Absence of direct delivery for single transmembrane apical proteins or their "Secretory" forms in polarized hepatic cells.

The absence of a direct route to the apical plasma membrane (PM) for single transmembrane domain (TMD) proteins in polarized hepatic cells has been inferred but never directly demonstrated. The genes encoding three pairs of apical PM proteins, whose extracellular domains are targeted exclusively to the apical milieu in Madin-Darby canine kidney cells, were packaged into recombinant adenovirus and delivered to WIF-B cells in vitro and liver hepatocytes in vivo. By immunofluorescence and pulse-chase metabolic labeling, we found that the soluble constructs were overwhelmingly secreted into the basolateral milieu, which in vivo is the blood and in vitro is the culture medium. The full-length proteins were first delivered to the basolateral surface but then concentrated in the apical PM. Our results imply that hepatic cells lack trans-Golgi network (TGN)-based machinery for directly sorting single transmembrane domain apical proteins and raise interesting questions about current models of PM protein sorting in polarized and nonpolarized cells.

Krüppel-like factor 4 (gut-enriched Krüppel-like factor) inhibits cell proliferation by blocking G1/S progression of the cell cycle.

Krüppel-like factor 4 (KLF4) is an epithelial cell-enriched, zinc finger-containing transcription factor, the expression of which is associated with growth arrest. Previous studies show that constitutive expression of KLF4 inhibits DNA synthesis but the manner by which KLF4 exerts this effect is unclear. In the present study, we developed a system in which expression of KLF4 is controlled by a promoter that is induced upon treatment of cells containing the receptors for the insect hormone, ecdysone, with ponasterone A, an ecdysone analogue. The rate of proliferation of a stably transfected colon cancer cell line, RKO, was significantly decreased following addition of ponasterone A when compared with untreated cells. Flow cytometric analyses indicated that the inducible expression of KLF4 caused a block in the G(1)/S phase of the cell cycle. A similar block was observed when ecdysone receptor-containing RKO cells were infected with a replication-defective recombinant adenovirus containing an inducible KLF4 and treated with ponasterone A. Results of these studies provide evidence that the inhibitory effect of KLF4 on cell proliferation is mainly exerted at the G(1)/S boundary of the cell cycle.

Constitutively active phosphatidylinositol 3-kinase and AKT are sufficient to stimulate the epithelial Na+/H+ exchanger 3.

Phosphatidylinositol 3-kinase (PI 3-kinase) is a cytoplasmic signaling molecule that is recruited to activated growth factor receptors and has been shown to be involved in regulation of stimulated exocytosis and endocytosis. One of the downstream signaling molecules activated by PI 3-kinase is the protein kinase Akt. Previous studies have indicated that PI 3-kinase is necessary for basal Na(+)/H(+) exchanger 3 (NHE3) transport and for fibroblast growth factor-stimulated NHE3 activity in PS120 fibroblasts. However, it is not known whether activation of PI 3-kinase is sufficient to stimulate NHE3 activity or whether Akt is involved in this PI 3-kinase effect. We used an adenoviral infection system to test the possibility that activation of PI 3-kinase or Akt alone is sufficient to stimulate NHE3 activity. This hypothesis was investigated in PS120 fibroblasts stably expressing NHE3 after somatic gene transfer using a replication-deficient recombinant adenovirus containing constitutively active catalytic subunit of PI 3-kinase or constitutively active Akt. The adenovirus construct used was engineered with an upstream ecdysone promoter to allow time-regulated expression. Adenoviral infection was nearly 100% at 48 h after infection. Forty-eight hours after infection (24 h after activation of the ecdysone promoter), PI 3-kinase and Akt amount and activity were increased. Increases in both PI 3-kinase activity and Akt activity stimulated NHE3 transport. In addition, a membrane-permeant synthetic 10-mer peptide that binds polyphosphoinositides and increases PI 3-kinase activity similarly enhanced NHE3 transport activity and also increased the percentage of NHE3 on the plasma membrane. The magnitudes of stimulation of NHE3 by constitutively active PI 3-kinase, PI 3-kinase peptide, and constitutively active Akt were similar to each other. These results demonstrate that activation of PI 3-kinase or Akt is sufficient to stimulate NHE3 transport activity in PS120/NHE3 cells.

