neonatal rat ventricular myocytes in low osmolarity (LO) medium and then recorded Kv1.5 current (IKv1.5) in a normal, isotonic solution. We observed that mechanical stretch increased IKv1.5, and this increase required the intact, long, proline-rich extracellular S1-S2 linker of the Kv1.5 Channel. The low osmolarity-induced IKv1.5 increase also required an intact intracellular N terminus, which contains the binding motif for endogenous Src tyrosine kinase that constitutively inhibits IKv1.5 Disrupting the Src-binding motif of Kv1.5 through N-terminal truncation or mutagenesis abolished the mechanical stretch-mediated increase in IKv1.5 Our results further showed that the extracellular S1-S2 linker of Kv1.5 communicates with the intracellular N terminus. Although the S1-S2 linker of WT Kv1.5 could be cleaved by extracellularly applied proteinase K (PK), an N-terminal truncation up to amino acid residue 209 altered the conformation of the S1-S2 linker and made it no longer susceptible to proteinase K-mediated cleavage. In summary, the findings of our study indicate that the S1-S2 linker of Kv1.5 represents a mechanosensor that regulates the activity of this Channel. By targeting the S1-S2 linker, mechanical stretch may induce a change in the N-terminal conformation of Kv1.5 that relieves Src-mediated tonic Channel inhibition and results in an increase in IKv1.5.
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Mechanical stretch increases Kv1.5 current through an interaction between the S1–S2 linker and N-terminus of the Channel
The Journal of biological chemistry, 2020Co-Authors: Alexandria O Milton, Tingzhong Wang, Jun Guo, Shetuan ZhangAbstract:The voltage-gated Potassium Channel Kv1.5 plays important roles in atrial repolarization and regulation of vascular tone. In the present study, we investigated the effects of mechanical stretch on Kv1.5 Channels. We induced mechanical stretch by centrifuging or culturing Kv1.5-expressing HEK 293 cells and neonatal rat ventricular myocytes in low osmolarity (LO) medium and then recorded Kv1.5 current (IKv1.5) in a normal, isotonic solution. We observed that mechanical stretch increased IKv1.5, and this increase required the intact, long, proline-rich extracellular S1–S2 linker of the Kv1.5 Channel. The low osmolarity–induced IKv1.5 increase also required an intact intracellular N terminus, which contains the binding motif for endogenous Src tyrosine kinase that constitutively inhibits IKv1.5. Disrupting the Src-binding motif of Kv1.5 through N-terminal truncation or mutagenesis abolished the mechanical stretch-mediated increase in IKv1.5. Our results further showed that the extracellular S1–S2 linker of Kv1.5 communicates with the intracellular N terminus. Although the S1–S2 linker of WT Kv1.5 could be cleaved by extracellularly applied proteinase K (PK), an N-terminal truncation up to amino acid residue 209 altered the conformation of the S1–S2 linker and made it no longer susceptible to proteinase K–mediated cleavage. In summary, the findings of our study indicate that the S1–S2 linker of Kv1.5 represents a mechanosensor that regulates the activity of this Channel. By targeting the S1–S2 linker, mechanical stretch may induce a change in the N-terminal conformation of Kv1.5 that relieves Src-mediated tonic Channel inhibition and results in an increase in IKv1.5.
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The N terminus and transmembrane segment S1 of Kv1.5 can coassemble with the rest of the Channel independently of the S1-S2 linkage.
The Journal of biological chemistry, 2018Co-Authors: Shawn M. Lamothe, Jun Guo, Tonghua Yang, Aja E Hogan-cann, Jared N. Tschirhart, Shetuan ZhangAbstract:The voltage-gated Potassium Channel Kv1.5 belongs to the Shaker superfamily. Kv1.5 is composed of four subunits, each comprising 613 amino acids, which make up the N terminus, six transmembrane segments (S1-S6), and the C terminus. We recently demonstrated that, in HEK cells, extracellularly applied proteinase K (PK) cleaves Kv1.5 Channels at a single site in the S1-S2 linker. This cleavage separates Kv1.5 into an N-fragment (N terminus to S1) and a C-fragment (S2 to C terminus). Interestingly, the cleavage does not impair Channel function. Here, we investigated the role of the N terminus and S1 in Kv1.5 expression and function by creating plasmids encoding various fragments, including those that mimic PK-cleaved products. Our results disclosed that although expression of the pore-containing fragment (Frag(304-613)) alone could not produce current, coexpression with Frag(1-303) generated a functional Channel. Immunofluorescence and biotinylation analyses uncovered that Frag(1-303) was required for Frag(304-613) to traffic to the plasma membrane. Biochemical analysis revealed that the two fragments interacted throughout Channel trafficking and maturation. In Frag(1-303)+(304-613)-coassembled Channels, which lack a covalent linkage between S1 and S2, amino acid residues 1-209 were important for association with Frag(304-613), and residues 210-303 were necessary for mediating trafficking of coassembled Channels to the plasma membrane. We conclude that the N terminus and S1 of Kv1.5 can attract and coassemble with the rest of the Channel (i.e. Frag(304-613)) to form a functional Channel independently of the S1-S2 linkage.
