Scott A. John

Connexin-43 Hemichannels Opened by Metabolic Inhibition

The cause of altered ionic homeostasis leading to cell death during ischemia and metabolic inhibition is unclear. Hemichannels, which are precursors to gap junctions, are nonselective ion channels that are permeable to molecules of less thanM r 1000. We show that hemichannels open upon exposure to calcium-free solutions when they are either heterologously overexpressed in HEK293 cells or endogenously expressed in cardiac ventricular myocytes. In the presence of normal extracellular calcium, hemichannels open during metabolic inhibition. During ischemia and other forms of metabolic inhibition, activation of relatively few hemichannels will seriously compromise the cell’s ability to maintain ionic homeostasis, which is an essential step promoting cell death.

A Novel Role for Connexin Hemichannel in Oxidative Stress and Smoking-Induced Cell Injury

Oxidative stress is linked to many pathological conditions, including ischemia, atherosclerosis and neurodegenerative disorders. The molecular mechanisms of oxidative stress induced pathophysiology and cell death are currently poorly understood. Our present work demonstrates that oxidative stress induced by reactive oxygen species and cigarette smoke extract depolarize the cell membrane and open connexin hemichannels. Under oxidative stress, connexin expression and connexin silencing resulted in increased and reduced cell deaths, respectively. Morphological and live/dead assays indicate that cell death is likely through apoptosis. Our studies provide new insights into the mechanistic role of hemichannels in oxidative stress induced cell injury.

The Na+/Ca2+ Exchange Molecule

Abstract: An overview of the molecular physiology of the Na+/Ca2+ exchanger is presented. This includes information on the variety of exchangers that have been described and their regulatory properties. Molecular insight is most detailed for the cardiac Na+/Ca2+ exchanger (NCX1). Parts of the NCS1 molecule involved in regulation and ion transport have been elucidated, and initial information on the topology and structure is available.

Subcellular Localization of Hexokinases I and II Directs the Metabolic Fate of Glucose

Background The first step in glucose metabolism is conversion of glucose to glucose 6-phosphate (G-6-P) by hexokinases (HKs), a family with 4 isoforms. The two most common isoforms, HKI and HKII, have overlapping tissue expression, but different subcellular distributions, with HKI associated mainly with mitochondria and HKII associated with both mitochondrial and cytoplasmic compartments. Here we tested the hypothesis that these different subcellular distributions are associated with different metabolic roles, with mitochondrially-bound HKs channeling G-6-P towards glycolysis (catabolic use), and cytoplasmic HKII regulating glycogen formation (anabolic use). Methodology/Principal Findings To study subcellular translocation of HKs in living cells, we expressed HKI and HKII linked to YFP in CHO cells. We concomitantly recorded the effects on glucose handling using the FRET based intracellular glucose biosensor, FLIPglu-600 mM, and glycogen formation using a glycogen-associated protein, PTG, tagged with GFP. Our results demonstrate that HKI remains strongly bound to mitochondria, whereas HKII translocates between mitochondria and the cytosol in response to glucose, G-6-P and Akt, but not ATP. Metabolic measurements suggest that HKI exclusively promotes glycolysis, whereas HKII has a more complex role, promoting glycolysis when bound to mitochondria and glycogen synthesis when located in the cytosol. Glycogen breakdown upon glucose removal leads to HKII inhibition and dissociation from mitochondria, probably mediated by increases in glycogen-derived G-6-P. Conclusions/Significance These findings show that the catabolic versus anabolic fate of glucose is dynamically regulated by extracellular glucose via signaling molecules such as intracellular glucose, G-6-P and Akt through regulation and subcellular translocation of HKII. In contrast, HKI, which activity and regulation is much less sensitive to these factors, is mainly committed to glycolysis. This may be an important mechanism by which HKs allow cells to adapt to changing metabolic conditions to maintain energy balance and avoid injury.

The sulphonylurea receptor SUR1 regulates ATP-sensitive mouse Kir6.2 K+ channels linked to the green fluorescent protein in human embryonic kidney cells (HEK 293).

