Characterization of novel Pannexin 1 isoforms from rat pituitary cells and their association with ATP-gated P2X channels

Our previous studies have showed that Pannexin 1 (Panx1), a member of a recently discovered family of gap junction proteins, is expressed in the pituitary gland. Here we investigated the presence and expression pattern of Panx1 isoforms in pituitary cells, their roles in ATP release, and their association with purinergic P2X receptor subtypes that are native to pituitary cells. In addition to the full-size Panx1, termed Panx1a, pituitary cells also express two novel shorter isoforms, termed Panx1c and Panx1d, which formation reflects the existence of alternative splicing sites in exons 2 and 4, respectively. Panx1c is lacking the Phe108-Gln180 sequence and P2X1d is missing the Val307-Cys426 C-terminal end sequence. Confocal microscopy and biotin labeling revealed that Panx1a is expressed in the plasma membrane, whereas Panx1c and Panx1d show the cytoplasmic localization when expressed as homomeric proteins. The three Panx1 isoforms and Panx2 form homomeric and heteromeric complexes in any combination. These splice forms can also physically associate with ATP-gated P2X2, P2X3, P2X4, and P2X7 receptor channels. The Panx1a-mediated ATP release in AtT-20 immortalized pituitary cells is attenuated when co-expressed with Panx1c or Panx1d. These results suggest that Panx1c and Panx1d may serve as dominant-negative effectors to modulate the functions of Panx1a through formation of heteromeric channels. The complex patterns of Panx1 expression and association could also define the P2X-dependent roles of these channels in cell types co-expressing both proteins.     

Expression of cytochrome P450-4A isoforms in the rat cremaster muscle microcirculation

OBJECTIVE: This study sought to identify any specific cytochrome P450 (CYP450) -4A enzyme isoforms expressed in arterioles and/or the surrounding parenchymal tissue of the rat cremaster muscle. METHODS: RT-PCR was used to detect the presence of specific CYP450-4A isoforms in isolated muscle fibers and arterioles from the cremaster muscle of Sprague-Dawley rats; CYP450-4A protein expression was determined by Western blotting. RESULTS: CYP450-4A3 mRNA was expressed in isolated muscle fibers and in cremasteric arterioles, while CYP450-4A8 mRNA was expressed only in cremasteric arterioles. CYP450-4A1 and CYP450-4A2 mRNA were not expressed in arterioles and skeletal muscle cells, although all four isoforms were strongly expressed in the liver. CYP450-4A protein was detected in both the isolated muscle fibers and in the isolated arterioles. CONCLUSIONS: The present study identifies the specific pattern of cytochrome P450-4A isoform expression in arterioles and parenchymal cells of the skeletal muscle microcirculation, and supports the hypothesis that the cytochrome P-450 enzymes may play a role in the regulation of microvascular function in the skeletal muscle microcirculation.     

Balloon catheterization induces arterial expression of new Tenascin-C isoform

Migration of smooth muscle cells (SMCs) across the internal elastic lamina is a key step in the development of atherosclerotic or restenotic plaques. Cell movement is a complex and highly dynamic phenomenon, involving the continuous formation and breakage of attachments with the underlying substratum. Tenascin-C (Tn-C), a counter-adhesive extracellular matrix protein, is comprised of several isoforms with distinct biological activities. Neither the structure nor function of these isoforms in SMCs has been defined. We have used primers and RT-PCR to fully identify Tn-C isoforms expressed by SMCs. Cloning and sequence analysis of the PCR product indicated that SMCs express a Tn-C isoform with only repeats A1 and A2 of fibronectin type III repeats. Using A1A2-specific antibodies, cDNA probes and RNase mapping, we observed that the A1A2 isoform is predominantly expressed by cultured SMCs derived from aorta of newborn rats, and its expression is up-regulated by PDGF-BB. In contrast, the expression of this isoform is markedly down-regulated in the SMCs derived from adult rat aorta. Western and Northern blots of injured rat carotid arteries revealed that the A1A2-isoform is expressed in response to injury. Using cultured SMCs, we found that the recombinant A1A2 protein that was found in the newly discovered Tn-C isoform promotes SMC chemotaxis. We conclude that Tn-C isoforms are expressed in a regulated fashion in vascular system. Our findings suggest a new role of Tn-C isoforms in the remodeling of vascular wall.     