Functional consequences of the arrhythmogenic G306R KvLQT1 K+ channel mutant probed by viral gene transfer in cardiomyocytes.

IKs, the slow component of the delayed rectifier potassium current, figures prominently in the repolarization of heart cells. The K+ channel gene KvLQT1 is mutated in the heritable long QT (LQT) syndrome. Heterologous coexpression of KvLQT1 and the accessory protein minK yields an IKs-like current. Nevertheless, the links between KvLQT1 and cardiac IKs are largely inferential. Since the LQT syndrome mutant KvLQT1-G306R suppresses channel activity when coexpressed with wild-type KvLQT1 in a heterologous system, overexpression of this mutant in cardiomyocytes should reduce or eliminate native IKs if KvLQT1 is indeed the major molecular component of this current. To test this idea, we created the adenovirus AdRMGI-KvLQT1-G306R, which overexpresses KvLQT1-G306R channels. In > 60 % of neonatal mouse myocytes, a sizable IKs could be measured using perforated-patch recordings (8.0 +/- 1.6 pA pF-1, n = 13). IKs was increased by forskolin and blocked by clofilium or indapamide but not by E-4031. While cells infected with a reporter virus expressing only green fluorescent protein (GFP) displayed IKs similar to that in uninfected cells, AdRMGI-KvLQT1-G306R-infected cells showed a significantly reduced IKs (2.4 +/- 1.1 pA pF-1, n = 10, P < 0.01) when measured 60-72 h after infection. Similar results were observed in adult guinea-pig myocytes (5.9 +/- 1.2 pA pF-1, n = 9, for control vs. 0.1 +/- 0.1 pA pF-1, n = 5, for AdRMGI-KvLQT1-G306R-infected cells). We conclude that KvLQT1 is the major molecular component of IKs. Our results further establish a dominant-negative mechanism for the G306R LQT syndrome mutation.

Distinct gene-specific mechanisms of arrhythmia revealed by cardiac gene transfer of two long QT disease genes, HERG and KCNE1.

The long QT syndrome (LQTS) is a heritable disorder that predisposes to sudden cardiac death. LQTS is caused by mutations in ion channel genes including HERG and KCNE1, but the precise mechanisms remain unclear. To clarify this situation we injected adenoviral vectors expressing wild-type or LQT mutants of HERG and KCNE1 into guinea pig myocardium. End points at 48-72 h included electrophysiology in isolated myocytes and electrocardiography in vivo. HERG increased the rapid component, I(Kr), of the delayed rectifier current, thereby accelerating repolarization, increasing refractoriness, and diminishing beat-to-beat action potential variability. Conversely, HERG-G628S suppressed I(Kr) without significantly delaying repolarization. Nevertheless, HERG-G628S abbreviated refractoriness and increased beat-to-beat variability, leading to early afterdepolarizations (EADs). KCNE1 increased the slow component of the delayed rectifier, I(Ks), without clear phenotypic sequelae. In contrast, KCNE1-D76N suppressed I(Ks) and markedly slowed repolarization, leading to frequent EADs and electrocardiographic QT prolongation. Thus, the two genes predispose to sudden death by distinct mechanisms: the KCNE1 mutant flagrantly undermines cardiac repolarization, and HERG-G628S subtly facilitates the genesis and propagation of premature beats. Our ability to produce electrocardiographic long QT in vivo with a clinical KCNE1 mutation demonstrates the utility of somatic gene transfer in creating genotype-specific disease models.

A functional assay for paralytic shellfish toxins that uses recombinant sodium channels.

Saxitoxin (STX) and its derivatives are highly toxic natural compounds produced by dinoflagellates commonly present in marine phytoplankton. During algal blooms ("red tides"), shellfish accumulate saxitoxins leading to paralytic shellfish poisoning (PSP) in human consumers. PSP is a consequence of the high-affinity block of voltage-dependent Na channels in neuronal and muscle cells. PSP poses a significant public health threat and an enormous economic challenge to the shellfish industry worldwide. The standard screening method for marine toxins is the mouse mortality bioassay that is ethically problematic, costly and time-consuming. We report here an alternative, functional assay based on electrical recordings in cultured cells stably expressing a PSP target molecule, the STX-sensitive skeletal muscle Na channel. STX-equivalent concentration in the extracts was calibrated by comparison with purified STX, yielding a highly significant correlation (R=0.95; N=30) between electrophysiological determinations and the values obtained by conventional methods. This simple, economical, and reproducible assay obviates the need to sacrifice millions of animals in mandatory paralytic shellfish toxin screening programs.

