Channel Expression is Regulated by Auxiliary Regulatory Proteins

An emerging idea in the area of regulation of fonction and expression of Kv channels is the direct interaction of the pore-forming a subunits with K+ channel-associated proteins (KChAPs) and K+ channel-interacting proteins (KChlPs), regulators different from the Kv channel P subunits. These proteins have predominantly been described in the brain and heart and play an important role in regulating Kv channel function and expression in these tissues; little data exist regarding their role on channel expression in the pulmonary vasculature. KChAPs function mainly as chaperones (39, 40) for specific Kv channels (e.g. Kv1.3 and Kv4.3), leading to enhanced protein expression of the channels. However, this interaction is transient and does not affect channel activity.

Three KChlPs have been identified (KChIPl-3) that modulate /K(V). In terms of channel kinetics, KChIPs appear to work oppositely to the subunit in that total current is modified, activation is slowed, and the chaperone proteins accelerate the channel recovery from inactivation (4). In addition, all KChIPs co-localize and co-immunoprecipitate with Kv channel a subunits, suggesting that they may be part of the channel complex (4). KChlPs have four EF-hand-like domains, each consisting of two perpendicular 10 to 12 residue a-helices with a 12-residue loop region, forming a single Ca2+-binding site. The KChlP-induced modulation of Kv channel activity is sensitive to Ca2+ levels (72). This suggests that a possible mechanism by which the cell senses and responds to a transient rise in [Ca2+]cy, would be the modulation of Kv channel activity by Ca2+ regulation of KChIPs or KChIP-Kv channel interaction.

Figure 7. KChAP mRNA expression in PASMC and MASMC is differentially modulated by hypoxia. A: PCR-amplified products for KChAP and P-actin in PASMC and MASMC incubated under normoxic (N) and hypoxic (H) conditions. B: Summarized mRNA levels (normalized to P-actin control) from the data presented in A. ** PO.Ol vs. normoxia.

Figure 7. KChAP mRNA expression in PASMC and MASMC is differentially modulated by hypoxia. A: PCR-amplified products for KChAP and P-actin in PASMC and MASMC incubated under normoxic (N) and hypoxic (H) conditions. B: Summarized mRNA levels (normalized to P-actin control) from the data presented in A. ** PO.Ol vs. normoxia.

Studies in PASMC involving the characterization of KChAPs and KChIPs are limited with respect to hypoxia. Our preliminary data showed that chronic hypoxia downregulated KChAP in PASMC (Fig. 7). Inhibition of these intracellular regulators of Kv channels may result in decreased expression of Kv channels or inability of Kv channels to open. A chronically hypoxic environment would result in long-term effects on these proteins that then contribute to pulmonary vascular remodeling and subsequent pulmonary hypertension.

7. Role of Plasmalemmal K+ Channels in Regulating Apoptosis in PASMC

The precise control of the balance between PASMC proliferation and apoptosis is important in maintaining the structural and functional integrity of the pulmonary vasculature. As was stated earlier, increased PASMC proliferation and/or decreased apoptosis, result in pulmonary vascular wall thickening that elevates pulmonary vascular resistance, a process that is partially responsible in the development of hypoxic pulmonary hypertension.

Apoptotic volume decrease or cell shrinkage is the early hallmarks of apoptosis (47, 112). Maintenance of a high concentration of cytoplasmic K+ ([K+]cyt) is essential to the regulation of normal ceU volume. Thus, apoptotic ceU shrinkage may result partly from a decrease in [K+]cyt due to increased K+efflux through opened K+ channels (11, 41, 47, 112). In addition to its role in the control of ceU volume, a high [K+]cyt is required for suppression of caspases and nucleases (34), the final mediators of apoptosis (85, 103). Accordingly, activation of K+ channels in the plasma membrane would induce apoptotic volume decrease and apoptosis by enhancing K+ efflux or loss, whereas inhibition of K+ channel activity would attenuate apoptotic volume decrease by maintaining sufficient [K+] in the cytoplasm to inhibit apoptosis.

