The voltage-dependent anion channel (VDAC) constitutes the major pathway for the entry and exit of metabolites across the outer membrane of the mitochondria and can serve as a scaffold for molecules that modulate the organelle. We report the crystal structure of a ␤-barrel eukaryotic membrane protein, the murine VDAC1 (mVDAC1) at 2.3 Å resolution, revealing a high-resolution image of its architecture formed by 19 ␤-strands. Unlike the recent NMR structure of human VDAC1, the position of the voltagesensing N-terminal segment is clearly resolved. The ␣-helix of the N-terminal segment is oriented against the interior wall, causing a partial narrowing at the center of the pore. This segment is ideally positioned to regulate the conductance of ions and metabolites passing through the VDAC pore.␤-barrel ͉ mitochondria ͉ outer membrane protein ͉ ATP flux A ll eukaryotic cells require efficient exchange of metabolites between the cytoplasm and the mitochondria. This exchange is mediated by the most abundant protein in the outer mitochondrial membrane, the voltage-dependent anion channel (VDAC), which facilitates movement of ions and metabolites between the cytoplasm and the intermembrane space of the mitochondria. VDAC was first discovered in 1976 (1) and has since been extensively studied by a number of biochemical and biophysical techniques demonstrating its conserved properties of voltage gating and ion selectivity and its ability to act as a scaffold for modulator proteins from both sides of the outer membrane (2, 3).Single-channel conductance experiments on VDAC1 at low membrane potential (10 mV) show a high conductance indicative of a large pore, often referred to as the open state of the channel (2). As voltage is increased (Ͼ30 mV) in either a positive or negative direction, a lower conductance, ostensibly the closed state, is obtained. Endogenous potentials caused by chemical gradients across the outer membrane [Donnan potentials (4)] may thus be sufficient to regulate this channel. Although the nature of either of these states is unknown in the absence of structural data, transition between them presumably involves conformational changes constituting a gating action that hinders the passage of metabolites such as adenine nucleotides. Furthermore, this transition is associated with altered ion selectivity because the channel shifts from weakly anion selective to weakly cation selective as it moves from open to closed. This complex gating behavior has driven numerous investigations that have provided sometimes contradictory findings; however, the role of VDAC to regulate metabolite traffic across the outer membrane is firmly established.As the major pathway into and out of mitochondria, VDAC mediates an intimate dichotomy between metabolism and death in all cells (5). Mitochondrial-dependent cell death involves numerous proteins [including hexokinase (6) and the Bcl-2 family of proteins (7), in particular] that alternatively promote or prevent mitochondrial dysfunction through interaction with, and potentially m...

Voltage-dependent and calcium-sensitive K ؉ (MaxiK) channels are key regulators of neuronal excitability, secretion, and vascular tone because of their ability to sense transmembrane voltage and intracellular Ca 2؉ . In most tissues, their stimulation results in a noninactivating hyperpolarizing K ؉ current that reduces excitability. In addition to noninactivating MaxiK currents, an inactivating MaxiK channel phenotype is found in cells like chromaffin cells and hippocampal neurons. The molecular determinants underlying inactivating MaxiK channels remain unknown. Herein, we report a transmembrane ␤ subunit (␤2) that yields inactivating MaxiK currents on coexpression with the pore-forming ␣ subunit of MaxiK channels. Intracellular application of trypsin as well as deletion of 19 N-terminal amino acids of the ␤2 subunit abolished inactivation of the ␣ subunit. Conversely, fusion of these N-terminal amino acids to the noninactivating smooth muscle ␤1 subunit leads to an inactivating phenotype of MaxiK channels. Furthermore, addition of a synthetic N-terminal peptide of the ␤2 subunit causes inactivation of the MaxiK channel ␣ subunit by occluding its K ؉ -conducting pore resembling the inactivation caused by the ''ball'' peptide in voltage-dependent K ؉ channels. Thus, the inactivating phenotype of MaxiK channels in native tissues can result from the association with different ␤ subunits. Ca 2ϩ-activated K ϩ channels, also known as BK, MaxiK, or slo channels, are key modulators of cellular excitability. They are characterized by their large single-channel conductance, intrinsic voltage dependence, Ca 2ϩ modulation, and blockade by charybdotoxin (CTX) and iberiotoxin (1-5). In most tissues, MaxiK channels produce noninactivating currents when activated by depolarization and͞or an increase in intracellular Ca 2ϩ . However, in chromaffin cells of the adrenal gland (6) and hippocampal neurons (7), inactivating MaxiK currents are also observed that otherwise resemble their noninactivating counterparts in their biophysical and pharmacological properties. The mechanism of inactivation in MaxiK channels has been investigated in detail in rat chromaffin cells, which express both inactivating and noninactivating MaxiK channels (8). Inactivation is removed by trypsin application to the cytosolic face of the membrane, suggesting the presence of an associated cytosolic inactivating particle (6, 7). A model developed to explain the biophysical and pharmacological properties of inactivating channels in chromaffin cells suggests that these channels are formed by a tetrameric assembly of inactivating and noninactivating subunits. Interestingly, inactivating channels in chromaffin cells are less sensitive to CTX, and heteromeric channels consisting of inactivating and noninactivating isoforms seem to have intermediate toxin sensitivities (9). Identification of a MaxiK channel variant or a subunit capable of producing fast inactivating MaxiK channel currents has so far been elusive (10).Here, we report a human ␤ subunit (␤2) ...

