Ligia Toro

The crystal structure of mouse VDAC1 at 2.3 Å resolution reveals mechanistic insights into metabolite gating

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 β-barrel eukaryotic membrane protein, the murine VDAC1 (mVDAC1) at 2.3 Å resolution, revealing a high-resolution image of its architecture formed by 19 β-strands. Unlike the recent NMR structure of human VDAC1, the position of the voltage-sensing N-terminal segment is clearly resolved. The α-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.

A calcium switch for the functional coupling between α (hslo) and β subunits (KV, Caβ) of maxi K channels

K V , Ca β subunit dramatically increases the apparent calcium sensitivity of the α subunit of MaxlK channels when probed in the micromolar [Ca2+]i range. Analysis in a wide range of [Ca2+]i revealed that this functional coupling is exquisitely modulated by [Ca2+]i. Ca2+ ions switch MaxiK α+β complex into a functionally coupled state at concentrations beyond resting [Ca2+]i. At [Ca2+] ≤ 100 nM, MaxiK activity becomes independent of Ca2+, is purely voltage‐activated, and its functional coupling with its β subunit is released. The functional switch develops at [Ca2+]i that occur during cellular excitation, providing the molecular basis of how MaxiK channels regulate smooth muscle excitability and neurotransmitter release.

Molecular constituents of maxi KCa channels in human coronary smooth muscle: predominant α+β subunit complexes

1 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. 2 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. 3 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. 4 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. 5 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.

MaxiK channel partners: physiological impact

The basic functional unit of the large‐conductance, voltage‐ and Ca2+‐activated K+ (MaxiK, BK, BKCa) channel is a tetramer of the pore‐forming α‐subunit (MaxiKα) encoded by a single gene, Slo, holding multiple alternative exons. Depending on the tissue, MaxiKα can associate with modulatory β‐subunits (β1–β4) increasing its functional diversity. As MaxiK senses and regulates membrane voltage and intracellular Ca2+, it links cell excitability with cell signalling and metabolism. Thus, MaxiK is a key regulator of vital body functions, like blood flow, uresis, immunity and neurotransmission. Epilepsy with paroxysmal dyskinesia syndrome has been recognized as a MaxiKα‐related disorder caused by a gain‐of‐function C‐terminus mutation. This channel region is also emerging as a key recognition module containing sequences for MaxiKα interaction with its surrounding signalling partners, and its targeting to cell‐specific microdomains. The growing list of interacting proteins highlights the possibility that associations with the C‐terminus of MaxiKα are dynamic and depending on each cellular environment. We speculate that the molecular multiplicity of the C‐terminus (and intracellular loops) dictated by alternative exons may modulate or create additional interacting sites in a tissue‐specific manner. A challenge is the dissection of MaxiK macromolecular signalling complexes in different tissues and their temporal association/dissociation according to the stimulus.

Molecular and Functional Signature of Heart Hypertrophy During Pregnancy

During pregnancy, the heart develops a reversible physiological hypertrophic growth in response to mechanical stress and increased cardiac output; however, underlying molecular mechanisms remain unknown. Here, we investigated pregnancy-related changes in heart structure, function, and gene expression of known markers of pathological hypertrophy and cell stretching in mice hearts. In late pregnancy, hearts show eccentric hypertrophy, as expected for a response to volume overload, with normal left ventricular diastolic function and a moderate reduction in systolic function. Pregnancy-related physiological heart hypertrophy does not induce expression changes of known markers of pathological hypertrophy like: &agr;- and &bgr;-myosin heavy chain, atrial natriuretic factor, phospholamban, and sarcoplasmic reticulum Ca2+-ATPase. Instead, it induces the remodeling of Kv4.3 channel and increased c-Src tyrosine kinase activity, a stretch-responsive kinase. Cardiac Kv4.3 channel gene expression was downregulated by ≈3- to 5-fold, both at the mRNA and protein levels, and was paralleled by a reduction in transient outward K+ currents, a longer action potential and by prolongation of the QT interval. Downregulation of cardiac Kv4.3 transcripts was mimicked by estrogen treatment in ovariectomized mice, and was prevented by the estrogen receptor antagonist ICI 182,780. c-Src activity increased by ≈2-fold in late pregnancy and after estrogen treatment. We propose that, in addition to mechanical stress, the rise of estrogen toward the end of pregnancy contributes to pregnancy-related heart hypertrophy by increased c-Src activity and that the rise of estrogen is one factor that down regulates cardiac Kv4.3 gene expression providing a molecular correlate for a longer QT interval in pregnancy.