Substance P is released from the endothelium of normal and capsaicin-treated rat hind-limb vasculature, in vivo, by increased flow.

The rat hind-limb vasculature releases substance P when subjected to a rapid increase in flow through the vascular bed. This release also occurs during high flow after rats have been capsaicinized, when loss of substance P-containing nerve fibers was verified by immunohistochemistry. Air treatment, a procedure shown by transmission electron microscopy to have removed endothelial cells from the arteries but not arterioles or capillaries of the hind-limb preparations, eliminated this release. Thus, the substance P released is unlikely to arise from perivascular nerves but rather from arterial endothelial cells.     

Metabotropic Ca(2+) channel-induced Ca(2+) release and ATP-dependent facilitation of arterial myocyte contraction

Voltage-gated Ca(2+) channels in arterial myocytes can mediate Ca(2+) release from the sarcoplasmic reticulum and, thus, induce contraction without the need of extracellular Ca(2+) influx. This metabotropic action of Ca(2+) channels (denoted as calcium-channel-induced calcium release or CCICR) involves activation of G proteins and the phospholipase C-inositol 1,4,5-trisphosphate pathway. Here, we show a form of vascular tone regulation by extracellular ATP that depends on the modulation of CCICR. In isolated arterial myocytes, ATP produced facilitation of Ca(2+)-channel activation and, subsequently, a strong potentiation of CCICR. The facilitation of L-type channel still occurred after full blockade of purinergic receptors and inhibition of G proteins with GDPbetaS, thus suggesting that ATP directly interacts with Ca(2+) channels. The effects of ATP appear to be highly selective, because they were not mimicked by other nucleotides (ADP or UTP) or vasoactive agents, such as norepinephrine, acetylcholine, or endothelin-1. We have also shown that CCICR can trigger arterial cerebral vasoconstriction in the absence of extracellular calcium and that this phenomenon is greatly facilitated by extracellular ATP. Although, at low concentrations, ATP does not induce arterial contraction per se, this agent markedly potentiates contractility of partially depolarized or primed arteries. Hence, the metabotropic action of L-type Ca(2+) channels could have a high impact on vascular pathophysiology, because, even in the absence of Ca(2+) channel opening, it might mediate elevations of cytosolic Ca(2+) and contraction in partially depolarized vascular smooth muscle cells exposed to small concentrations of agonists.     

Mechanisms of ATP release and signalling in the blood vessel wall

The nucleotide adenosine 5'-triphosphate (ATP) has classically been considered the cell's primary energy currency. Importantly, a novel role for ATP as an extracellular autocrine and/or paracrine signalling molecule has evolved over the past century and extensive work has been conducted to characterize the ATP-sensitive purinergic receptors expressed on almost all cell types in the body. Extracellular ATP elicits potent effects on vascular cells to regulate blood vessel tone but can also be involved in vascular pathologies such as atherosclerosis. While the effects of purinergic signalling in the vasculature have been well documented, the mechanism(s) mediating the regulated release of ATP from cells in the blood vessel wall and circulation are now a key target of investigation. The aim of this review is to examine the current proposed mechanisms of ATP release from vascular cells, with a special emphasis on the transporters and channels involved in ATP release from vascular smooth muscle cells, endothelial cells, circulating red blood cells, and perivascular sympathetic nerves, including vesicular exocytosis, plasma membrane F(1)/F(0)-ATP synthase, ATP-binding cassette (ABC) transporters, connexin hemichannels, and pannexin channels.     