Jun Guo - One of the best experts on this subject based on the ideXlab platform.
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Kv1.5 Channels are regulated by PKC-mediated endocytic degradation.
The Journal of biological chemistry, 2021Co-Authors: Tingzhong Wang, Jun Guo, Tonghua Yang, Mark Szendrey, Shetuan ZhangAbstract:The voltage-gated Potassium Channel Kv1.5 plays important roles in the repolarization of atrial action potentials and regulation of the vascular tone. While the modulation of Kv1.5 function has been well studied, less is known about how the protein levels of Kv1.5 on the cell membrane are regulated. Here, through electrophysiological and biochemical analyses of Kv1.5 Channels heterologously expressed in HEK293 cells and neonatal rat ventricular myocytes, as well as native Kv1.5 in human induced pluripotent stem cell (iPSC)-derived atrial cardiomyocytes, we found that activation of protein kinase C (PKC) with phorbol 12-myristate 13-acetate (PMA, 10 nM) diminished Kv1.5 current (IKv1.5) and protein levels of Kv1.5 in the plasma membrane. Mechanistically, PKC activation led to monoubiquitination and degradation of the mature Kv1.5 proteins. Overexpression of Vps24, a protein that sorts transmembrane proteins into lysosomes via the multivesicular body (MVB) pathway, accelerated, whereas the lysosome inhibitor bafilomycin A1 completely prevented PKC-mediated Kv1.5 degradation. Kv1.5, but not Kv1.1, Kv1.2, Kv1.3, or Kv1.4, was uniquely sensitive to PMA treatment. Sequence alignments suggested that residues within the N terminus of Kv1.5 are essential for PKC-mediated Kv1.5 reduction. Using N-terminal truncation as well as site-directed mutagenesis, we identified that Thr15 is the target site for PKC that mediates endocytic degradation of Kv1.5 Channels. These findings indicate that alteration of protein levels in the plasma membrane represents an important regulatory mechanism of Kv1.5 Channel function under PKC activation conditions.
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mechanical stretch increases Kv1.5 current through an interaction between the s1 s2 linker and n terminus of the Channel
Journal of Biological Chemistry, 2020Co-Authors: Alexandria O Milton, Tingzhong Wang, Jun Guo, Shetuan ZhangAbstract:The voltage-gated Potassium Channel Kv1.5 plays important roles in atrial repolarization and regulation of vascular tone. In the present study, we investigated the effects of mechanical stretch on Kv1.5 Channels. We induced mechanical stretch by centrifuging or culturing Kv1.5-expressing HEK 293 cells and neonatal rat ventricular myocytes in low osmolarity (LO) medium and then recorded Kv1.5 current (IKv1.5) in a normal, isotonic solution. We observed that mechanical stretch increased IKv1.5, and this increase required the intact, long, proline-rich extracellular S1-S2 linker of the Kv1.5 Channel. The low osmolarity-induced IKv1.5 increase also required an intact intracellular N terminus, which contains the binding motif for endogenous Src tyrosine kinase that constitutively inhibits IKv1.5 Disrupting the Src-binding motif of Kv1.5 through N-terminal truncation or mutagenesis abolished the mechanical stretch-mediated increase in IKv1.5 Our results further showed that the extracellular S1-S2 linker of Kv1.5 communicates with the intracellular N terminus. Although the S1-S2 linker of WT Kv1.5 could be cleaved by extracellularly applied proteinase K (PK), an N-terminal truncation up to amino acid residue 209 altered the conformation of the S1-S2 linker and made it no longer susceptible to proteinase K-mediated cleavage. In summary, the findings of our study indicate that the S1-S2 linker of Kv1.5 represents a mechanosensor that regulates the activity of this Channel. By targeting the S1-S2 linker, mechanical stretch may induce a change in the N-terminal conformation of Kv1.5 that relieves Src-mediated tonic Channel inhibition and results in an increase in IKv1.5.
