| ATP-sensitive potassium channels (KATP channels) link the cellular energy levels to membrane potential and excitability in various cell types. They control many important functions like insulin secretion in pancreatic beta-cells; vascular tone in vascular smooth muscle cells; and action potential duration in cardiac myocytes and neurons under ischemic conditions. In cardiac myocytes, it has been shown that KATP channels on plasma membrane are composed of Kir6.2 and SUR2A subunits in 4:4 stoichiometry. Though many regulators of KATP channels and signal transduction mechanisms regulating opening or closing of KATP channels have been identified, much less is known about the subcellular localization of KATP channels which can have a profound impact on the temporal and spatial regulation by its signaling modulators. Many studies have localized KATP channels to nucleus and mitochondria, besides cellular plasma membrane. However, it not known if these are the same cell surface KATP channels, which are also targeted to mitochondria, or some other isoform of KATP channel. The plasma membrane itself is not homogenous through out. It is interspersed by sub-domains rich in sterols and glycosphingolipids. In majority of cases, this sub-domain organization is orchestrated by special proteins called as caveolins, which are the main structural components of caveolae. Caveolae are small (50 to 100 nm), cholesterol and sphingolipid enriched "cave"-like invaginations of the surface membrane. These specialized lipid microdomains have the ability to selectively compartmentalize many signaling molecules, including many modulators of KATP channels. Since little is known about the subcellular locations of KATP channel protein, we designed our studies to characterize the localization of KATP channel protein in cardiac myocytes.;We first endeavored to look at KATP channel localization along the plasma membrane in rat cardiac myocytes. Using a variety of different techniques on isolated murine cardiomyocytes, we found that majority of cardiac KATP channels are localized to caveolin-enriched membrane microdomains. Further, whole-cell voltage-clamp recording in both adult and neonatal cardiac myocytes confirmed our hypothesis that caveolae integrity is essential for activation of KATP channel by its modulator adenosine (Chapter 1). Adenosine released from ischemic myocardium is a very important modulator of KATP channels. These findings have significant implications for cardioprotective role of KATP channels during ischemic conditions.;A signaling function for caveolins either on their own (direct) or by acting as scaffolding proteins (indirect) has also been described. To test its relevance for KATP channels, we employed HEK293T cells transfected with recombinant cardiac KATP channels (Kir6.2/SUR2A) with or without caveolin-3 (Cav-3, a muscle-specific caveolin isoform). We found that Cav-3 has significant inhibitory effect on KATP channel current density which can be reversed by a scaffolding domain peptide from caveolin-3 protein sequence (CSD) (Chapter 2). This crucial experiment indicates a very interesting, dual regulation of KATP channels by caveolins and caveolae. Though caveolae structure ensures that KATP channel modulators are close to the channel proteins for efficient signal transduction, caveolin-3 protein through direct or indirect interaction with channel proteins makes sure that they remain inhibited until required.;Whether Kir6.2 containing KATP channel are present in mitochondria or not is controversial; nevertheless no one has ever studied the trafficking aspect of KATP channel to mitochondria. In our effort to elucidate the subcellular localization of cardiac KATP channels, we hypothesized that localization of Kir6.2-containing KATP in mitochondria can be increased by activation of protein kinase C (PKC). Utilizing KATP -deficient COS-7 cells, we reported a novel finding that a specific protein kinase C (PKC) isoform, PKCepsilon, promotes mitochondrial import of Kir6.2-containing KATP channels from cytosol. These findings were further corroborated with functional data using mitochondrial potential measurement studies.;Collectively, our data demonstrated that besides bulk plasma membrane, Kir6.2 containing cardiac KATP channels are localized to two distinct sub-cellular locations, namely caveolae and mitochondria. Their localization can be modulated by specific regulatory pathways, and furthermore their regulation can be affected by their sub-cellular localization. This provides valuable insight into the mechanisms regulating KATP channels, which have been implicated for cardioprotection under ischemic conditions. |
