Pharmacological comparison of native mitochondrial K(ATP) channels with molecularly defined surface K(ATP) channels.

Many mammalian cells have two distinct types of ATP-sensitive potassium (K(ATP)) channels: the classic ones in the surface membrane (sK(ATP)) and others in the mitochondrial inner membrane (mitoK(ATP)). Cardiac mitoK(ATP) channels play a pivotal role in ischemic preconditioning, and thus represent interesting drug targets. Unfortunately, the molecular structure of mitoK(ATP) channels is unknown, in contrast to sK(ATP) channels, which are composed of a pore-forming subunit (Kir6.1 or Kir6.2) and a sulfonylurea receptor (SUR1, SUR2A, or SUR2B). As a means of probing the molecular makeup of mitoK(ATP) channels, we compared the pharmacology of native cardiac mitoK(ATP) channels with that of molecularly defined sK(ATP) channels expressed heterologously in human embryonic kidney 293 cells. Using mitochondrial oxidation to index mitoK(ATP) channel activity in rabbit ventricular myocytes, we found that pinacidil and diazoxide open mitoK(ATP) channels, but P-1075 does not. On the other hand, 5-hydroxydecanoic acid (5HD), but not HMR-1098, blocks mitoK(ATP) channels. Although pinacidil is a nonselective activator of expressed sK(ATP) channels, diazoxide did not open channels formed by Kir6.1/SUR2A, Kir6.2/SUR2A (known components of cardiac sK(ATP) channels) or Kir6.2/SUR2B. P-1075 activated all the K(ATP) channels, except Kir6.1/SUR1 channels. Glybenclamide potently blocked all sK(ATP) channels, but 5HD only blocked channels formed by SUR1/Kir6.1 or Kir6.2 (IC(50)s of 66 and 81 microM, respectively). This potency is similar to that for block of mitoK(ATP) channels (IC(50) = 95 microM). In addition, HMR-1098 potently blocked Kir6.2/SUR2A channels (IC(50) = 1.5 microM), but was 67 times less potent in blocking Kir6.1/SUR1 channels (IC(50) = 100 microM). Our results demonstrate that mitoK(ATP) channels closely resemble Kir6.1/SUR1 sK(ATP) channels in their pharmacological profiles.

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.

Selective pharmacological agents implicate mitochondrial but not sarcolemmal K(ATP) channels in ischemic cardioprotection.

BACKGROUND: Pharmacological evidence has implicated ATP-sensitive K(+) (K(ATP)) channels as the effectors of cardioprotection, but the relative roles of mitochondrial (mitoK(ATP)) and sarcolemmal (surfaceK(ATP)) channels remain controversial. METHODS AND RESULTS: We examined the effects of the K(ATP) channel blocker HMR1098 and the K(ATP) channel opener P-1075 on surfaceK(ATP) and mitoK(ATP) channels in rabbit ventricular myocytes. HMR1098 (30 micromol/L) inhibited the surfaceK(ATP) current activated by metabolic inhibition, whereas the drug did not blunt diazoxide (100 micromol/L)-induced flavoprotein oxidation, an index of mitoK(ATP) channel activity. P-1075 (30 micromol/L) did not increase flavoprotein oxidation but did elicit a robust surfaceK(ATP) current that was completely inhibited by HMR1098. These results indicate that HMR1098 selectively inhibits surfaceK(ATP) channels, whereas P-1075 selectively activates surface K(ATP) channels. In a cellular model of simulated ischemia, the mitoK(ATP) channel opener diazoxide (100 micromol/L), but not P-1075, blunted cellular injury. The cardioprotection afforded by diazoxide or by preconditioning was prevented by the mitoK(ATP) channel blocker 5-hydroxydecanoate (500 micromol/L) but not by the surfaceK(ATP) channel blocker HMR1098 (30 micromol/L). CONCLUSIONS: The cellular effects of mitochondria- or surface-selective agents provide further support for the emerging consensus that mitoK(ATP) channels rather than surfaceK(ATP) channels are the likely effectors of cardioprotection.

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.

Mitochondrial ATP-dependent potassium channels. Viable candidate effectors of ischemic preconditioning.