INAUGURAL ARTICLE by a Recently Elected Academy Member:A physiologic rise in cytoplasmic calcium ion signal increases pannexin1 channel activity via a C-terminus phosphorylation by CaMKII

Pannexin1 (Panx1) channels are ubiquitously expressed in vertebrate cells and are widely accepted as adenosine triphosphate (ATP)-releasing membrane channels. Activation of Panx1 has been associated with phosphorylation in a specific tyrosine residue or cleavage of its C-terminal domains. In the present work, we identified a residue (S394) as a putative phosphorylation site by Ca²⁺/calmodulin-dependent kinase II (CaMKII). In HeLa cells transfected with rat Panx1 (rPanx1), membrane stretch (MS)-induced activation—measured by changes in DAPI uptake rate—was drastically reduced by either knockdown of Piezo1 or pharmacological inhibition of calmodulin or CaMKII. By site-directed mutagenesis we generated rPanx1S394A-EGFP (enhanced green fluorescent protein), which lost its sensitivity to MS, and rPanx1S394D-EGFP, mimicking phosphorylation, which shows high DAPI uptake rate without MS stimulation or cleavage of the C terminus. Using whole-cell patch-clamp and outside-out excised patch configurations, we found that rPanx1-EGFP and rPanx1S394D-EGFP channels showed current at all voltages between ±100 mV, similar single channel currents with outward rectification, and unitary conductance (∼30 to 70 pS). However, using cell-attached configuration we found that rPanx1S394D-EGFP channels show increased spontaneous unitary events independent of MS stimulation. In silico studies revealed that phosphorylation of S394 caused conformational changes in the selectivity filter and increased the average volume of lateral tunnels, allowing ATP to be released via these conduits and DAPI uptake directly from the channel mouth to the cytoplasmic space. These results could explain one possible mechanism for activation of rPanx1 upon increase in cytoplasmic Ca²⁺ signal elicited by diverse physiological conditions in which the C-terminal domain is not cleaved.

Pannexin1 (Panx1) is a glycoprotein ubiquitously expressed by vertebrate cells (1, 2). It oligomerizes as a heptamer forming a nonjunctional membrane channel with distinct open conformations and multimodal activation (3–5). Without stimulation, Panx1 channels adopt a conformation that appears to be sensitive to changes in membrane potential (strong depolarization) and selective for chloride ions (6, 7). However, Panx1 channels undergo conformational changes upon different types of stimulation, e.g., low oxygen tension, high extracellular K⁺ concentration ([K⁺]_o), membrane stretch (MS), different types of receptor activation, and elevated cytoplasmic free Ca²⁺ concentration [Ca²⁺]_i, affecting the probability of dwelling in several distinct channel states with unique conductance and/or permeability properties (8–14). After stimulation, Panx1 channels are “activated” and allow the passage of small molecules (i.e., glucose and adenosine triphosphate [ATP]), monovalent cations (15), and small cationic molecules such as DAPI (16).The understanding about electrophysiological and permeability properties of Panx1 channels has changed over the years (3, 4). The current view of the Panx1 channel under normal conditions is that it behaves as a constitutively active and selective chloride channel, with outward rectifying properties and no (or very low) permeability to ATP or cationic dyes (6, 17–19). This channel conformation exhibits unitary conductance from ∼15 pS at negative potentials to ∼90 pS at positive potentials (unitary conductance varies depending on ion concentration and composition of recording solutions and cell type). Another consensus is that Panx1 channels need to be “activated” to become permeable to ATP and cationic dyes. However, it is a matter of debate whether this “activation” requires a big conformational change to allow ATP passage (3, 20).Under various conditions including high [K⁺]_o, oxygen–glucose deprivation, hypertonic conditions, or exposure to caffeine, a nonrectifying large-conductance (>300 pS) ATP-releasing channel identified as a Panx1 channel has been reported (8, 9, 21, 22). Nonetheless, removal of the C terminus and activation of α1D-adrenoreceptors also “activate” Panx1 channels and induce ATP release and cationic dye uptake without a mayor change in single-channel conductance and rectifying properties (20). A rise in [Ca²⁺]_i is a converging point of numerous cellular stimuli. Notably, a graded increase in Panx1 channel activity with increasing [Ca²⁺]_i has been demonstrated in Xenopus oocytes (20), but the molecular mechanism remains largely unknown. In diverse cell types, rises in [Ca²⁺]_i have been related to purinergic signaling induced by extracellular ATP, which activates metabotropic or ionotropic P2 receptors (8, 23–25). A putative stretch sensitivity of Panx1 channels has been challenged since they were not activated by swelling in Xenopus oocytes (26). Nonetheless, the participation of a well-accepted stretch-activated membrane channel, such as Piezo1, might explain the MS-induced rise in [Ca²⁺]_i and its subsequent Panx1 channel activation. At least two examples favor this hypothesis. In urothelial cell cultures Piezo1 is involved in stretch-evoked Ca²⁺ influx and ATP release via Panx1 channels (27), whereas in endothelial cells Piezo1 controls blood pressure by mediating flow-induced ATP release via Panx1 channels (28).Here, we describe a phosphorylation-dependent mechanism activated by a transient and localized rise in cytosolic Ca²⁺ signal which involves upstream activation of Piezo1 channels, and downstream activation of calmodulin and CaMKII, resulting in phosphorylation of rat Panx1 (rPanx1) and conformational changes of lateral tunnels and selectivity filter. The outcome of these events is elevated Panx1 channel activity and permeability to DAPI, without significant modifications in single-channel conductance, outward rectifying properties, or length of the C-terminal end of Panx1. These findings indicate that rPanx1 channels are not directly sensitive to MS, and their permeability properties are modulated by phosphorylation-dependent signaling cascades under physiological conditions.     