In human PASMC, treatment with staurosporine, a potent inducer of apoptosis, increased (Fig. 8Aa) (37), caused cell shrinkage (Fig. 8Ab), and increased the percentage of the cells undergoing apoptosis (Fig. 8Ac). The time courses for staurosporine-mediated effects on /K(V), ceU volume change and apoptosis were, however, quite different. The staurosporine-mediated increase in /K(V) took place at first foUowed by apoptotic cell shrinkage. The correlated increase in /K(V) and ceU shrinkage far preceded the increase in the percentage of apoptotic cells (Fig. 8Ac). Blockade of Kv channels with 4-aminopyridine inhibited staurosporine-induced increase in /K(V) (Fig. 8Ba) and attenuated staurosporine-induced apoptosis (Fig. 8B&). These results suggest that the earliest event in apoptosis involves an increase in Kv channel activity which subsequently results in efflux and cell volume decrease, and ultimately causes apoptosis.

Bcl-2 is an antiapoptotic membrane protein which enhances cell survival by preventing cytochrome c release from the mitochondrial intermembrane space to the cytosol. In keeping with the known importance of transmembrane movement in modulating cell death, it is not surprising to observe that the overexpression of human Bcl-2 in rat PASMC downregulated the expression of Kv channel a subunits (particularly Kvl.l, Kvl.5, and Kv2.1) (Fig. 8Ca) and decreased /K(V) (Fig. 8Cb). Additionally, staurosporine-induced augmentation in /K(V) and apoptosis were both attenuated in the Bcl-2-transfected PASMC (Fig. 8Cc) (22).

In summary, function and expression of K+ channels play an important role in apoptosis in PASMC. Decreased activity of voltage-dependent K+ channels due to decreased expression or function of K+ channels during hypoxia leads to maintenance of a high [K+]cyl, which opposes apoptosis by preventing apoptotic cell shrinkage and by suppression of cytoplasmic caspases and nucleases. Therefore, decreased expression or function of Kv channels in PASMC may be an important anti-apoptotic mechanism and, hence, may significantly contribute to pulmonary vascular remodeling.

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Figure 8. Kv channel activity in PASMC is modulated by pro- and anti-apoptotic agents. A: Staurosporine (ST), a pro-apoptotic agent, enhances /K(V) (a) and causes apoptotic cell shrinkage (b) in rat PASMC. Time-courses of the ST-induced /K(V) increase (c, circles), cell volume decrease (c, triangles), and the percentage of apoptotic cells (c, bars). Note that increased /K(V) precedes cell shrinkage and apoptosis. B: Inhibition of Kv channels with 4-AP (5 mM) attenuates ST-induced apoptosis in human PASMC. C: Overexpression (+bcl-2) of Bcl-2 downregulates mRNA expression of Kvl.l, Kvl.5, and Kv2.1 (a), decreases amplitude of /K(V) (b), and inhibits ST-induced apoptosis (e). ***P<0.001 vs. control {-bcl-2) (Reproduced from Refs. 23,37, and 81).

Figure 8. Kv channel activity in PASMC is modulated by pro- and anti-apoptotic agents. A: Staurosporine (ST), a pro-apoptotic agent, enhances /K(V) (a) and causes apoptotic cell shrinkage (b) in rat PASMC. Time-courses of the ST-induced /K(V) increase (c, circles), cell volume decrease (c, triangles), and the percentage of apoptotic cells (c, bars). Note that increased /K(V) precedes cell shrinkage and apoptosis. B: Inhibition of Kv channels with 4-AP (5 mM) attenuates ST-induced apoptosis in human PASMC. C: Overexpression (+bcl-2) of Bcl-2 downregulates mRNA expression of Kvl.l, Kvl.5, and Kv2.1 (a), decreases amplitude of /K(V) (b), and inhibits ST-induced apoptosis (e). ***P<0.001 vs. control {-bcl-2) (Reproduced from Refs. 23,37, and 81).

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