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Mechanical stretch increases Kv1.5 current through an interaction between the S1–S2 linker and N-terminus of the Channel
The Journal of biological chemistry, 2020Co-Authors: Alexandria O Milton, Tingzhong Wang, Jun Guo, Shetuan ZhangAbstract:The voltage-gated Potassium Channel Kv1.5 plays important roles in atrial repolarization and regulation of vascular tone. In the present study, we investigated the effects of mechanical stretch on Kv1.5 Channels. We induced mechanical stretch by centrifuging or culturing Kv1.5-expressing HEK 293 cells and neonatal rat ventricular myocytes in low osmolarity (LO) medium and then recorded Kv1.5 current (IKv1.5) in a normal, isotonic solution. We observed that mechanical stretch increased IKv1.5, and this increase required the intact, long, proline-rich extracellular S1–S2 linker of the Kv1.5 Channel. The low osmolarity–induced IKv1.5 increase also required an intact intracellular N terminus, which contains the binding motif for endogenous Src tyrosine kinase that constitutively inhibits IKv1.5. Disrupting the Src-binding motif of Kv1.5 through N-terminal truncation or mutagenesis abolished the mechanical stretch-mediated increase in IKv1.5. Our results further showed that the extracellular S1–S2 linker of Kv1.5 communicates with the intracellular N terminus. Although the S1–S2 linker of WT Kv1.5 could be cleaved by extracellularly applied proteinase K (PK), an N-terminal truncation up to amino acid residue 209 altered the conformation of the S1–S2 linker and made it no longer susceptible to proteinase K–mediated cleavage. In summary, the findings of our study indicate that the S1–S2 linker of Kv1.5 represents a mechanosensor that regulates the activity of this Channel. By targeting the S1–S2 linker, mechanical stretch may induce a change in the N-terminal conformation of Kv1.5 that relieves Src-mediated tonic Channel inhibition and results in an increase in IKv1.5.
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The N terminus and transmembrane segment S1 of Kv1.5 can coassemble with the rest of the Channel independently of the S1-S2 linkage.
The Journal of biological chemistry, 2018Co-Authors: Shawn M. Lamothe, Jun Guo, Tonghua Yang, Aja E Hogan-cann, Jared N. Tschirhart, Shetuan ZhangAbstract:The voltage-gated Potassium Channel Kv1.5 belongs to the Shaker superfamily. Kv1.5 is composed of four subunits, each comprising 613 amino acids, which make up the N terminus, six transmembrane segments (S1-S6), and the C terminus. We recently demonstrated that, in HEK cells, extracellularly applied proteinase K (PK) cleaves Kv1.5 Channels at a single site in the S1-S2 linker. This cleavage separates Kv1.5 into an N-fragment (N terminus to S1) and a C-fragment (S2 to C terminus). Interestingly, the cleavage does not impair Channel function. Here, we investigated the role of the N terminus and S1 in Kv1.5 expression and function by creating plasmids encoding various fragments, including those that mimic PK-cleaved products. Our results disclosed that although expression of the pore-containing fragment (Frag(304-613)) alone could not produce current, coexpression with Frag(1-303) generated a functional Channel. Immunofluorescence and biotinylation analyses uncovered that Frag(1-303) was required for Frag(304-613) to traffic to the plasma membrane. Biochemical analysis revealed that the two fragments interacted throughout Channel trafficking and maturation. In Frag(1-303)+(304-613)-coassembled Channels, which lack a covalent linkage between S1 and S2, amino acid residues 1-209 were important for association with Frag(304-613), and residues 210-303 were necessary for mediating trafficking of coassembled Channels to the plasma membrane. We conclude that the N terminus and S1 of Kv1.5 can attract and coassemble with the rest of the Channel (i.e. Frag(304-613)) to form a functional Channel independently of the S1-S2 linkage.
Tingzhong Wang - One of the best experts on this subject based on the ideXlab platform.