Probing the interaction between inactivation gating and Dd-sotalol block of HERG.

Potassium channels encoded by HERG underlie I:(Kr), a sensitive target for most class III antiarrhythmic drugs, including methanesulfonanilides such as Dd-sotalol. Recently it was shown that these drugs are trapped in the channel as it closes during hyperpolarization. At the same time, HERG channels rapidly open and inactivate when depolarized, and methanesulfonanilide block is known to develop in a use-dependent manner, suggesting a potential role for inactivation in drug binding. However, the role of HERG inactivation in class III drug action is uncertain: pore mutations that remove inactivation reduce block, yet many of these mutations also modify the channel permeation properties and could alter drug affinity through gating-independent mechanisms. In the present study, we identify a definitive role for inactivation gating in Dd-sotalol block of HERG, using interventions complementary to mutagenesis. These interventions (addition of extracellular Cd(2+), removal of extracellular Na(+)) modify the voltage dependence of inactivation but not activation. In normal extracellular solutions, block of HERG current by 300 micromol/L Dd-sotalol reached 80% after a 10-minute period of repetitive depolarization to +20 mV. Maneuvers that impeded steady-state inactivation also reduced Dd-sotalol block of HERG: 100 micromol/L Cd(2+) reduced steady-state block to 55% at +20 mV (P:<0.05); removing extracellular Na(+) reduced block to 44% (P:<0.05). An inactivation-disabling mutation (G628C-S631C) reduced Dd-sotalol block to only 11% (P:<0.05 versus wild type). However, increasing the rate of channel inactivation by depolarizing to +60 mV reduced Dd-sotalol block to 49% (P:<0.05 versus +20 mV), suggesting that the drug does not primarily bind to the inactivated state. Coexpression of MiRP1 with HERG had no effect on inactivation gating and did not modify Dd-sotalol block. We postulate that Dd-sotalol accesses its receptor in the open pore, and the drug-receptor interaction is then stabilized by inactivation. Whereas deactivation traps the bound methanesulfonanilide during hyperpolarization, we propose that HERG inactivation stabilizes the drug-receptor interaction during membrane depolarization.

Adenovirus-mediated inducible gene expression in vivo by a hybrid ecdysone receptor.

Precise control of transgene expression would markedly facilitate certain applications of gene therapy. To regulate expression of a transferred gene in response to an exogenous compound in vivo, we modified the ecdysone-responsive system. We combined the advantages of the Drosophila (DmEcR) and the Bombyx ecdysone receptor (BmEcR) by creating a chimeric Drosophila/Bombyx ecdysone receptor (DB-EcR) that preserved the ability to bind to the modified ecdysone promoter without exogenous retinoid X receptor (RXR). In cultured cells, DB-EcR effectively mediates ligand-dependent transactivation of a reporter gene at lower concentrations of the chemical ecdysone agonist GS-E than VgRXR (DmEcR + RXR). Transgene delivery in vivo was achieved by intramyocardial injection of recombinant adenovirus vectors in adult rats. Upon stimulation with GS-E, DB-EcR potently (>40-fold induction) activated gene expression in vivo while VgRXR was not induced. This hybrid ecdysone receptor represents an important new tool for in vivo transgene regulation with potentially diverse applications in somatic and germline transfer.

Viral gene transfer of dominant-negative Kv4 construct suppresses an O2-sensitive K+ current in chemoreceptor cells.