1 Using a chimeric protein comprising the green fluorescent protein (GFP) linked to the C‐terminus of the K+ channel protein mouse Kir6.2 (Kir6.2‐C‐GFP), the interactions between the sulphonylurea receptor SUR1 and Kir6.2 were investigated in transfected human embryonic kidney cells (HEK 293) by combined imaging and patch clamp techniques. 2 HEK 293 cells transfected with mouse Kir6.2‐C‐GFP and wild‐type Kir6.2 exhibited functional K+ channels independently of SUR1. These channels were inhibited by ATP (IC50= 150 μM), but were not responsive to stimulation by ADP or inhibition by sulphonylureas. Typically, 15 ± 7 active channels were found in an excised patch. 3 The distribution of Kir6.2‐C‐GFP protein was investigated by imaging of GFP fluorescence. There was a lamellar pattern of fluorescence labelling inside the cytoplasm (presumably associated with the endoplasmic reticulum and the Golgi apparatus) and intense punctate labelling near the cell membrane, but little fluorescence was associated with the plasma membrane. 4 In contrast, cells co‐transfected with Kir6.2‐C‐GFP and SUR1 exhibited intense uniform plasma membrane labelling, and the lamellar and punctate labelling seen without SUR1 was no longer prominent. 5 In cells co‐transfected with Kir6.2‐C‐GFP and SUR1, strong membrane labelling was associated with very high channel activity, with 484 ± 311 active channels per excised patch. These K+ channels were sensitive to inhibition by ATP (IC50= 17 μM), stimulated by ADP and inhibited by sulphonylureas. 6 We conclude that co‐expression of SUR1 and Kir6.2 generates channels with the properties of native KATP channels. In addition, SUR1 promotes uniform insertion of Kir6.2‐C‐GFP into the plasma membrane and a 35‐fold increase in channel activity, suggesting that SUR1 facilitates protein trafficking of Kir6.2 into the plasma membrane.

Spermine Block of the Strong Inward Rectifier Potassium Channel Kir2.1: Dual Roles of Surface Charge Screening and Pore Block

Inward rectification in strong inward rectifiers such as Kir2.1 is attributed to voltage-dependent block by intracellular polyamines and Mg2+. Block by the polyamine spermine has a complex voltage dependence with shallow and steep components and complex concentration dependence. To understand the mechanism, we measured macroscopic Kir2.1 currents in excised inside-out giant patches from Xenopus oocytes expressing Kir2.1, and single channel currents in the inside-out patches from COS7 cells transfected with Kir2.1. We found that as spermine concentration or voltage increased, the shallow voltage-dependent component of spermine block at more negative voltages was caused by progressive reduction in the single channel current amplitude, without a decrease in open probability. We attributed this effect to spermine screening negative surface charges involving E224 and E299 near the inner vestibule of the channel, thereby reducing K ion permeation rate. This idea was further supported by experiments in which increasing ionic strength also decreased Kir2.1 single channel amplitude, and by mutagenesis experiments showing that this component of spermine block decreased when E224 and E299, but not D172, were neutralized. The steep voltage-dependent component of block at more depolarized voltages was attributed to spermine migrating deeper into the pore and causing fast open channel block. A quantitative model incorporating both features showed excellent agreement with the steady-state and kinetic data. In addition, this model accounts for previously described substate behavior induced by a variety of Kir2.1 channel blockers.

Ca2+-dependent structural rearrangements within Na+–Ca2+ exchanger dimers

Cytoplasmic Ca2+ is known to regulate Na+–Ca2+ exchanger (NCX) activity by binding to two adjacent Ca2+-binding domains (CBD1 and CBD2) located in the large intracellular loop between transmembrane segments 5 and 6. We investigated Ca2+-dependent movements as changes in FRET between exchanger proteins tagged with CFP or YFP at position 266 within the large cytoplasmic loop. Data indicate that the exchanger assembles as a dimer in the plasma membrane. Addition of Ca2+ decreases the distance between the cytoplasmic loops of NCX pairs. The Ca2+-dependent movements detected between paired NCXs were abolished by mutating the Ca2+ coordination sites in CBD1 (D421A, E451A, and D500V), whereas disruption of the primary Ca2+ coordination site in CBD2 (E516L) had no effect. Thus, the Ca2+-induced conformational changes of NCX dimers arise from the movement of CBD1. FRET studies of CBD1, CBD2, and CBD1–CBD2 peptides displayed Ca2+-dependent movements with different apparent affinities. CBD1–CBD2 showed a Ca2+-dependent phenotype mirroring full-length NCX but distinct from both CBD1 and CBD2.