Angiotensin II-activated protein kinase C targets caveolae to inhibit aortic ATP-sensitive potassium channels

OBJECTIVE: The vasoconstrictor angiotensin II (Ang II) acts at G(q/11)-coupled receptors to suppress ATP-sensitive potassium (K(ATP)) channel activity via activation of protein kinase C (PKC). The aim of this study was to determine the PKC isoforms involved in the Ang II-induced inhibition of aortic K(ATP) channel activity and to investigate potential mechanisms by which these isoforms specifically target these ion channels. METHODS AND RESULTS: We show that the inhibitory effect of Ang II on pinacidil-evoked whole-cell rat aortic K(ATP) currents persists in the presence of G?6976, an inhibitor of the conventional PKC isoforms, but is abolished by intracellular dialysis of a selective PKCepsilon translocation inhibitor peptide. This suggests that PKC-dependent inhibition of aortic K(ATP) channels by Ang II arises exclusively from the activation and translocation of PKCepsilon. Using discontinuous sucrose density gradients and Western blot analysis, we show that Ang II induces the translocation of PKCepsilon to cholesterol-enriched rat aortic smooth muscle membrane fractions containing both caveolin, a protein found exclusively in caveolae, and Kir6.1, the pore-forming subunit of the vascular K(ATP) channel. Immunogold electron microscopy of rat aortic smooth muscle plasma membrane sheets confirms both the presence of Kir6.1 in morphologically identifiable regions of the membrane rich in caveolin and Ang II-evoked migration of PKCepsilon to these membrane compartments. CONCLUSIONS: Ang II induces the recruitment of the novel PKC isoform, PKCepsilon, to arterial smooth muscle caveolae. This translocation allows PKCepsilon access to K(ATP) channels compartmentalized within these specialized membrane microdomains and highlights a potential role for caveolae in targeting PKC isozymes to an ion channel effector.     