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Kv1.5 Channels are regulated by PKC-mediated endocytic degradation.
The Journal of biological chemistry, 2021Co-Authors: Tingzhong Wang, Jun Guo, Tonghua Yang, Mark Szendrey, Shetuan ZhangAbstract:The voltage-gated Potassium Channel Kv1.5 plays important roles in the repolarization of atrial action potentials and regulation of the vascular tone. While the modulation of Kv1.5 function has been well studied, less is known about how the protein levels of Kv1.5 on the cell membrane are regulated. Here, through electrophysiological and biochemical analyses of Kv1.5 Channels heterologously expressed in HEK293 cells and neonatal rat ventricular myocytes, as well as native Kv1.5 in human induced pluripotent stem cell (iPSC)-derived atrial cardiomyocytes, we found that activation of protein kinase C (PKC) with phorbol 12-myristate 13-acetate (PMA, 10 nM) diminished Kv1.5 current (IKv1.5) and protein levels of Kv1.5 in the plasma membrane. Mechanistically, PKC activation led to monoubiquitination and degradation of the mature Kv1.5 proteins. Overexpression of Vps24, a protein that sorts transmembrane proteins into lysosomes via the multivesicular body (MVB) pathway, accelerated, whereas the lysosome inhibitor bafilomycin A1 completely prevented PKC-mediated Kv1.5 degradation. Kv1.5, but not Kv1.1, Kv1.2, Kv1.3, or Kv1.4, was uniquely sensitive to PMA treatment. Sequence alignments suggested that residues within the N terminus of Kv1.5 are essential for PKC-mediated Kv1.5 reduction. Using N-terminal truncation as well as site-directed mutagenesis, we identified that Thr15 is the target site for PKC that mediates endocytic degradation of Kv1.5 Channels. These findings indicate that alteration of protein levels in the plasma membrane represents an important regulatory mechanism of Kv1.5 Channel function under PKC activation conditions.
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mechanical stretch increases Kv1.5 current through an interaction between the s1 s2 linker and n terminus of the Channel
Journal of Biological Chemistry, 2020Co-Authors: Alexandria O Milton, Tingzhong Wang, Jun Guo, Shetuan ZhangAbstract:The voltage-gated Potassium Channel Kv1.5 plays important roles in atrial repolarization and regulation of vascular tone. In the present study, we investigated the effects of mechanical stretch on Kv1.5 Channels. We induced mechanical stretch by centrifuging or culturing Kv1.5-expressing HEK 293 cells and neonatal rat ventricular myocytes in low osmolarity (LO) medium and then recorded Kv1.5 current (IKv1.5) in a normal, isotonic solution. We observed that mechanical stretch increased IKv1.5, and this increase required the intact, long, proline-rich extracellular S1-S2 linker of the Kv1.5 Channel. The low osmolarity-induced IKv1.5 increase also required an intact intracellular N terminus, which contains the binding motif for endogenous Src tyrosine kinase that constitutively inhibits IKv1.5 Disrupting the Src-binding motif of Kv1.5 through N-terminal truncation or mutagenesis abolished the mechanical stretch-mediated increase in IKv1.5 Our results further showed that the extracellular S1-S2 linker of Kv1.5 communicates with the intracellular N terminus. Although the S1-S2 linker of WT Kv1.5 could be cleaved by extracellularly applied proteinase K (PK), an N-terminal truncation up to amino acid residue 209 altered the conformation of the S1-S2 linker and made it no longer susceptible to proteinase K-mediated cleavage. In summary, the findings of our study indicate that the S1-S2 linker of Kv1.5 represents a mechanosensor that regulates the activity of this Channel. By targeting the S1-S2 linker, mechanical stretch may induce a change in the N-terminal conformation of Kv1.5 that relieves Src-mediated tonic Channel inhibition and results in an increase in IKv1.5.