Hypoxia initiates the neurosecretory response of the carotid body (CB) by inhibiting one or more potassium channels in the chemoreceptor cells. Oxygen-sensitive K(+) channels were first described in rabbit CB chemoreceptor cells, in which a transient outward K(+) current was reported to be reversibly inhibited by hypoxia. Although progress has been made to characterize this current with electrophysiological and pharmacological tools, no attempts have been made to identify which Kv channel proteins are expressed in rabbit CB chemoreceptor cells and to determine their contribution to the native O(2)-sensitive K(+) current. To probe the molecular identity of this current, we have used dominant-negative constructs to block the expression of functional Kv channels of the Shaker (Kv1.xDN) or the Shal (Kv4.xDN) subfamilies, because members of these two subfamilies contribute to the transient outward K(+) currents in other preparations. Delivery of the constructs into chemoreceptor cells has been achieved with adenoviruses that enabled ecdysone-inducible expression of the dominant-negative constructs and reporter genes in polycistronic vectors. In voltage-clamp experiments, we found that, whereas adenoviral infections of chemoreceptor cells with Kv1.xDN did not modify the O(2)-sensitive K(+) current, infections with Kv4.xDN suppressed the transient outward current in a time-dependent manner, significantly depolarized the cells, and abolished the depolarization induced by hypoxia. Our work demonstrate that genes of the Shal K(+) channels underlie the transient outward, O(2)-sensitive, K(+) current of rabbit CB chemoreceptor cells and that this current contributes to the cell depolarization in response to low pO(2).

Evidence against functional heteromultimerization of the KATP channel subunits Kir6.1 and Kir6.2.

K(ATP) channels consist of pore-forming potassium inward rectifier (Kir6.x) subunits and sulfonylurea receptors (SURs). Although Kir6.1 or Kir6.2 coassemble with different SUR isoforms to form heteromultimeric functional K(ATP) channels, it is not known whether Kir6.1 and Kir6.2 coassemble with each other. To define the molecular identity of K(ATP) channels, we used adenoviral gene transfer to express wild-type and dominant-negative constructs of Kir6.1 and Kir6.2 in a heterologous expression system (A549 cells) and in native cells (rabbit ventricular myocytes). Dominant-negative (DN) Kir6.2 gene transfer suppressed current through heterologously expressed SUR2A + Kir6.2 channels. Conversely, DN Kir6.1 suppressed SUR2B + Kir6.1 current but had no effect on coexpressed SUR2A + Kir6. 2. We next probed the ability of Kir6.1 and Kir6.2 to affect endogenous K(ATP) channels in adult rabbit ventricular myocytes, using adenoviral vectors to achieve efficient gene transfer. Infection with the DN Kir6.2 virus for 72 h suppressed pinacidil-inducible K(ATP) current density measured by whole-cell patch clamp. However, there was no effect of infection with the DN Kir6.1 on the K(ATP) current. Based on these functional assays, we conclude that Kir6.1 and Kir6.2 do not heteromultimerize with each other and that Kir6.2 is the sole K(ATP) pore-forming subunit in the surface membrane of heart cells.

Molecular dissection of cardiac repolarization by in vivo Kv4.3 gene transfer.

Heart failure leads to marked suppression of the Ca(2+)-independent transient outward current (I(to1)), but it is not clear whether I(to1) downregulation suffices to explain the concomitant action potential prolongation. To investigate the role of I(to1) in cardiac repolarization while circumventing culture-related action potential alterations, we injected adenovirus vectors in vivo to overexpress or to suppress I(to1) in guinea pigs and rats, respectively. Myocytes were isolated 72 hours after intramyocardial injection and stimulation of the ecdysone-inducible vectors with intraperitoneal injection of an ecdysone analog. Kv4.3-infected guinea pig myocytes exhibited robust transient outward currents. Increasing density of I(to1) progressively depressed the plateau potential in Kv4. 3-infected guinea pig myocytes and abbreviated action potential duration (APD). In vivo infection with a dominant-negative Kv4. 3-W362F construct suppressed peak I(to1) in rat ventriculocytes, elevated the plateau height, significantly prolonged the APD, and resulted in a prolongation by about 30% of the QT interval in surface electrocardiogram recordings. These results indicate that I(to1) plays a crucial role in setting the plateau potential and overall APD, supporting a causative role for suppression of this current in the electrophysiological alterations of heart failure. The electrocardiographic findings indicate that somatic gene transfer can be used to create gene-specific animal models of the long QT syndrome.

A revised view of cardiac sodium channel "blockade" in the long-QT syndrome.