Direct evidence of leukocyte adhesion in arterioles by angiotensin II

Although leukocytes adhere in arteries in various vascular diseases, to date no endogenous proinflammatory molecule has been identified to initiate leukocyte adhesion in the arterial vasculature. This study was undertaken to assess angiotensin II (Ang II)-induced leukocyte adhesion in arterioles in vivo. Rats received intraperitoneal injections of Ang II; 4 hours later, leukocyte recruitment in mesenteric microcirculation was examined using intravital microscopy. Ang II (1 nM) produced significant arteriolar leukocyte adhesion of mononuclear cells. Using function-blocking monoclonal antibodies (mAbs) against different rat cell adhesion molecules (CAMs), we discovered that this effect was dependent on P-selectin and beta(2)-integrin. In postcapillary venules, Ang II also induced leukocyte infiltration, which was reduced by P-selectin and by beta(2)- and alpha(4)-integrin blockade. Interestingly, neutrophils were the primary cells recruited in venules. Although beta(2)-integrin expression in peripheral leukocytes of Ang II-treated animals was not altered, it was increased in peritoneal cells. Immunohistochemical studies revealed increased P-selectin, E-selectin, intercellular cell adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1) expression in response to Ang II in arterioles and venules. These findings provide the first evidence that Ang II causes leukocyte adhesion to the arterial endothelium in vivo at physiologically relevant doses. Therefore, Ang II may be a key molecule in cardiovascular diseases in which leukocyte adhesion to the arteries is a characteristic feature.     

Capillary perfusion and wall shear stress are restored in the coronary circulation of hypertrophic right ventricle

It has been shown that right ventricle (RV) hypertrophy involves significant compensatory vascular growth and remodeling. The objective of the present study was to determine the functional implications of the vascular growth and remodeling through a full flow analysis of arterial tree down to first capillary segments. A computer reconstruction of RV branches including the proximal right coronary artery to the posterior descending artery was established based on measured morphometric data in arrested, vasodilated porcine heart. The flows were computed throughout the reconstructed trees based on conservation of mass and momentum and appropriate pressure boundary conditions. It was found that the flow rate was significantly increased in large epicardial coronary arteries in hypertrophic as compared with control hearts but normalized in the intramyocardial coronary arteries and smaller vessels in RV hypertrophy primarily because of the significant increase in number of arterioles. Furthermore, the wall shear stress was restored to nearly homeostatic levels throughout most of the vasculature after 5 weeks of RV hypertrophy. The compensatory remodeling in RV hypertrophy functionally restores the perfusion at the arteriolar and capillary level and wall shear stress in most of larger vessels. This is the first full analysis of coronary arterial tree, with millions of vessels, in cardiac hypertrophy that reveals the compensatory adaptation of structure to function.     

Epithelial expression of angiogenic growth factors modulate arterial vasculogenesis in human liver development

Intrahepatic bile ducts maintain a close anatomical relationship with hepatic arteries. During liver ontogenesis, the development of the hepatic artery appears to be modulated by unknown signals originating from the bile duct. Given the capability of cholangiocytes to produce angiogenic growth factors and influence peribiliary vascularization, we studied the immunohistochemical expression of vascular endothelial growth factor (VEGF), angiopoietin-1, angiopoietin-2, and their cognate receptors (VEGFR-1, VEGFR-2, Tie-2) in fetal human livers at different gestational ages and in mice characterized by defective biliary morphogenesis (Hnf6(-/-)). The results showed that throughout the different developmental stages, VEGF was expressed by developing bile ducts and angiopoietin-1 by hepatoblasts, whereas their cognate receptors were variably expressed by vascular cells according to the different maturational stages. Precursors of endothelial and mural cells expressed VEGFR-2 and Tie-2, respectively. In immature hepatic arteries, endothelial cells expressed VEGFR-1, whereas mural cells expressed both Tie-2 and Angiopoietin-2. In mature hepatic arteries, endothelial cells expressed Tie-2 along with VEGFR-1. In early postnatal Hnf6(-/-) mice, VEGF-expressing ductal plates failed to incorporate into the portal mesenchyma, resulting in severely altered arterial vasculogenesis. CONCLUSION: The reciprocal expression of angiogenic growth factors and receptors during development supports their involvement in the cross talk between liver epithelial cells and the portal vasculature. Cholangiocytes generate a VEGF gradient that is crucial during the migratory stage, when it determines arterial vasculogenesis in their vicinity, whereas angiopoietin-1 signaling from hepatoblasts contributes to the remodeling of the hepatic artery necessary to meet the demands of the developing epithelium.     