Pharmacological evidence has implicated ATP-dependent potassium (KATP) channels in the mechanism of ischemic preconditioning; however, the effects of sarcolemmal KATP channels on excitability cannot account for the protection. KATP channels also exist in mitochondrial inner membrane. To test whether such channels play a role in cardioprotection, we simultaneously measured flavoprotein fluorescence, an index of mitochondrial redox state, and sarcolemmal KATP currents in intact rabbit ventricular myocytes. Our results show that diazoxide, a KATP channel opener, induced reversible oxidation of flavoproteins, but did not activate sarcolemmal KATP channels. This effect of diazoxide was blocked by 5-hydroxydecanoic acid (5-HD). We further verified that 5-HD is a selective blocker of the mitochondrial KATP channels. These methods have enabled us to demonstrate that the activity of mitochondrial KATP channels can be regulated by protein kinase C. In a cellular model of simulated ischemia, inclusion of diazoxide decreased the rate of cell death to about half of that in control. Such protection is inhibited by 5-HD. In conclusion, our results demonstrate that diazoxide targets mitochondrial but not sarcolemmal KATP channels, and imply that mitochondrial KATP channels may mediate preconditioning.

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.

CNTF potentiates peripheral nerve regeneration.

The effect of ciliary neurotrophic factor (CNTF) on peripheral nerve regeneration was studied in 6-week-old rats following sciatic nerve transection and juxtaposition of proximal and distal stumps. Rats received intraperitoneal recombinant human CNTF, 1 mg/kg every other day for 12 days. In all CNTF-treated animals, the distribution of the anterograde transport of [3H]leucine-labeled material within the distal stump showed a distinct peak activity 21-24 mm distal to the nerve transection site. In contrast, the radioactivity in control nerves declined rapidly, distributed as proximodistal gradient without a distinct peak. In addition, the amount of radioactivity returning from the growing axon tips in CNTF was significantly greater than controls. The correlative morphologic studies showed a well-advanced stage of myelination in regenerating axons in CNTF compared to controls. These combined in vivo correlative kinetic and morphological studies indicate that CNTF dramatically potentiates axonal regeneration by promoting a greater number of elongating axon tips into the distal stump.

Gene delivery to spinal motor neurons.

This study demonstrates the direct delivery of plasmid gene constructs into spinal motor neurons utilizing retrograde axoplasmic transport. The plasmid vectors contained the Lac Z gene under the control of both the Rous sarcoma virus (RSV) and Simian virus (SV)40 promoters. beta-Galactosidase expression was observed in alpha and gamma motor neurons by histochemical staining following direct injection into the sciatic nerve or gastrocnemius muscle. The presence of LacZ gene constructs was confirmed by the polymerase chain reaction (PCR). The ability to introduce gene constructs into motor neurons allows for the study of gene regulation and permits the development of gene therapy strategies for motor neuron diseases including the spinal muscular atrophies (SMA) and amyotrophic lateral sclerosis (ALS).

Human embryonic/atrial myosin alkali light chain gene: characterization, sequence, and chromosomal location.

We have isolated and sequenced the gene encoding the human embryonic/atrial myosin alkali light chain isoform (MLC-1emb/A). The gene is split into seven exons by six introns; the last exon, as in all MLC isoform genes sequenced to date, is completely 3' untranslated sequence. Comparison of the MLC-1emb/A isoform gene with the other MLC-1 genes showed that the exon-intron arrangement of the human MLC-1emb/A isoform gene is analogous to that of the other MLC-1 type isoform genes. We have also mapped the human MLC-1emb/A isoform gene to the long arm of chromosome 17; the corresponding mouse gene has been mapped to chromosome 11. This gene, together with a number of others such as the collagen(I) alpha 1, galactokinase, and thymidine kinase genes, is part of the largest syntenic group between mouse and man.

Human ventricular/slow twitch myosin alkali light chain gene characterization, sequence, and chromosomal location.

The gene coding for the human ventricular/slow twitch myosin alkali light chain isoform was isolated and sequenced. It was found to contain a total of seven exons, the last of which is completely 3'-untranslated sequence. Comparison of this gene sequence with that of the various fast twitch skeletal isoform gene sequences revealed that the exon-intron arrangement is conserved within the myosin alkali light chain gene family. In fact the introns are in exactly the same positions within analogous codons. Comparison of the derived amino acid sequence from the human ventricular/slow twitch isoform gene with that of other isoform protein sequences indicated that the protein encoded by this gene is more homologous to the chicken cardiac isoform protein sequence than to any of the other protein sequences. These results indicate that the gene duplication which gave rise to the ventricular/slow twitch and fast twitch isoform genes must have occurred prior to the divergence of mammals and avians. We have also localized the human ventricular/slow twitch isoform gene to the short arm of human chromosome 3. Interestingly the corresponding mouse gene has been mapped to the distal region of mouse chromosome 9 which contains a conserved syntenic group of genes that map to the short arm of human chromosome 3.