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Prolonged depolarization induces a slow inactivation process in some K+ channels. We have studied ionic and gating currents during long depolarizations in the mutant Shaker H4-Δ(6–46) K+ channel and in the nonconducting mutant (Shaker H4-Δ(6–46)-W434F). These channels lack the amino terminus that confers the fast (N-type) inactivation (Hoshi, T., W.N. Zagotta, and R.W. Aldrich. 1991. Neuron. 7:547–556). Channels were expressed in oocytes and currents were measured with the cut-open-oocyte and patch-clamp techniques. In both clones, the curves describing the voltage dependence of the charge movement were shifted toward more negative potentials when the holding potential was maintained at depolarized potentials. The evidences that this new voltage dependence of the charge movement in the depolarized condition is associated with the process of slow inactivation are the following: (a) the installation of both the slow inactivation of the ionic current and the inactivation of the charge in response to a sustained 1-min depolarization to 0 mV followed the same time course; and (b) the recovery from inactivation of both ionic and gating currents (induced by repolarizations to −90 mV after a 1-min inactivating pulse at 0 mV) also followed a similar time course. Although prolonged depolarizations induce inactivation of the majority of the channels, a small fraction remains non–slow inactivated. The voltage dependence of this fraction of channels remained unaltered, suggesting that their activation pathway was unmodified by prolonged depolarization. The data could be fitted to a sequential model for Shaker K+ channels (Bezanilla, F., E. Perozo, and E. Stefani. 1994. Biophys. J. 66:1011–1021), with the addition of a series of parallel nonconducting (inactivated) states that become populated during prolonged depolarization. The data suggest that prolonged depolarization modifies the conformation of the voltage sensor and that this change can be associated with the process of slow inactivation.

The large-conductance Ca 2+ - and voltage-activated K + channel (BK Ca, MaxiK), which is encoded by the Kcnma1 gene, is generally expressed at the plasma membrane of excitable and nonexcitable cells. However, in adult cardiomyocytes, a BK Ca -like channel activity has been reported in the mitochondria but not at the plasma membrane. The putative opening of this channel with the BK Ca agonist, NS1619, protects the heart from ischemic insult. However, the molecular origin of mitochondrial BK Ca (mitoBK Ca ) is unknown because its linkage to Kcnma1 has been questioned on biochemical and molecular grounds. Here, we unequivocally demonstrate that the molecular correlate of mitoBK Ca is the Kcnma1 gene, which produces a protein that migrates at ∼140 kDa and arranges in clusters of ∼50 nm in purified mitochondria. Physiological experiments further support the origin of mitoBK Ca as a Kcnma1 product because NS1619-mediated cardioprotection was absent in Kcnma1 knockout mice. Finally, BK Ca transcript analysis and expression in adult cardiomyocytes led to the discovery of a 50-aa C-terminal splice insert as essential for the mitochondrial targeting of mitoBK Ca .