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Mechanical stretch increases Kv1.5 current through an interaction between the S1–S2 linker and N-terminus of the Channel
The Journal of biological chemistry, 2020Co-Authors: Alexandria O Milton, Tingzhong Wang, Jun Guo, Shetuan ZhangAbstract:The voltage-gated Potassium Channel Kv1.5 plays important roles in atrial repolarization and regulation of vascular tone. In the present study, we investigated the effects of mechanical stretch on Kv1.5 Channels. We induced mechanical stretch by centrifuging or culturing Kv1.5-expressing HEK 293 cells and neonatal rat ventricular myocytes in low osmolarity (LO) medium and then recorded Kv1.5 current (IKv1.5) in a normal, isotonic solution. We observed that mechanical stretch increased IKv1.5, and this increase required the intact, long, proline-rich extracellular S1–S2 linker of the Kv1.5 Channel. The low osmolarity–induced IKv1.5 increase also required an intact intracellular N terminus, which contains the binding motif for endogenous Src tyrosine kinase that constitutively inhibits IKv1.5. Disrupting the Src-binding motif of Kv1.5 through N-terminal truncation or mutagenesis abolished the mechanical stretch-mediated increase in IKv1.5. Our results further showed that the extracellular S1–S2 linker of Kv1.5 communicates with the intracellular N terminus. Although the S1–S2 linker of WT Kv1.5 could be cleaved by extracellularly applied proteinase K (PK), an N-terminal truncation up to amino acid residue 209 altered the conformation of the S1–S2 linker and made it no longer susceptible to proteinase K–mediated cleavage. In summary, the findings of our study indicate that the S1–S2 linker of Kv1.5 represents a mechanosensor that regulates the activity of this Channel. By targeting the S1–S2 linker, mechanical stretch may induce a change in the N-terminal conformation of Kv1.5 that relieves Src-mediated tonic Channel inhibition and results in an increase in IKv1.5.
Alexandria O Milton - One of the best experts on this subject based on the ideXlab platform.
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mechanical stretch increases Kv1.5 current through an interaction between the s1 s2 linker and n terminus of the Channel
Journal of Biological Chemistry, 2020Co-Authors: Alexandria O Milton, Tingzhong Wang, Jun Guo, Shetuan ZhangAbstract:The voltage-gated Potassium Channel Kv1.5 plays important roles in atrial repolarization and regulation of vascular tone. In the present study, we investigated the effects of mechanical stretch on Kv1.5 Channels. We induced mechanical stretch by centrifuging or culturing Kv1.5-expressing HEK 293 cells and neonatal rat ventricular myocytes in low osmolarity (LO) medium and then recorded Kv1.5 current (IKv1.5) in a normal, isotonic solution. We observed that mechanical stretch increased IKv1.5, and this increase required the intact, long, proline-rich extracellular S1-S2 linker of the Kv1.5 Channel. The low osmolarity-induced IKv1.5 increase also required an intact intracellular N terminus, which contains the binding motif for endogenous Src tyrosine kinase that constitutively inhibits IKv1.5 Disrupting the Src-binding motif of Kv1.5 through N-terminal truncation or mutagenesis abolished the mechanical stretch-mediated increase in IKv1.5 Our results further showed that the extracellular S1-S2 linker of Kv1.5 communicates with the intracellular N terminus. Although the S1-S2 linker of WT Kv1.5 could be cleaved by extracellularly applied proteinase K (PK), an N-terminal truncation up to amino acid residue 209 altered the conformation of the S1-S2 linker and made it no longer susceptible to proteinase K-mediated cleavage. In summary, the findings of our study indicate that the S1-S2 linker of Kv1.5 represents a mechanosensor that regulates the activity of this Channel. By targeting the S1-S2 linker, mechanical stretch may induce a change in the N-terminal conformation of Kv1.5 that relieves Src-mediated tonic Channel inhibition and results in an increase in IKv1.5.