Identification of phospholipase C beta isoforms and their location in cultured vascular smooth muscle cells of pig, human and rat

OBJECTIVES: Four phospholipase C (PLC) beta isoforms have been described in pig aortic vascular smooth muscle. The aim was to determine if all four PLC beta isoforms are commonly expressed in vascular smooth muscle cells (VSMC) of three species, i.e. pig, human and rat, and if the individual isoforms had distinctive intracellular distributions. METHODS: Vascular smooth muscle cell cultures were derived from explants of porcine and rat aorta and a human renal artery cell line. PLC beta isoform content was resolved using Western blotting. Intracellular location was determined by immunocytochemistry and confocal microscopy. RESULTS: All three species expressed PLCs, beta 1, beta 2, beta 3 and beta 4. In all species, PLC beta 1 demonstrated foci of concentration throughout the cytoplasm; PLC beta 2 demonstrated a punctate pattern that was principally at the cell periphery or was in the Golgi, depending upon the antibody used; PLC beta 3 was also cytoplasmic but showed a different pattern from PLC beta 1 and PLC beta 4 was cytoplasmic, except in pig quiescent cells, where it was associated with filamentous structures at the intersection with the plasma membrane. CONCLUSIONS: VSMCs of three different species express all four PLC beta isoforms. Each isoform has a unique and consistent signature of distribution that is generally common to all species.     

Diversity among tight junctions in rat kidney: glomerular slit diaphragms and endothelial junctions express only one isoform of the tight junction protein ZO-1.

ZO-1 is a 225-kDa peripheral membrane protein present in all tight junctions. It was recently shown to consist of two isoforms that differ in the presence of an internal 80-amino acid domain termed motif-alpha. To obtain information on their distribution and potential functional significance we have localized the two isoforms in rat kidney by using antibodies that recognize either both ZO-1 isoforms or the larger, motif-alpha-containing isoform. By immunofluorescence, staining with both antibodies was demonstrated at all tight junctions of tubular epithelial cells and the epithelial cells of Bowman's capsule. In contrast, the motif-alpha-containing isoform was absent from the slit diaphragms of the glomerular epithelium and the tight junctions of glomerular and peritubular capillary endothelial cells. This restricted isoform expression was confirmed by immunoblot analysis comparing proteins from purified glomeruli with those from kidney cortex or medulla. Thus, while both isoforms are expressed in typical epithelial tight junctions, only a single isoform, lacking motif-alpha, is expressed in the highly specialized slit diaphragms, where the intercellular spaces are normally open, and in endothelial junctions, which are readily opened by physiologic signals. The differential expression of ZO-1 isoforms in structurally and functionally distinct junctions in the kidney suggests that they may contribute to defining the variable functional properties, in particular the lability of these intercellular junctions.     

Induction of Cyclooxygenase-2 Following Anoxic Stress in Piglet Cerebral Arteries

Objective: Ischemic stress causes damage to cerebrovascular endothelium and alters arteriolar responses to prostanoid-dependent stimuli. However, effects of ischemic stress on cyclooxygenase (COX) levels in endothelium are unclear. We examined the effect of ischemia and reperfusion and asphyxia and reventilation on production of COX isoforms in cerebral vascular endothelium. Methods: Neonatal pigs were exposed to global ischemia (n= 4) or asphyxia (n= 3) for 5–10 min. Following 2–6 h of recovery, the animals were killed, and the cerebral arteries and arterioles were removed. Cerebral arteries and arterioles were also removed from untreated control animals (n= 1) and from time control animals (n= 3). Cerebral vessels were fixed in 4% formalin and paraffin embedded, and constitutive and inducible COX (COX-1 and COX-2, respectively) levels were assessed using indirect immunofluorescence. Results: Hemotoxylin and eosin staining indicated that anoxic stress leads to enlargement of endothelial cells. Immunofluorescence for COX-1 in endothelium was minimal in cerebral arteries and arterioles from control animals and did not show an increase in animals exposed to anoxic stress. Similarly, cerebral vessels from control animals showed little immunostaining for COX-2. In contrast, immunofluorescence for COX-2 was greatly increased in cerebral arteries and arterioles from animals exposed to asphyxia or ischemia. Conclusions: We conclude that anoxic stress increases COX-2 but not COX-1 levels in cerebral endothelium.     