Mutations in SCN5A, encoding the cardiac sodium (Na) channel, are linked to a form of the congenital long-QT syndrome (LQT3) that provokes lethal ventricular arrhythmias. These autosomal dominant mutations disrupt Na channel function, inhibiting channel inactivation, thereby causing a sustained ionic current that delays cardiac repolarization. Sodium channel-blocking antiarrhythmics, such as lidocaine, potently inhibit this pathologic Na current (I(Na)) and are being evaluated in patients with LQT3. The mechanism underlying this effect is unknown, although high-affinity "block" of the open Na channel pore has been proposed. Here we report that a recently identified LQT3 mutation (R1623Q) imparts unusual lidocaine sensitivity to the Na channel that is attributable to its altered functional behavior. Studies of lidocaine on individual R1623Q single-channel openings indicate that the open-time distribution is not changed, indicating the drug does not block the open pore as proposed previously. Rather, the mutant channels have a propensity to inactivate without ever opening ("closed-state inactivation"), and lidocaine augments this gating behavior. An allosteric gating model incorporating closed-state inactivation recapitulates the effects of lidocaine on pathologic I(Na). These findings explain the unusual drug sensitivity of R1623Q and provide a general and unanticipated mechanism for understanding how Na channel-blocking agents may suppress the pathologic, sustained Na current induced by LQT3 mutations.

Molecular composition of mitochondrial ATP-sensitive potassium channels probed by viral Kir gene transfer.

Heart cells contain ATP-sensitive potassium (KATP) channels in both the sarcolemma and the inner mitochondrial membrane. The sarcolemmal channels are believed to be heteromultimeric complexes of sulfonylurea receptors (SUR) and potassium inward rectifier (Kir) gene products, but the molecular identity of mitochondrial KATP (mitoKATP) channels remains unclear. To probe the molecular composition of KATP channels, we used adenoviral gene transfer to express wild-type (WT) and dominant-negative (AFA) constructs of Kir6.1 and 6.2 in rabbit ventricular myocytes. None of the Kir6.1 or 6.2 constructs affected mitoKATPchannel activity as assayed by confocal imaging of flavoprotein fluorescence, contradicting the proposal, based on subcellular antibody localization, that Kir6.1 forms part of mitoKATP channels. As previously reported, dominant-negative Kir6.2 gene transfer suppressed sarcolemmal KATP current, while Kir6.1 constructs had no effect on sarcolemmal activity. Immunohistochemistry with an anti-Kir6.1 antibody revealed expression of this protein in heart but no apparent co-localization with mitochondria. Thus, the available evidence indicates that both Kir6.1 and 6.2 are expressed in ventricular myocytes, but neither plays a discernible functional role in the mitoKATP channel.

Ex vivo cultured megakaryocytes express functional glycoprotein IIb-IIIa receptors and are capable of adenovirus-mediated transgene expression.

Investigation of the molecular basis of megakaryocyte (MK) and platelet biology has been limited by an inadequate source of genetically manipulable cells exhibiting physiologic MK and platelet functions. We hypothesized that ex vivo cultured MKs would exhibit agonist inducible glycoprotein (GP) IIb-IIIa activation characteristic of blood platelets and that these cultured MKs would be capable of transgene expression. Microscopic and flow cytometric analyses confirmed that human hematopoietic stem cells cultured in the presence of pegylated recombinant human MK growth and development factor (PEG-rHuMGDF) differentiated into morphologic and phenotypic MKs over 2 weeks. Cultured MKs expressed functional GPIIb-IIIa receptors as assessed by agonist inducible soluble fibrinogen and PAC1 binding. The specificity and kinetics of fibrinogen binding to MK GPIIb-IIIa receptors were similar to those described for blood platelets. The reversibility and internalization of ligands bound to MK GPIIb-IIIa also shared similarities with those observed in platelets. Cultured MKs were transduced with an adenoviral vector encoding green fluorescence protein (GFP) or beta-galactosidase (beta-gal). Efficiency of gene transfer increased with increasing multiplicities of infection and incubation time, with 45% of MKs expressing GFP 72 hours after viral infection. Transduced MKs remained capable of agonist induced GPIIb-IIIa activation. Thus, ex vivo cultured MKs (1) express agonist responsive GPIIb-IIIa receptors, (2) are capable of expressing transgenes, and (3) may prove useful for investigation of the molecular basis of MK differentiation and GPIIb-IIIa function.