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Mechanical stretch increases Kv1.5 current through an interaction between the S1–S2 linker and N-terminus of the Channel
The Journal of biological chemistry, 2020Co-Authors: Alexandria O Milton, Tingzhong Wang, Jun Guo, Shetuan ZhangAbstract:The voltage-gated Potassium Channel Kv1.5 plays important roles in atrial repolarization and regulation of vascular tone. In the present study, we investigated the effects of mechanical stretch on Kv1.5 Channels. We induced mechanical stretch by centrifuging or culturing Kv1.5-expressing HEK 293 cells and neonatal rat ventricular myocytes in low osmolarity (LO) medium and then recorded Kv1.5 current (IKv1.5) in a normal, isotonic solution. We observed that mechanical stretch increased IKv1.5, and this increase required the intact, long, proline-rich extracellular S1–S2 linker of the Kv1.5 Channel. The low osmolarity–induced IKv1.5 increase also required an intact intracellular N terminus, which contains the binding motif for endogenous Src tyrosine kinase that constitutively inhibits IKv1.5. Disrupting the Src-binding motif of Kv1.5 through N-terminal truncation or mutagenesis abolished the mechanical stretch-mediated increase in IKv1.5. Our results further showed that the extracellular S1–S2 linker of Kv1.5 communicates with the intracellular N terminus. Although the S1–S2 linker of WT Kv1.5 could be cleaved by extracellularly applied proteinase K (PK), an N-terminal truncation up to amino acid residue 209 altered the conformation of the S1–S2 linker and made it no longer susceptible to proteinase K–mediated cleavage. In summary, the findings of our study indicate that the S1–S2 linker of Kv1.5 represents a mechanosensor that regulates the activity of this Channel. By targeting the S1–S2 linker, mechanical stretch may induce a change in the N-terminal conformation of Kv1.5 that relieves Src-mediated tonic Channel inhibition and results in an increase in IKv1.5.
David Fedida - One of the best experts on this subject based on the ideXlab platform.
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kif5b is an essential forward trafficking motor for the Kv1.5 cardiac Potassium Channel
The Journal of Physiology, 2009Co-Authors: Alireza Dehghani Zadeh, Yvonne Cheng, Nathan Wong, Charitha L. Goonasekara, David F Steele, Zhuren Wang, Hongjian Xu, David FedidaAbstract:We have investigated the role of the kinesin I isoform Kif5b in the trafficking of a cardiac voltage-gated Potassium Channel, Kv1.5. In Kv1.5-expressing HEK293 cells and H9c2 cardiomyoblasts, current densities were increased from control levels of 389 ± 50.0 and 317 ± 50.3 pA pF−1, respectively, to 614 ± 74.3 and 580 ± 90.9 pA pF−1 in cells overexpressing the Kif5b motor. Overexpression of the Kif5b motor increased Kv1.5 expression additively with several manipulations that reduce Channel internalization, suggesting that it is involved in the delivery of the Channel to the cell surface. In contrast, expression of a Kif5b dominant negative (Kif5bDN) construct increased Kv1.5 expression non-additively with these manipulations. Thus, the dominant negative acts by indirectly inhibiting endocytosis. The increase in Kv1.5 currents induced by wild-type Kif5b was dependent on Golgi function; a 6 h treatment with Brefeldin A reduced Kv1.5 currents to control levels in Kif5b-overexpressing cells but had little effect on the increase associated with Kif5bDN expression. Finally, expression of the Kif5bDN prior to induction of Kv1.5 in a tetracycline inducible system blocked surface expression of the Channel in both HEK293 cells and H9c2 cardiomyoblasts. Thus, Kif5b is essential to anterograde trafficking of a cardiac voltage-gated Potassium Channel.
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localization of Kv1.5 Channels in rat and canine myocyte sarcolemma
FEBS Letters, 2006Co-Authors: Jodene Eldstrom, David R Van Wagoner, Edwin D W Moore, David FedidaAbstract:Abstract Voltage-gated Potassium (Kv) Channel subtypes localize to the plasma membrane of a number of cell types, and the sarcolemma in myocytes. Because many signaling molecules concentrate in subdomains of the plasma membrane, the localization of Kv Channels to these sites may have important implications for Channel function and regulation. In this study, the association of the voltage-gated Potassium Channel Kv1.5 with a specific subtype of lipid rafts, caveolae, in rat and canine cardiac myocytes has been investigated. Interactions between caveolin-3 and β-dystroglycan or eNOS, as well as between Kv1.5 and α-actinin were readily detected in co-immunoprecipitation experiments, whereas no association between Kv1.5 and caveolin-3 was evident. Wide-field microscopy and deconvolution techniques revealed that the percent co-localization of Kv1.5 with caveolin-3 was extremely low in atrial myocytes from rat and canine hearts (8 ± 1% and 12.2 ± 2%, respectively), and limited in ventricular myocytes (11 ± 4% and 20 ± 3% in rat and canine, respectively). Immunoelectron microscopic imaging of rat atrial and ventricular tissues showed that Kv1.5 and caveolin-3 labeling generally did not overlap. In HEK293 cells stably expressing the Channel, Kv1.5 did not target to the low buoyant density raft fraction along with flotillin but instead fractionated along with the non-raft associated transferrin receptor. Taken together, these results suggest that Kv1.5 is not present in caveolae of rat and canine heart.
