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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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Dose dependence of photoablation. (A) Dose response of αKv4.2•1-mediated photoablation. Circles, relative peak currents from Kv4.2 CHO cells during 0-mV pulses with the indicated concentration of αKv4.2•1. Purple bar indicates illumination. Gray circles, 0 nM control; rate = 0.00015 ± 0.00004 s−1 and f = 1; black circles: 0.5 nM, rate = 0.019 ± 0.003 s−1 and f = 0.30 ± 0.02; 2.5 nM, rate = 0.021 ± 0.002 s−1 and f = 0.86 ± 0.02; 10 nM, rate = 0.052 ± 0.001 s−1 and f = 0.953 ± 0.004. (B) Summary data of αKv4.2•1 concentration versus photoinhibitory rates derived from exponential fitting. Circles, individual cells. 0 nM, n = 10; 2.5 nM, n = 5; 10 nM, n = 6.
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fig7: Dose dependence of photoablation. (A) Dose response of αKv4.2•1-mediated photoablation. Circles, relative peak currents from Kv4.2 CHO cells during 0-mV pulses with the indicated concentration of αKv4.2•1. Purple bar indicates illumination. Gray circles, 0 nM control; rate = 0.00015 ± 0.00004 s−1 and f = 1; black circles: 0.5 nM, rate = 0.019 ± 0.003 s−1 and f = 0.30 ± 0.02; 2.5 nM, rate = 0.021 ± 0.002 s−1 and f = 0.86 ± 0.02; 10 nM, rate = 0.052 ± 0.001 s−1 and f = 0.953 ± 0.004. (B) Summary data of αKv4.2•1 concentration versus photoinhibitory rates derived from exponential fitting. Circles, individual cells. 0 nM, n = 10; 2.5 nM, n = 5; 10 nM, n = 6.

Mentions: The details of mAb-guided photoablation were investigated to better understand how K current elimination occurs. The degree of photoablation was found to be dependent on the concentration of K57/1 antibody–porphyrin conjugate applied. Increased concentration resulted in photoablation of a greater fraction of K current (Fig. 7 A). During photostimulation, the rate of Kv4.2 current decay did not reveal a consistent difference between the conjugate concentrations tested (Fig. 7 B). The dynamics of remaining current elicited from cells after partial photoablation was similar to control. The sigmoid activation kinetics of Kv4.2 were similar to currents elicited before illumination, as were decaying tail currents from deactivation at negative voltages (Fig. 8 A), and inactivation (Fig. 3 A). The normal behavior of current remaining after photoablation contrasts with the progressive alteration of the channel gating machinery, as is seen with cyclic nucleotide–gated channels (Middendorf and Aldrich, 2000; Middendorf et al., 2000). The extent of Kv photoablation could be halted at a given level by the termination of illumination (Fig. 8 B), indicating that the physical processes leading to irreversible current loss are rapid events, rather than the triggering of a slow decay process.


Antibody-guided photoablation of voltage-gated potassium currents.

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

Dose dependence of photoablation. (A) Dose response of αKv4.2•1-mediated photoablation. Circles, relative peak currents from Kv4.2 CHO cells during 0-mV pulses with the indicated concentration of αKv4.2•1. Purple bar indicates illumination. Gray circles, 0 nM control; rate = 0.00015 ± 0.00004 s−1 and f = 1; black circles: 0.5 nM, rate = 0.019 ± 0.003 s−1 and f = 0.30 ± 0.02; 2.5 nM, rate = 0.021 ± 0.002 s−1 and f = 0.86 ± 0.02; 10 nM, rate = 0.052 ± 0.001 s−1 and f = 0.953 ± 0.004. (B) Summary data of αKv4.2•1 concentration versus photoinhibitory rates derived from exponential fitting. Circles, individual cells. 0 nM, n = 10; 2.5 nM, n = 5; 10 nM, n = 6.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig7: Dose dependence of photoablation. (A) Dose response of αKv4.2•1-mediated photoablation. Circles, relative peak currents from Kv4.2 CHO cells during 0-mV pulses with the indicated concentration of αKv4.2•1. Purple bar indicates illumination. Gray circles, 0 nM control; rate = 0.00015 ± 0.00004 s−1 and f = 1; black circles: 0.5 nM, rate = 0.019 ± 0.003 s−1 and f = 0.30 ± 0.02; 2.5 nM, rate = 0.021 ± 0.002 s−1 and f = 0.86 ± 0.02; 10 nM, rate = 0.052 ± 0.001 s−1 and f = 0.953 ± 0.004. (B) Summary data of αKv4.2•1 concentration versus photoinhibitory rates derived from exponential fitting. Circles, individual cells. 0 nM, n = 10; 2.5 nM, n = 5; 10 nM, n = 6.
Mentions: The details of mAb-guided photoablation were investigated to better understand how K current elimination occurs. The degree of photoablation was found to be dependent on the concentration of K57/1 antibody–porphyrin conjugate applied. Increased concentration resulted in photoablation of a greater fraction of K current (Fig. 7 A). During photostimulation, the rate of Kv4.2 current decay did not reveal a consistent difference between the conjugate concentrations tested (Fig. 7 B). The dynamics of remaining current elicited from cells after partial photoablation was similar to control. The sigmoid activation kinetics of Kv4.2 were similar to currents elicited before illumination, as were decaying tail currents from deactivation at negative voltages (Fig. 8 A), and inactivation (Fig. 3 A). The normal behavior of current remaining after photoablation contrasts with the progressive alteration of the channel gating machinery, as is seen with cyclic nucleotide–gated channels (Middendorf and Aldrich, 2000; Middendorf et al., 2000). The extent of Kv photoablation could be halted at a given level by the termination of illumination (Fig. 8 B), indicating that the physical processes leading to irreversible current loss are rapid events, rather than the triggering of a slow decay process.

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