Decreased Expression of Voltage- and Ca2+-Activated K+ Channels in Coronary Smooth Muscle During Aging

Abstract— Aging is the main risk factor for coronary artery disease. One characteristic of aging coronary arteries is their enhanced contractile responses to endothelial vasoconstricting factors, which increase the risk of coronary vasospasm in older people. Because large-conductance voltage- and Ca2+-activated K+ channels (MaxiK) are key regulators of vascular tone, we explored the possibility that this class of channels is diminished with increasing age. Using site-directed antibodies recognizing the pore-forming &agr; subunit and electrophysiological methods, we demonstrate that the number of MaxiK channels is dramatically diminished in aged coronary arteries from old F344 rats. Channel density was reduced from 52±9 channels/pF (3 months old) to 18±5 channels/pF (25 to 30 months old), which represents a 65% reduction in the older population. Pixel intensity of Western blots was also diminished by ≈50%. Moreover, the age-related decrease in the channel protein expression was also evident in humans, which showed ≈80% reduction in 61- to 70-year-old subjects compared with 3- to 18-year-old youngsters and ≈45% reduction compared with 19- to 56-year-old adults. In agreement with a reduction of MaxiK channel numbers in aging coronary arteries, old coronary arteries from F344 rats contract less effectively (≈70% reduction) than young coronary arteries when exposed to the MaxiK channel blocker iberiotoxin. The contraction studies indicate that under physiological conditions, MaxiK channels are tonically active, serving as a hyperpolarizing force that opposes contraction. Thus, reduced expression of MaxiK channels in aged coronary arteries would lead to a decreased vasodilating capacity and increased risk of coronary spasm and myocardial ischemia in older people.

Coupling of c-Src to large conductance voltage- and Ca2+-activated K+ channels as a new mechanism of agonist-induced vasoconstriction

The voltage-dependent and Ca2+-activated K+ channel (MaxiK, BK) and the cellular proto-oncogene pp60c-Src (c-Src) are abundant proteins in vascular smooth muscle. The role of MaxiK channels as a vasorelaxing force is well established, but their role in vasoconstriction is unclear. Because Src participates in regulating vasoconstriction, we investigated whether c-Src inhibits MaxiK as a mechanism for agonist-induced vasoconstriction. Functional experiments in human and rat show that inhibitors of Src (Lavendustin A, PP2) but not inactive compounds (Lavendustin B, PP3) induce a pronounced relaxation of coronary or aortic smooth muscle precontracted with 5-hydroxytriptamine, phenylephrine, or Angiotensin II. Iberiotoxin, a MaxiK blocker, antagonizes the relaxation induced by Lavendustin A or PP2, indicating that c-Src inhibits the Iberiotoxin-sensitive component, likely MaxiK channels. In agreement, coronary muscle MaxiK currents were enhanced by Lavendustin A. To investigate the molecular mechanism of c-Src action on MaxiK channels, we transiently expressed its α subunit, hSlo, with or without c-Src in HEK293T cells. The voltage sensitivity of hSlo was right-shifted by ≈16 mV. hSlo inhibition by c-Src is due to channel direct phosphorylation because: (i) excised patches exposed to protein tyrosine phosphatase (CD45) resulted in a partial reversal of the inhibitory effect by ≈10 mV, and (ii) immunoprecipitated hSlo channels were recognized by an anti-phosphotyrosine Ab. Furthermore, coexpression of hSlo and c-Src demonstrate a striking colocalization in HEK293T cells. We propose that MaxiK channels via direct c-Src-dependent phosphorylation play a significant role supporting vasoconstriction after activation of G protein-coupled receptors by vasoactive substances and neurotransmitters.