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Single Channel Analysis Reveals Different Modes of Kv1.5 Gating Behavior Regulated by Changes of External pH
Biophysical journal, 2005Co-Authors: Daniel C. H. Kwan, David Fedida, Steven J. KehlAbstract:In the voltage-gated Potassium Channel Kv1.5, extracellular acidification decreases the peak macroscopic conductance and accelerates slow inactivation. To better understand the mechanistic basis for these two effects, we recorded unitary currents of Kv1.5 expressed in a mouse cell line (ltk−) using the voltage clamp technique both in cell-attached and excised outside-out patches. Single Channel current amplitude at 100 mV (1.7 ± 0.2 pA at pH 7.4, 1.7 ± 0.2 pA at pH 6.4) and the single Channel conductance between 0 and 100 mV (11.8 ± 0.6 pS at pH 7.4 and 11.3 ± 0.8 pS at pH 6.4) did not change significantly with pH. External acidification significantly decreased the number of active sweeps, and this reduction in Channel availability accounted for most of the reduction of the peak macroscopic current. The results of runs analyses suggested the null sweeps occur in clusters, and the rate constants for the transition between clusters of null and active sweeps at pH 6.4 were slow (0.12 and 0.18 s−1, to and from the active clusters, respectively). We propose that low pH facilitates a shift from an available mode (mode A) into an unavailable mode of gating (mode U). In addition to promoting mode U gating, external acidification accelerates depolarization-induced inactivation, which is manifest at the single Channel level as a reduction of the mean burst length and an apparent increase of the interburst interval. These effects of external acidification, which are thought to reflect the protonation of a histidine residue in the turret (H-463), point to an important role for the turret in the regulation of Channel availability and inactivation.
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Kv1.5 Surface Expression Is Modulated by Retrograde Trafficking of Newly Endocytosed Channels by the Dynein Motor
Circulation research, 2005Co-Authors: Woo Sung Choi, David F Steele, Anu Khurana, Rajesh Mathur, Vijay Viswanathan, David FedidaAbstract:In this article we have investigated the mechanisms by which retrograde trafficking regulates the surface expression of the voltage-gated Potassium Channel, Kv1.5. Overexpression of p50/dynamitin, known to disrupt the dynein–dynactin complex responsible for carrying vesicle cargo, substantially increased outward K+ currents in HEK293 cells stably expressing Kv1.5 (0.57±0.07 nA/pF, n=12; to 1.18±0.2 nA/pF, n=12, P
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Slow gating charge immobilization in the human Potassium Channel Kv1.5 and its prevention by 4-aminopyridine.
The Journal of Physiology, 1996Co-Authors: David Fedida, R Bouchard, F. S. P. ChenAbstract:1. The relationship between ionic current inactivation and immobilization of 'off'-gating charge in human Kv1.5 Channels expressed in human embryonic kidney (HEK293) cells was studied using 4-aminopyridine (4-AP) and tetraethylammonium chloride (TEA-Cl). 2. The charge transferred during short ( 1.2 ms). For +80 mV pulses longer than 50 ms, Q(off) at 20 ms was less than Q(on) (Q(off)/Q(on) ratio was 0.26 +/- 0.06 at 450 ms). We attribute this to a relative 'immobilization' of gating charge during long depolarizations. 3. 4-AP (0.1-1 mM) prevented slowing of off-Ig, allowing saturation of peak off-Ig. 4-AP also completely prevented immobilization of off-Ig after long depolarizations. In 1 mM 4-AP, off-Ig waveforms decayed rapidly and the charge ratio Q(off)/Q(on) remained at 1.0. 4. In addition to its effects on Ig, 1 mM 4-AP prevented the slow inactivation of ionic current seen during strong depolarizations. An initial block was caused by 4-AP or 1 mM intracellular TEA internally applied. However, only 4-AP prevented the slower, later development of C-type inactivation. 5. We suggest that slow current inactivation is accompanied by a gating charge immobilization in Kv1.5. 4-AP potently inhibits the changes in Q(off)/Q(on0, off-Ig, and ionic currents that underlie slow inactivation. Some actions of 4-AP appear independent of its properties as a blocker of open K+ Channels, and are not mimicked by internal TEA.