Molecular Properties of Ko2 Channels in PC 12 Cells

Further molecular biological experiments have indicated that the Ko2 channel in PC 12 cells belongs to the KV1 subfamily (6, 7). Kv channels are tetrameric arrangements of 4 separate pore-forming a subunits and auxiliary P subunits (5). The genes that encode functional Kv a subunits are classified in 4 major subfamilies: Kvl-Kv4. New subfamilies (Kv5.1-Kv10) are recently added. The molecular composition of Ko2 channels is still under investigation. The Kv a subunits implicated informing Ko2 channels in PA are: Kv2.1 (homomultimer or heteromultimer in combination with the electrically silent Kv 9.3 a subunit), Kv1.5, Kv1.2, Kv3.1b (21). Kv4.1 and Kv4.3 have been proposed in rabbit CB (26). We have identified the Kv1.2 a subunit as an important component of the Ko2 channel in PC 12 cells (6, 7). The 02-sensitivity ofKv1.2 homomultimers was also confirmed in recombinant studies in L cells or Xenopus oocytes (6,16).

Figure 3. Expression of genes encoding for the a subunit of delayed-rectifier type of K+ channels in PC12 cells. A: RT-PCR products for Kv1.2, Kv1.3, Kv2.1, Kv3.1, and Kv3.2 in PC12 cells maintained for 18 hrs in a normoxic (C) or hypoxic (H, 10% 02)) incubator. The relative intensity (in arbitrary units) of each band, averaged from 7 experiments for Kv1.2 and 4 experiments for other Kv channels, is shown in the bar graph. B: 02-sensitivity of PC 12 cells after prolonged exposure to hypoxia (18 hr, 10% 02). Whole-cell K+ currents (IK) elicited by a 800 ms test pulse from -70 to +50 mV were measured before (C) and after 1 -min exposure to hypoxia (H). Hypoxic inhibition of IK is significantly greater in cells maintained in hypoxia than in cells maintained in normoxia (19 % vs 35% in hypoxic cells; P<0.05) (Modified from Refs. 6 and 7).

Figure 3. Expression of genes encoding for the a subunit of delayed-rectifier type of K+ channels in PC12 cells. A: RT-PCR products for Kv1.2, Kv1.3, Kv2.1, Kv3.1, and Kv3.2 in PC12 cells maintained for 18 hrs in a normoxic (C) or hypoxic (H, 10% 02)) incubator. The relative intensity (in arbitrary units) of each band, averaged from 7 experiments for Kv1.2 and 4 experiments for other Kv channels, is shown in the bar graph. B: 02-sensitivity of PC 12 cells after prolonged exposure to hypoxia (18 hr, 10% 02). Whole-cell K+ currents (IK) elicited by a 800 ms test pulse from -70 to +50 mV were measured before (C) and after 1 -min exposure to hypoxia (H). Hypoxic inhibition of IK is significantly greater in cells maintained in hypoxia than in cells maintained in normoxia (19 % vs 35% in hypoxic cells; P<0.05) (Modified from Refs. 6 and 7).

PC 12 express different genes that may encode the a subunits of delayed rectifier types of K+channels: Kv1.2, Kv 1.3 ,KVTo identify the Kv a subunits that form the Ko2 channel in PC 12 cells, we studied the regulation of Kv gene expression by chronic hypoxia. It is known that 02-sensitive tissues (e.g., the carotid bodies) enhance their chemosensitivity in the process of adaptation to chronic hypoxia (29). Regulation of the Ko2 channel expression by chronic hypoxia has also been shown to occur in other cell types (2, 8, 25). We found that expression of the Kv1.2, but not other Kv genes, was increased by -40% after prolonged exposure to hypoxia (Fig. 3A). The increased expression of the

Kv1.2 gene correlated with an enhanced acute response to hypoxia in those cells pre-exposed to 10% 02 for 18 hrs (Fig. 3B) (7). These data provided the first evidence that the Ko2 channel in PC 12 cells is a homo- or heterotetramer of Kv1.2 subunit, as the pharmacological and electrophysiological studies described above had suggested. Indeed, it was shown previously that expression of Kv1.2 gene in a mammalian cell line leads to a delayed rectifier K+ channel of 18 pS conductance (13). The conductance and gating properties ofthis channel are very similar to those of the K02 channel we identified in PC 12 cells. Expression of other Kv a genes is associated with K+channels of much different conductance (e.g., 27 pSfor Kv3.1) (13).

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