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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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Anionic porphyrin conjugates do not facilitate selective photoablation. Data points are relative peak currents during 0-mV pulses to patch-clamped whole cells. Photon flux per unit area is approximately four times greater than in all other figures because of the 20× objective used. (A) Antibody–porphyrin 2 conjugate (20 nM) produced only nonselective photoablation of Kv currents. Black circles, Kv4.2 from HEK cells; rate = 0.012 ± 0.001 s−1 and f = 0.62 ± 0.04; gray circles, Kv2.1 from CHO cells; rate = 0.014 ± 0.004 s−1 and f = 0.48 ± 0.07. (B) Antibody–porphyrin 3 conjugate (20 nM) produced only nonselective photoablation of Kv currents. Black circles, Kv4.2; rate = 0.016 ± 0.001 s−1 and f = 0.75 ± 0.02; gray circles, Kv2.1; rate = 0.023 ± 0.004 s−1 and f = 0.49 ± 0.03.
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fig5: Anionic porphyrin conjugates do not facilitate selective photoablation. Data points are relative peak currents during 0-mV pulses to patch-clamped whole cells. Photon flux per unit area is approximately four times greater than in all other figures because of the 20× objective used. (A) Antibody–porphyrin 2 conjugate (20 nM) produced only nonselective photoablation of Kv currents. Black circles, Kv4.2 from HEK cells; rate = 0.012 ± 0.001 s−1 and f = 0.62 ± 0.04; gray circles, Kv2.1 from CHO cells; rate = 0.014 ± 0.004 s−1 and f = 0.48 ± 0.07. (B) Antibody–porphyrin 3 conjugate (20 nM) produced only nonselective photoablation of Kv currents. Black circles, Kv4.2; rate = 0.016 ± 0.001 s−1 and f = 0.75 ± 0.02; gray circles, Kv2.1; rate = 0.023 ± 0.004 s−1 and f = 0.49 ± 0.03.

Mentions: The αKv4.2•1 porphyrin–mAb conjugates allowed selective photoablation of the targeted Kv channels. There was little photoablation of Kv4.3 or Kv2.1 in response to photon doses that eliminated Kv4.2 currents, suggesting that antibody targeting could direct the photoablation effect (Fig. 4). The effect of increasing photon dose was quantified as a photoablation rate, corresponding to the inverse time constant of current loss under our illumination conditions (Fig. 4 E). With prolonged illumination, current was gradually lost from the off-target Kv2.1 and Kv4.3 channels. This side effect indicated that although the anti-Kv antibody could direct photoablation to channels of interest, collateral damage also occurred. Alternate porphyrin chemistries were not as successful at enabling targeted photoablation. The mAb–porphyrin conjugates of the two anionic porphyrins synthesized (2 and 3) did not selectively ablate currents from the targeted Kv4.2 channel. Under more intense illumination, high photon doses yielded nonselective photoablation against all channels tested (Fig. 5). This suggests that the ability of the K57/1 antibody to recognize its epitope is compromised by attachment of the anionic conjugates. It is not clear why the anionic conjugates were not targeted as effectively as their cationic counterpart, αKv4.2•1. The weaker, nonselective effects of αKv4.2• and αKv4.2•3, together with the off-target effects of αKv4.2•1 on Kv2.1 and Kv4.3, demonstrate that a mechanism beyond antibody–epitope binding can also lead to photoablation. Yet, with this technique, nonselective side effects were dwarfed by selective antibody-guided photoablation of Kv currents.


Antibody-guided photoablation of voltage-gated potassium currents.

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

Anionic porphyrin conjugates do not facilitate selective photoablation. Data points are relative peak currents during 0-mV pulses to patch-clamped whole cells. Photon flux per unit area is approximately four times greater than in all other figures because of the 20× objective used. (A) Antibody–porphyrin 2 conjugate (20 nM) produced only nonselective photoablation of Kv currents. Black circles, Kv4.2 from HEK cells; rate = 0.012 ± 0.001 s−1 and f = 0.62 ± 0.04; gray circles, Kv2.1 from CHO cells; rate = 0.014 ± 0.004 s−1 and f = 0.48 ± 0.07. (B) Antibody–porphyrin 3 conjugate (20 nM) produced only nonselective photoablation of Kv currents. Black circles, Kv4.2; rate = 0.016 ± 0.001 s−1 and f = 0.75 ± 0.02; gray circles, Kv2.1; rate = 0.023 ± 0.004 s−1 and f = 0.49 ± 0.03.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig5: Anionic porphyrin conjugates do not facilitate selective photoablation. Data points are relative peak currents during 0-mV pulses to patch-clamped whole cells. Photon flux per unit area is approximately four times greater than in all other figures because of the 20× objective used. (A) Antibody–porphyrin 2 conjugate (20 nM) produced only nonselective photoablation of Kv currents. Black circles, Kv4.2 from HEK cells; rate = 0.012 ± 0.001 s−1 and f = 0.62 ± 0.04; gray circles, Kv2.1 from CHO cells; rate = 0.014 ± 0.004 s−1 and f = 0.48 ± 0.07. (B) Antibody–porphyrin 3 conjugate (20 nM) produced only nonselective photoablation of Kv currents. Black circles, Kv4.2; rate = 0.016 ± 0.001 s−1 and f = 0.75 ± 0.02; gray circles, Kv2.1; rate = 0.023 ± 0.004 s−1 and f = 0.49 ± 0.03.
Mentions: The αKv4.2•1 porphyrin–mAb conjugates allowed selective photoablation of the targeted Kv channels. There was little photoablation of Kv4.3 or Kv2.1 in response to photon doses that eliminated Kv4.2 currents, suggesting that antibody targeting could direct the photoablation effect (Fig. 4). The effect of increasing photon dose was quantified as a photoablation rate, corresponding to the inverse time constant of current loss under our illumination conditions (Fig. 4 E). With prolonged illumination, current was gradually lost from the off-target Kv2.1 and Kv4.3 channels. This side effect indicated that although the anti-Kv antibody could direct photoablation to channels of interest, collateral damage also occurred. Alternate porphyrin chemistries were not as successful at enabling targeted photoablation. The mAb–porphyrin conjugates of the two anionic porphyrins synthesized (2 and 3) did not selectively ablate currents from the targeted Kv4.2 channel. Under more intense illumination, high photon doses yielded nonselective photoablation against all channels tested (Fig. 5). This suggests that the ability of the K57/1 antibody to recognize its epitope is compromised by attachment of the anionic conjugates. It is not clear why the anionic conjugates were not targeted as effectively as their cationic counterpart, αKv4.2•1. The weaker, nonselective effects of αKv4.2• and αKv4.2•3, together with the off-target effects of αKv4.2•1 on Kv2.1 and Kv4.3, demonstrate that a mechanism beyond antibody–epitope binding can also lead to photoablation. Yet, with this technique, nonselective side effects were dwarfed by selective antibody-guided photoablation of Kv currents.

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