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Figure 4. Effect of acute hypoxia on K+ currents after selectively blocking Kv1.2 and Kv2.1 channels with specific antibodies. A. Schematic diagram showing how the antibodies (Ab) are delivered to the cytosol through the pipette (left), and block K+ efflux by binding to the C-terminal epitope of the channel polypeptide (right). B: Hypoxia-induced inhibition of K+ currents (IK) in cells dialyzed with an anti-Kv1.2 antibody (Ab), an anti-Kv2.1 Ab, or an irrelevant antibody (IgG). * Ps0.001 vs. Kv1.2 Ab (Modified from Refs. 6 and 7).

In addition to Kv1.2, PC 12 cells also express a Kv2.1 a subunit. Kv2.1 channels have been proposed as possible Ko2 channels in PA (2, 24). Although the Kv2.1 gene is not regulated by chronic hypoxia in PC12 cells, it is still possible that afunctional Ko2 channel formed by the Kv2.1 subunit might be also expressed. It is known that Kv channel regulation can post-transcriptionally occur, independently on mRNA levels. In PC12 cells Kv2.1 protein levels are increased by exposure to nerve growth factor, with no change in steady-state levels of Kv2.1 mRNA (28). The molecular nature of the functional Ko2 channel/s in PC 12 cells was established by selective block of the channel activity with the corresponding antibody (Ab) (6). The anti-Kv antibodies used are prepared against the C-terminal part of their corresponding Kv channel protein. The anti-Kv Ab dialyzes into the cell through the patch pipette, binds to the corresponding antigenic portion of the Kvv a subunit and blocks, possibly by steric hindrance, the flow ofK+ ions through the channel pore (Fig. 4A). Using antibodies against the Kv1.2 polypeptide to block Kv1.2 channels and by comparing the obtained results with similar experiments performed in the presence of anti-Kv2.1 Ab, we have confirmed that the K02 channel in PC 12 cells is composed of Kv1.2 a subunits (Fig. 4B). The specificity of the anti-Kv1.2

Figure 4. Effect of acute hypoxia on K+ currents after selectively blocking Kv1.2 and Kv2.1 channels with specific antibodies. A. Schematic diagram showing how the antibodies (Ab) are delivered to the cytosol through the pipette (left), and block K+ efflux by binding to the C-terminal epitope of the channel polypeptide (right). B: Hypoxia-induced inhibition of K+ currents (IK) in cells dialyzed with an anti-Kv1.2 antibody (Ab), an anti-Kv2.1 Ab, or an irrelevant antibody (IgG). * Ps0.001 vs. Kv1.2 Ab (Modified from Refs. 6 and 7).

antibody was established by immunohistochemical and western blot experiments. We have also established the feasibility of using the anti-Kv1.2 antibody to selectively block the K+ current carried by Kv1.2 channels (6). The use of Kv channel antibodies as experimental tools to assess the molecular nature of the Ko2 channels has been done by others and elegantly applied recently by Sanchez and colleagues (2, 26). Dialysis of PC12 cells with specific antibodies against the Kvl.2 a subunit prevented the hypoxia-induced inhibition of voltage-activated K+ currents. Cells dialyzed with anti-Kv2.1 Ab maintained their response to hypoxia, as shown in Fig. 4B. This important finding confirmed that, in PC 12 cells, afunctional Kv1.2 a subunit is necessary for the response of the Ko2 channel to hypoxia and that the Kv2.1 channels are not 02-sensitive.

Figure 5. A model for hypoxic modulation of Ko2 channels in PC 12 cells and its functional consequences. PC 12 cells express at least four types of voltage-dependent K+ channels: delayed rectifier (Kdr), transient (Ktr), small conductance (Ksm) and Ca2+-activated (KCa) K+ channels. Exposure to acute hypoxia selectively inhibits the Kdr channel. The subsequent decrease in K+ currents through Kdr channels results in membrane depolarization, activation of voltage-dependent Ca2+ channels, increase in [Ca2+]|, and transmitter release. Kdr channels are encoded by different genes. Chronic hypoxia increases the expression of 02-sensitive Kv1.2 channels.

Figure 5. A model for hypoxic modulation of Ko2 channels in PC 12 cells and its functional consequences. PC 12 cells express at least four types of voltage-dependent K+ channels: delayed rectifier (Kdr), transient (Ktr), small conductance (Ksm) and Ca2+-activated (KCa) K+ channels. Exposure to acute hypoxia selectively inhibits the Kdr channel. The subsequent decrease in K+ currents through Kdr channels results in membrane depolarization, activation of voltage-dependent Ca2+ channels, increase in [Ca2+]|, and transmitter release. Kdr channels are encoded by different genes. Chronic hypoxia increases the expression of 02-sensitive Kv1.2 channels.

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