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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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Anti-Kv4.2 mAb–porphyrin conjugate facilitates photoablation of K currents. (A) Kv4.2 currents during photoablation with 10 nM αKv4.2•1. Stimulatory pulses to 0 mV, with illumination beginning after time = 0. Data from CHO cell. (B) Circles, peak currents from cell in A. Purple bar indicates period of illumination. Line is fit of Eq. 1; rate = 0.053 ± 0.001 s−1 and f = 0.953 ± 0.004. (C) Peak Kv4.2 currents from cell similar to that in B, except conjugate was removed from bath solution for 2 min before illumination. Line is fit of Eq. 1; rate = 0.035 ± 0.003 s−1 and f = 0.82 ± 0.02.
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fig3: Anti-Kv4.2 mAb–porphyrin conjugate facilitates photoablation of K currents. (A) Kv4.2 currents during photoablation with 10 nM αKv4.2•1. Stimulatory pulses to 0 mV, with illumination beginning after time = 0. Data from CHO cell. (B) Circles, peak currents from cell in A. Purple bar indicates period of illumination. Line is fit of Eq. 1; rate = 0.053 ± 0.001 s−1 and f = 0.953 ± 0.004. (C) Peak Kv4.2 currents from cell similar to that in B, except conjugate was removed from bath solution for 2 min before illumination. Line is fit of Eq. 1; rate = 0.035 ± 0.003 s−1 and f = 0.82 ± 0.02.

Mentions: Antibody–porphyrin conjugates were added to the external solution of patch-clamped cells heterologously expressing Kv channels. After a brief incubation period to allow antibodies to bind channel targets, photostimulation of the highest extinction coefficient absorption (Soret) band of the anti-Kv4.2 mAb–porphyrin 1 conjugate, αKv4.2•1 (Fig. 2 C), resulted in progressive inhibition of Kv4.2 current with photon dose (Fig. 3, A and B). This irreversible photoablation is consistent with oxidative damage to channels from diffusible reactive oxygen species generated by photostimulated porphyrin, such as singlet oxygen, as schematized (Fig. 2 D). Photoablation was also apparent after washout of mAb conjugate (Fig. 3 C), consistent with the conjugate channel complex having a slow dissociation rate from the targeted channels.


Antibody-guided photoablation of voltage-gated potassium currents.

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

Anti-Kv4.2 mAb–porphyrin conjugate facilitates photoablation of K currents. (A) Kv4.2 currents during photoablation with 10 nM αKv4.2•1. Stimulatory pulses to 0 mV, with illumination beginning after time = 0. Data from CHO cell. (B) Circles, peak currents from cell in A. Purple bar indicates period of illumination. Line is fit of Eq. 1; rate = 0.053 ± 0.001 s−1 and f = 0.953 ± 0.004. (C) Peak Kv4.2 currents from cell similar to that in B, except conjugate was removed from bath solution for 2 min before illumination. Line is fit of Eq. 1; rate = 0.035 ± 0.003 s−1 and f = 0.82 ± 0.02.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig3: Anti-Kv4.2 mAb–porphyrin conjugate facilitates photoablation of K currents. (A) Kv4.2 currents during photoablation with 10 nM αKv4.2•1. Stimulatory pulses to 0 mV, with illumination beginning after time = 0. Data from CHO cell. (B) Circles, peak currents from cell in A. Purple bar indicates period of illumination. Line is fit of Eq. 1; rate = 0.053 ± 0.001 s−1 and f = 0.953 ± 0.004. (C) Peak Kv4.2 currents from cell similar to that in B, except conjugate was removed from bath solution for 2 min before illumination. Line is fit of Eq. 1; rate = 0.035 ± 0.003 s−1 and f = 0.82 ± 0.02.
Mentions: Antibody–porphyrin conjugates were added to the external solution of patch-clamped cells heterologously expressing Kv channels. After a brief incubation period to allow antibodies to bind channel targets, photostimulation of the highest extinction coefficient absorption (Soret) band of the anti-Kv4.2 mAb–porphyrin 1 conjugate, αKv4.2•1 (Fig. 2 C), resulted in progressive inhibition of Kv4.2 current with photon dose (Fig. 3, A and B). This irreversible photoablation is consistent with oxidative damage to channels from diffusible reactive oxygen species generated by photostimulated porphyrin, such as singlet oxygen, as schematized (Fig. 2 D). Photoablation was also apparent after washout of mAb conjugate (Fig. 3 C), consistent with the conjugate channel complex having a slow dissociation rate from the targeted channels.

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