The pore-forming ␣ subunit of large conductance voltage-and Ca 2؉ -sensitive K (MaxiK) channels is regulated by a ␤ subunit that has two membrane-spanning regions separated by an extracellular loop. To investigate the structural determinants in the pore-forming ␣ subunit necessary for ␤-subunit modulation, we made chimeric constructs between a human MaxiK channel and the Drosophila homologue, which we show is insensitive to ␤-subunit modulation, and analyzed the topology of the ␣ subunit. A comparison of multiple sequence alignments with hydrophobicity plots revealed that MaxiK channel ␣ subunits have a unique hydrophobic segment (S0) at the N terminus. This segment is in addition to the six putative transmembrane segments (S1-S6) usually found in voltage-dependent ion channels. High-conductance voltage-and Ca 2ϩ -sensitive potassium channels are found virtually in all excitable and nonexcitable tissues, with the exception of heart. As sensors of both voltage and intracellular calcium, they are responsible for membrane hyperpolarization, associated with phenomena like repetitive firing, spike shaping, transmitter release, and regulation of vascular and visceral smooth muscle contractility (1-4). Cloning of high-conductance voltage-activated and Ca 2ϩ -sensitive K ϩ (MaxiK) channels revealed that they belong to the S4 superfamily of ion channels (5) but carry a unique C terminus containing four hydrophobic, possibly membrane-spanning regions (S7-S10) with a nonconserved linker between regions S8 and S9 (6-8). The C-terminal region after the nonconserved linker shows the highest sequence conservation between the Drosophila (Dslo) and mammalian clones and includes hydrophobic regions S9 and S10. This region can be expressed as a separate domain and has been proposed to determine the Ca 2ϩ sensitivity of this channel (9). Alternative splicing rather than homologous genes seems to be responsible for the diversity of MaxiK channels (8,10,11).The common features of voltage-dependent K ϩ channels and individual domains of Na ϩ and Ca 2ϩ channels of the S4 superfamily are six putative transmembrane segments with a pore loop between transmembrane segments S5 and S6. The S4 region, which has been shown to move outward during depolarization and activation of these channels (12, 13), carries positive charges that are thought to interact with negative charges in regions S2 and S3 in Shaker K ϩ channels (14). By analyzing sequence alignments and hydrophobicity plots, we show that MaxiK channels may share these features, as initially proposed (7), but carry an additional hydrophobic region (S0) at the N terminus. Our data suggest that this hydrophobic region serves as a type I signal anchor directing the N terminus to the extracellular space.MaxiK channels purified from smooth muscle are tightly associated with an accessory ␤ subunit (15). Purification and cloning of this ␤ subunit revealed that it has two putative membrane-spanning regions and a large extracellular loop with two glycosylation sites (16,17). This ␤ subu...

Human large‐conductance voltage‐ and calcium‐sensitive K+ (maxi KCa) channels are composed of at least two subunits: the pore‐forming subunit, α, and a modulatory subunit, β. Expression of the β subunit induces dramatic changes in α subunit function. It increases the apparent Ca2+ sensitivity and it allows dehydrosoyasaponin I (DHS‐I) to upregulate the channel. The functional coupling of maxi KCa channel α and β subunits in freshly dissociated human coronary smooth muscle cells was assessed. To distinguish maxi KCa currents modulated by the β subunit, we examined (a) their apparent Ca2+ sensitivity, as judged from the voltage necessary to half‐activate the channel (V1/2), and (b) their activation by DHS‐I. In patches with unitary currents, the majority of channels were half‐activated near –85 mV at 18 μm Ca2+, a value similar to that obtained when the human KCa channel α (HSLO) and β (HKVCaβ) subunits are co‐expressed. A small number of channels half‐activated around 0 mV, suggesting the activity of the α subunit alone. The properties of macroscopic currents were consistent with the view that most pore‐forming α subunits were coupled to β subunits, since the majority of currents had values for V1/2 near to –90 mV, and currents were potentiated by DHS‐I. We conclude that in human coronary artery smooth muscle cells, most maxi KCa channels are composed of α and β subunits. The higher Ca2+ sensitivity of maxi KCa channels, resulting from their coupling to β subunits, suggests an important role of this channel in regulating coronary tone. Their massive activation by micromolar Ca2+ concentrations may lead to a large hyperpolarization causing profound changes in coronary blood flow and cardiac function.

Large-conductance, voltage-, and Ca(2+)-sensitive K(+) (maxi-K(Ca)) channels regulate neuronal and smooth muscle excitability. Their pore-forming alpha-subunit shows similarities with voltage-gated channels and indeed can open in the practical absence of Ca(2+). The NH(2) terminus is unique, with a seventh transmembrane segment involved in beta-subunit modulation. The long COOH terminus is implied in Ca(2+) modulation.