Differences in the ways sympathetic neurons and endocrine cells process, store, and secrete exogenous neuropeptides and peptide-processing enzymes.

Most neurons store peptides in large dense core vesicles (LDCVs) and release the neuropeptides in a regulated manner. Although LDCVs have been studied in endocrine cells, less is known about these storage organelles in neurons. In this study we use the endogenous peptide NPY (neuropeptide Y) and the endogenous peptide-processing enzyme PAM (peptidylglycine alpha-amidating monooxygenase) as tools to study the peptidergic system in cultured neurons from the superior cervical ganglion (SCG). Once mature, SCG neurons devote as much of their biosynthetic capabilities to neurotransmitter production as endocrine cells devote to hormone production. Unlike pituitary and atrium, SCG neurons cleave almost all of the bifunctional PAM protein they produce into soluble monofunctional enzymes. Very little PAM or NPY is secreted under basal conditions, and the addition of secretagogue dramatically stimulates the secretion of PAM and NPY to a similar extent. Although endocrine cells typically package "foreign" secretory products together with endogenous products, pro-opiomelanocortin- and PAM-derived products encoded by adenovirus in large part were excluded from the LDCVs of SCG neurons. When expressed in corticotrope tumor cells and primary anterior pituitary cultures, the same virally encoded products were metabolized normally. The differences that were observed could reflect differences in the properties of neuronal and endocrine peptidergic systems or differences in the ability of neurons and endocrine cells to express viral transcripts.

Overexpression of uncoupling protein 2 inhibits glucose-stimulated insulin secretion from rat islets.

Uncoupling protein 2 (UCP-2) mRNA expression has been shown to be altered by metabolic conditions such as obesity in humans, but its functional significance is unknown. The expression of UCP-2 mRNA and protein in normal rat islets was established by reverse transcriptase-polymerase chain reaction and immunocytochemistry in pancreatic islets and tissue, respectively. Intense immunostaining of UCP-2 correlated with insulin-positive ,-cells. Overexpression of UCP-2 in normal rat islets was accomplished by infection with an adenovirus (AdEGI-UCP-2) containing the full-length human UCP-2 coding sequence. Induction of the AdEGI-UCP-2 gene resulted in severe blunting of glucose-stimulated insulin secretion (GSIS) without affecting islet insulin content or the ability of the calcium ionophore A23187 to increase insulin secretion from AdEGI-UCP-2-expressing islets. Therefore, UCP-2 overexpression affects signal transduction proximal to Ca2+-mediated steps, including exocytosis. Insulin secretion from single beta-cells to 16.5 mmol/l glucose examined by reverse hemolytic plaque assay was nearly ablated if UCP-2 was overexpressed. Thus, a direct, causal relationship between overexpression of UCP-2 and inhibition of GSIS in normal islets has been established. These data suggest that increased expression of UCP-2 has the potential to cause the lack of a glucose effect on insulin secretion in type 2 diabetes.

Manipulation of cellular excitability by cell fusion: effects of rapid introduction of transient outward K+ current on the guinea pig action potential.

To investigate the still-undetermined role of the Ca2+-independent transient outward current (Ito1) on repolarization of the cardiac action potential, we used cell fusion to introduce Ito1 into guinea pig cardiomyocytes, which normally lack this current. This technique enables the rapid delivery of premade functional ion channels to cardiomyocytes within hours of isolation, thus eliminating the action potential alterations that complicate prolonged cell culture. Chinese hamster ovary (CHO) cells stably expressing Kv4.3 (CHO-Kv4. 3) were loaded with a fluorescent dye and fused to guinea pig cardiomyocytes using polyethylene glycol. As controls, nontransfected CHO cells were fused using the same protocol. Myocytes fused with CHO-Kv4.3 cells exhibited a robust Ito1 (16. 5+/-2.6 pA/pF at +40 mV; 37 degrees C; n=19), whereas controls had none. Ito1 accelerated the early repolarization velocity (r=-0.68; 3 ms after the overshoot) and progressively suppressed the voltage of the plateau phase (r=-0.90) with increasing Ito1 density. Reduction of the action potential duration to 50% repolarization (r=-0.76) and to 90% repolarization (r=-0.65) also correlated well with Ito1 density. Thus, Ito1 exerted a significant effect on the early repolarization phase and abbreviated action potential duration. Cell fusion is a valuable and generalizable technique to introduce preformed membrane proteins into native cells.

