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Antibody-guided photoablation of voltage-gated potassium currents.

Sack JT, Stephanopoulos N, Austin DC, Francis MB, Trimmer JS - J. Gen. Physiol. (2013)

Bottom Line: Guided by the exquisite selectivity of immune system interactions, we find potential for antibody conjugates as selective Kv inhibitors.Antibodies were conjugated to porphyrin compounds that upon photostimulation inflict localized oxidative damage.These findings demonstrate that subtype-specific mAbs that in themselves do not modulate ion channel function are capable of delivering functional payloads to specific ion channel targets.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Physiology and Membrane Biology, University of California, Davis, Davis, CA 95616, USA. jsack@ucdavis.edu

ABSTRACT
A family of 40 mammalian voltage-gated potassium (Kv) channels control membrane excitability in electrically excitable cells. The contribution of individual Kv channel types to electrophysiological signaling has been difficult to assign, as few selective inhibitors exist for individual Kv subunits. Guided by the exquisite selectivity of immune system interactions, we find potential for antibody conjugates as selective Kv inhibitors. Here, functionally benign anti-Kv channel monoclonal antibodies (mAbs) were chemically modified to facilitate photoablation of K currents. Antibodies were conjugated to porphyrin compounds that upon photostimulation inflict localized oxidative damage. Anti-Kv4.2 mAb-porphyrin conjugates facilitated photoablation of Kv4.2 currents. The degree of K current ablation was dependent on photon dose and conjugate concentration. Kv channel photoablation was selective for Kv4.2 over Kv4.3 or Kv2.1, yielding specificity not present in existing neurotoxins or other Kv channel inhibitors. We conclude that antibody-porphyrin conjugates are capable of selective photoablation of Kv currents. These findings demonstrate that subtype-specific mAbs that in themselves do not modulate ion channel function are capable of delivering functional payloads to specific ion channel targets.

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Validation of anti-Kv4.2 mAb. (A) Sequence alignment of relevant K channels indicating residues included in Kv4.2 immunogenic peptide. (B) Immunohistochemical analysis of antibody specificity in brain. Double-label immunofluorescence staining for anti-Kv4.2 mAb (red) and anti-Kv2.1 polyclonal antibody (green) in sections prepared from the brains of wild-type (top row) and Kv4.2 knockout (bottom row) mice. (C) Immunoblot analysis of antibody specificity in brain. Immunoblot of adult rat brain membranes (RBM), adult mouse brain membranes from Kv4.2 knockout (MBM-Kv4.2-KO), and wild-type (MBM-WT) mice probed with anti-Kv4.2 or anti-Kv2.1 mAbs as noted. Numbers to the left denote mobility of prestained molecular weight standards in kilodaltons. (D) Whole cell Kv4.2 current from a 0-mV pulse before and 8 min after the application of 1 µM anti-Kv4.2 mAb K57/1 to a HEK cell. P/5 subtraction was used to isolate K currents.
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fig1: Validation of anti-Kv4.2 mAb. (A) Sequence alignment of relevant K channels indicating residues included in Kv4.2 immunogenic peptide. (B) Immunohistochemical analysis of antibody specificity in brain. Double-label immunofluorescence staining for anti-Kv4.2 mAb (red) and anti-Kv2.1 polyclonal antibody (green) in sections prepared from the brains of wild-type (top row) and Kv4.2 knockout (bottom row) mice. (C) Immunoblot analysis of antibody specificity in brain. Immunoblot of adult rat brain membranes (RBM), adult mouse brain membranes from Kv4.2 knockout (MBM-Kv4.2-KO), and wild-type (MBM-WT) mice probed with anti-Kv4.2 or anti-Kv2.1 mAbs as noted. Numbers to the left denote mobility of prestained molecular weight standards in kilodaltons. (D) Whole cell Kv4.2 current from a 0-mV pulse before and 8 min after the application of 1 µM anti-Kv4.2 mAb K57/1 to a HEK cell. P/5 subtraction was used to isolate K currents.

Mentions: To target photoinhibitors to Kv channels, an extensively validated, subtype-selective anti-Kv mAb was chosen. The IgG1 clone K57/1 (Shibata et al., 2003) was found to be selective for mammalian Kv4.2 channels (Rhodes et al., 2004; Menegola and Trimmer, 2006), has been used for studies by multiple research groups (Burkhalter et al., 2006; Gardoni et al., 2007; Kim et al., 2007; Amarillo et al., 2008; Hammond et al., 2008), and is publicly available from the UC Davis/National Institutes of Health NeuroMab facility. The K57/1 mAb was generated against a synthetic peptide, “Kv4.2e,” that mimicked amino acids of the external loop between the S1 and S2 segment of the human Kv4.2 subunit (Fig. 1 A). The S1–S2 linker is the longest and most variable external region of Kv proteins and, as such, is an attractive target for generating subtype-specific externally acting antibodies. The Kv4.2e sequence is identical in human, rat, mouse, and other mammalian Kv4.2 orthologues. The K57/1 antibody is highly specific for Kv4.2 over its closely related family members Kv4.1 and Kv4.3 (Rhodes et al., 2004). K57/1 exhibits robust binding in immunohistochemical staining of the intact brains and in immunoblot analyses of denatured protein samples prepared from wild-type mice did not exhibit detectable signals in Kv4.2 knockout mice (Fig. 2, B and C) (Menegola and Trimmer, 2006). To date, all anti-Kv channel S1–S2 linker antibodies we have tested, including the K57/1 mAb, have proven ineffectual as inhibitors of their targeted Kv channels (Fig. 1 D).