Function and clustered expression of MaxiK channels in cerebral myocytes remain intact with aging

The incidence of stroke increases significantly in the aging population where stroke related deaths boost at >75 years and survivors are often permanently disabled. Aging is known to decrease cerebral blood flow likely due to an increase in arterial tone. Although MaxiK channels are key regulators of cerebral arterial tone their pattern of expression and function in cerebral blood vessels during aging is unknown. Using specific antibodies against the alpha-subunit of MaxiK channels and current recordings, we now demonstrate that in aging cerebral myocytes, MaxiK channels remain healthy. Furthermore, we show for the first time that in the vasculature, MaxiK channels are expressed in clusters. Clusters have an estimated radius of approximately 200 nm in young rats (3-5 month old Fisher 344 rats) which remains normal in old (25-30 month rats) cerebral myocytes. Consistent with a healthy MaxiK channel expression in old cerebral arteries, MaxiK current density, kinetics and Ca(2+) sensitivity were practically identical in young and old myocytes. Sensitivity to nanomolar concentrations of dehydrosoyasaponin-I that activates channels formed by alpha and beta subunits is also the same in young and old myocytes. These results demonstrate that MaxiK channels maintain normal expression during cerebral aging which is in sharp contrast to our previous finding of loss of expression in aging coronary arteries. It seems therefore, that cerebral myocytes have developed a protective anti-aging mechanism leading to the continued expression of MaxiK channels.     

Functional expression of the alpha 2 and alpha 3 isoforms of the Na,K-ATPase in baculovirus-infected insect cells.

Multiple isoforms of both the alpha and beta subunits of Na,K-ATPase have been identified. Elucidating their roles has been complicated by the fact that most tissues express multiple isoforms and purification techniques specific for each isoform have not been achieved. The baculovirus expression system, which uses the baculovirus Autographica californica to infect insect cells, is an ideal system for studying the Na,K-ATPase isoforms since high amounts of foreign proteins can be produced and some insect cell lines have low levels of endogenous Na,K-ATPase. Recombinant baculoviruses containing the cDNAs for the alpha 2, alpha 3, and beta 1 isoforms of the rat Na,K-ATPase were prepared and used to infect Sf-9 cells, an insect cell line derived from the ovary of the fall armyworm Spodoptera frugiperda. By using this system, Na,K-ATPase alpha 2 and alpha 3 subunits that were antigenically and electrophoretically indistinguishable from the native subunits were produced. When each subunit is expressed independently in the Sf-9 cells, it is primarily delivered to the plasma membrane. Although the isolated expression of each Na,K-ATPase subunit did not render active Na,K-ATPase molecules, the coexpression of alpha 2 or alpha 3 with beta 1 resulted in catalytically active molecules. This activity could be measured as a ouabain-sensitive ATPase activity or directly demonstrated using either [gamma-32P]ATP or 32Pi to identify the phosphorylated intermediates of the alpha 2 and alpha 3 isoforms. [3H]Ouabain binding studies showed that both isoforms are capable of binding the cardiotonic steroid with high affinity, alpha 3 being more sensitive to ouabain. These results demonstrate that the baculovirus system is suitable for the expression of the Na,K-ATPase isoforms and should provide a useful method for the characterization of the enzymatic properties of each isoform.