Pharmacological and histochemical distinctions between molecularly defined sarcolemmal KATP channels and native cardiac mitochondrial KATP channels.

A variety of direct and indirect techniques have revealed the existence of ATP-sensitive potassium (KATP) channels in the inner membranes of mitochondria. The molecular identity of these mitochondrial KATP (mitoKATP) channels remains unclear. We used a pharmacological approach to distinguish mitoKATP channels from classical, molecularly defined cardiac sarcolemmal KATP (surfaceKATP) channels encoded by the sulfonylurea receptor SUR2A and the pore-forming subunit Kir6.2. SUR2A and Kir6.2 were expressed in human embryonic kidney (HEK)293 cells, and their activities were measured by patch-clamp recordings of membrane current. SurfaceKATP channels are activated potently by 100 microM pinacidil but only weakly by 100 microM diazoxide; in addition, they are blocked by 10 microM glibenclamide, but are insensitive to 500 microM 5-hydroxydecanoate. This pharmacology, which was confirmed with patch-clamp recordings in intact rabbit ventricular myocytes, contrasts with that of mitoKATP channels as indexed by flavoprotein oxidation. MitoKATP channels in myocytes are activated equally by 100 microM diazoxide and 100 microM pinacidil. In contrast to its lack of effect on surfaceKATP channels, 5-hydroxydecanoate is an effective blocker of mitoKATP channels. Glibenclamide's effects on mitoKATP channels are difficult to assess, because it independently activates flavoprotein fluorescence, consistent with a previously described primary uncoupling effect. Confocal imaging of the subcellular distribution of expressed fluorescent Kir6.2 in HEK cells and in myocytes revealed no targeting of mitochondrial membranes. The differences in drug sensitivity and subcellular localization indicate that mitoKATP channels are distinct from surface KATP channels at a molecular level.

Functional expression of exogenous proteins in mammalian sensory hair cells infected with adenoviral vectors.

To understand the function of specific proteins in sensory hair cells, it is necessary to add or inactivate those proteins in a system where their physiological effects can be studied. Unfortunately, the usefulness of heterologous expression systems for the study of many hair cell proteins is limited by the inherent difficulty of reconstituting the hair cell's exquisite cytoarchitecture. Expression of exogenous proteins within hair cells themselves may provide an alternative approach. Because recombinant viruses were efficient vectors for gene delivery in other systems, we screened three viral vectors for their ability to express exogenous genes in hair cells of organotypic cultures from mouse auditory and vestibular organs. We observed no expression of the genes for beta-galactosidase or green fluorescent protein (GFP) with either herpes simplex virus or adeno-associated virus. On the other hand, we found robust expression of GFP in hair cells exposed to a recombinant, replication-deficient adenovirus that carried the gene for GFP driven by a cytomegalovirus promoter. Titers of 4 x 10(7) pfu/ml were sufficient for expression in 50% of the approximately 1,000 hair cells in the utricular epithelium; < 1% of the nonhair cells in the epithelium were GFP positive. Expression of GFP was evident as early as 12 h postinfection, was maximal at 4 days, and continued for at least 10 days. Over the first 36 h there was no evidence of toxicity. We recorded normal voltage-dependent and transduction currents from infected cells identified by GFP fluorescence. At longer times hair bundle integrity was compromised despite a cell body that appeared healthy. To assess the ability of adenovirus-mediated gene transfer to alter hair cell function we introduced the gene for the ion channel Kir2.1. We used an adenovirus vector encoding Kir2.1 fused to GFP under the control of an ecdysone promoter. Unlike the diffuse distribution within the cell body we observed with GFP, the ion channel-GFP fusion showed a pattern of fluorescence that was restricted to the cell membrane and a few extranuclear punctate regions. Patch-clamp recordings confirmed the expression of an inward rectifier with a conductance of 43 nS, over an order of magnitude larger than the endogenous inward rectifier. The zero-current potential in infected cells was shifted by -17 mV. These results demonstrate an efficient method for gene transfer into both vestibular and auditory hair cells in culture, which can be used to study the effects of gene products on hair cell function.