A novel estrogen receptor GPER inhibits mitochondria permeability transition pore opening and protects the heart against ischemia-reperfusion injury

Several studies have recently demonstrated that G protein-coupled receptor 30 (GPER) can directly bind to estrogen and mediate its action. We investigated the role and the mechanism of estrogen-induced cardioprotection after ischemia-reperfusion using a specific GPER agonist G1. Isolated hearts from male mice were perfused using Langendorff technique with oxygenated (95% O(2) and 5% CO(2)) Krebs Henseleit buffer (control), with G1 (1 microM), and G1 (1 microM) together with extracellular signal-regulated kinase (Erk) inhibitor PD-98059 (5 microM). After 20 min of perfusion, hearts were subjected to 20 min global normothermic (37 degrees C) ischemia followed by 40 min reperfusion. Cardiac function was measured, and myocardial necrosis was evaluated by triphenyltetrazolium chloride staining at the end of the reperfusion. Mitochondria were isolated after 10 min of reperfusion to assess the Ca(2+) load required to induce mitochondria permeability transition pore (mPTP) opening. G1-treated hearts developed better functional recovery with higher rate pressure product (RPP, 6140 +/- 264 vs. 2,640 +/- 334 beats mmHg(-1) min(-1), P < 0.05). The infarct size decreased significantly in G1-treated hearts (21 +/- 2 vs. 46 +/- 3%, P < 0.001), and the Ca(2+) load required to induce mPTP opening increased (2.4 +/- 0.06 vs. 1.6 +/- 0.11 microM/mg mitochondrial protein, P < 0.05) compared with the controls. The protective effect of G1 was abolished in the presence of PD-98059 [RPP: 4,120 +/- 46 beats mmHg(-1) min(-1), infarct size: 53 +/- 2%, and Ca(2+) retention capacity: 1.4 +/- 0.11 microM/mg mitochondrial protein (P < 0.05)]. These results suggest that GPER activation provides a cardioprotective effect after ischemia-reperfusion by inhibiting the mPTP opening, and this effect is mediated by the Erk pathway.

mitoBKCa is encoded by the Kcnma1 gene, and a splicing sequence defines its mitochondrial location

The large-conductance Ca2+- and voltage-activated K+ channel (BKCa, 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 BKCa-like channel activity has been reported in the mitochondria but not at the plasma membrane. The putative opening of this channel with the BKCa agonist, NS1619, protects the heart from ischemic insult. However, the molecular origin of mitochondrial BKCa (mitoBKCa) is unknown because its linkage to Kcnma1 has been questioned on biochemical and molecular grounds. Here, we unequivocally demonstrate that the molecular correlate of mitoBKCa 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 mitoBKCa as a Kcnma1 product because NS1619-mediated cardioprotection was absent in Kcnma1 knockout mice. Finally, BKCa 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 mitoBKCa.

β1 (KCNMB1) subunits mediate lithocholate activation of large-conductance Ca2+-activated K+ channels and dilation in small, resistance-size arteries

Among the nongenomic effects of steroids, control of vasomotion has received increasing attention. Lithocholate (LC) and other physiologically relevant cholane-derived steroids cause vasodilation, yet the molecular targets and mechanisms underlying this action remain largely unknown. We demonstrate that LC (45 μM) reversibly increases the diameter of pressurized resistance cerebral arteries by ∼10%, which would result in ∼30% increase in cerebral blood flow. LC action is independent of endothelial integrity, prevented by 55 nM iberiotoxin, and unmodified by 0.8 mM 4-aminopyridine, indicating that LC causes vasodilation via myocyte BK channels. Indeed, LC activates BK channels in isolated myocytes through a destabilization of channel long-closed states without modifying unitary conductance. LC channel activation occurs within a wide voltage range and at Ca2+ concentrations reached in the myocyte at rest and during contraction. Channel accessory β1 subunits, which are predominant in smooth muscle, are necessary for LC to modify channel activity. In contrast, β4 subunits, which are predominant in neuronal tissues, fail to evoke LC sensitivity. LC activation of cbv1+β1 and native BK channels display identical characteristics, including EC50 (46 μM) and Emax (≈300 μM) values, strongly suggesting that the cbv1+β1 complex is necessary and sufficient to evoke LC action. Finally, intact arteries from β1 subunit knockout mice fail to relax in response to LC, although they are able to respond to other vasodilators. This study pinpoints the BK β1 subunit as the molecule that senses LC, which results in myocyte BK channel activation and, thus, endothelial-independent relaxation of small, resistance-size arteries.