Antibody-guided photoablation of voltage-gated potassium currents.

Sack JT, Stephanopoulos N, Austin DC, Francis MB, Trimmer JS - J. Gen. Physiol. (2013)

Validation of anti-Kv4.2 mAb. (A) Sequence alignment of relevant K channels indicating residues included in Kv4.2 immunogenic peptide. (B) Immunohistochemical analysis of antibody specificity in brain. Double-label immunofluorescence staining for anti-Kv4.2 mAb (red) and anti-Kv2.1 polyclonal antibody (green) in sections prepared from the brains of wild-type (top row) and Kv4.2 knockout (bottom row) mice. (C) Immunoblot analysis of antibody specificity in brain. Immunoblot of adult rat brain membranes (RBM), adult mouse brain membranes from Kv4.2 knockout (MBM-Kv4.2-KO), and wild-type (MBM-WT) mice probed with anti-Kv4.2 or anti-Kv2.1 mAbs as noted. Numbers to the left denote mobility of prestained molecular weight standards in kilodaltons. (D) Whole cell Kv4.2 current from a 0-mV pulse before and 8 min after the application of 1 µM anti-Kv4.2 mAb K57/1 to a HEK cell. P/5 subtraction was used to isolate K currents.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3753605&req=5

fig1: Validation of anti-Kv4.2 mAb. (A) Sequence alignment of relevant K channels indicating residues included in Kv4.2 immunogenic peptide. (B) Immunohistochemical analysis of antibody specificity in brain. Double-label immunofluorescence staining for anti-Kv4.2 mAb (red) and anti-Kv2.1 polyclonal antibody (green) in sections prepared from the brains of wild-type (top row) and Kv4.2 knockout (bottom row) mice. (C) Immunoblot analysis of antibody specificity in brain. Immunoblot of adult rat brain membranes (RBM), adult mouse brain membranes from Kv4.2 knockout (MBM-Kv4.2-KO), and wild-type (MBM-WT) mice probed with anti-Kv4.2 or anti-Kv2.1 mAbs as noted. Numbers to the left denote mobility of prestained molecular weight standards in kilodaltons. (D) Whole cell Kv4.2 current from a 0-mV pulse before and 8 min after the application of 1 µM anti-Kv4.2 mAb K57/1 to a HEK cell. P/5 subtraction was used to isolate K currents.
Mentions: To target photoinhibitors to Kv channels, an extensively validated, subtype-selective anti-Kv mAb was chosen. The IgG1 clone K57/1 (Shibata et al., 2003) was found to be selective for mammalian Kv4.2 channels (Rhodes et al., 2004; Menegola and Trimmer, 2006), has been used for studies by multiple research groups (Burkhalter et al., 2006; Gardoni et al., 2007; Kim et al., 2007; Amarillo et al., 2008; Hammond et al., 2008), and is publicly available from the UC Davis/National Institutes of Health NeuroMab facility. The K57/1 mAb was generated against a synthetic peptide, “Kv4.2e,” that mimicked amino acids of the external loop between the S1 and S2 segment of the human Kv4.2 subunit (Fig. 1 A). The S1–S2 linker is the longest and most variable external region of Kv proteins and, as such, is an attractive target for generating subtype-specific externally acting antibodies. The Kv4.2e sequence is identical in human, rat, mouse, and other mammalian Kv4.2 orthologues. The K57/1 antibody is highly specific for Kv4.2 over its closely related family members Kv4.1 and Kv4.3 (Rhodes et al., 2004). K57/1 exhibits robust binding in immunohistochemical staining of the intact brains and in immunoblot analyses of denatured protein samples prepared from wild-type mice did not exhibit detectable signals in Kv4.2 knockout mice (Fig. 2, B and C) (Menegola and Trimmer, 2006). To date, all anti-Kv channel S1–S2 linker antibodies we have tested, including the K57/1 mAb, have proven ineffectual as inhibitors of their targeted Kv channels (Fig. 1 D).

Bottom Line: Guided by the exquisite selectivity of immune system interactions, we find potential for antibody conjugates as selective Kv inhibitors.Antibodies were conjugated to porphyrin compounds that upon photostimulation inflict localized oxidative damage.These findings demonstrate that subtype-specific mAbs that in themselves do not modulate ion channel function are capable of delivering functional payloads to specific ion channel targets.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Physiology and Membrane Biology, University of California, Davis, Davis, CA 95616, USA. jsack@ucdavis.edu

ABSTRACT
A family of 40 mammalian voltage-gated potassium (Kv) channels control membrane excitability in electrically excitable cells. The contribution of individual Kv channel types to electrophysiological signaling has been difficult to assign, as few selective inhibitors exist for individual Kv subunits. Guided by the exquisite selectivity of immune system interactions, we find potential for antibody conjugates as selective Kv inhibitors. Here, functionally benign anti-Kv channel monoclonal antibodies (mAbs) were chemically modified to facilitate photoablation of K currents. Antibodies were conjugated to porphyrin compounds that upon photostimulation inflict localized oxidative damage. Anti-Kv4.2 mAb-porphyrin conjugates facilitated photoablation of Kv4.2 currents. The degree of K current ablation was dependent on photon dose and conjugate concentration. Kv channel photoablation was selective for Kv4.2 over Kv4.3 or Kv2.1, yielding specificity not present in existing neurotoxins or other Kv channel inhibitors. We conclude that antibody-porphyrin conjugates are capable of selective photoablation of Kv currents. These findings demonstrate that subtype-specific mAbs that in themselves do not modulate ion channel function are capable of delivering functional payloads to specific ion channel targets.

Show MeSH
Related in: MedlinePlus