Phosphatidylinositol-4,5-bisphosphate (PIP2) regulation of strong inward rectifier Kir2.1 channels: multilevel positive cooperativity

Inwardly rectifying potassium (Kir) channels are gated by the interaction of their cytoplasmic regions with membrane‐bound phosphatidylinositol‐4,5‐bisphosphate (PIP2). In the present study, we examined how PIP2 interaction regulates channel availability and channel openings to various subconductance levels (sublevels) as well as the fully open state in the strong inward rectifier Kir2.1 channel. Various Kir2.1 channel constructs were expressed in Xenopus oocytes and single channel or macroscopic currents were recorded from inside‐out patches. The wild‐type (WT) channel rarely visited the subconductance levels under control conditions. However, upon reducing Kir2.1 channel interaction with PIP2 by a variety of interventions, including PIP2 antibodies, screening PIP2 with neomycin, or mutating PIP2 binding sites (e.g. K188Q), visitation to the sublevels was markedly increased before channels were converted to an unavailable mode in which they did not open. No channel activity was detected in channels with the double mutation K188A/R189A, a mutant which exhibits extremely weak interaction with PIP2. By linking subunits together in tandem dimers or tetramers containing mixtures of WT and K188A/R189A subunits, we demonstrate that one functional PIP2‐interacting WT subunit is sufficient to convert channels from the unavailable to the available mode with a high open probability dominated by the fully open state, with similar kinetics as tetrameric WT channels. Occasional openings to sublevels become progressively less frequent as the number of WT subunits increases. Quantitative analysis reveals that the interaction of PIP2 with WT subunits exerts strong positive cooperativity in both converting the channels from the unavailable to the available mode, and in promoting the fully open state over sublevels. We conclude that the interaction of PIP2 with only one Kir2.1 subunit is sufficient for the channel to become available and to open to its full conductance state. Interaction with additional subunits exerts positive cooperativity at multiple levels to further enhance channel availability and promote the fully open state.

Molecular mechanism for ATP-dependent closure of the K+ channel Kir6.2.

In the ATP‐dependent K+ (KATP) channel pore‐forming protein Kir6.2, mutation of three positively charged residues, R50, K185 and R201, impairs the ability of ATP to close the channel. The mutations do not change the channel open probability (Po) in the absence of ATP, supporting the involvement of these residues in ATP binding. We recently proposed that at least two of these positively charged residues, K185 and R201, interact with ATP phosphate groups to cause channel closure: the β phosphate group of ATP interacts with K185 to initiate closure, while the α phosphate interacts with R201 to stabilize the channels closed state. In the present study we replaced these three positive residues with residues of different charge, size and hydropathy. For K185 and R201, we found that charge, more than any other property, controls the interaction of ATP with Kir6.2. At these positions, replacement with another positive residue had minor effects on ATP sensitivity. In contrast, replacement of K185 with a negative residue (K185D/E) decreased ATP sensitivity much more than neutral substitutions, suggesting that an electrostatic interaction between the β phosphate group of ATP and K185 destabilizes the open state of the channel. At R201, replacement with a negative charge (R201E) had multiple effects, decreasing ATP sensitivity and preventing full channel closure at high concentrations. In contrast, the R50E mutation had a modest effect on ATP sensitivity, and only residues such as proline and glycine that affect protein structure caused major decreases in ATP sensitivity at the R50 position. Based on these results and the recently published structure of Kir3.1 cytoplasmic domain, we propose a scheme where binding of the β phosphate group of ATP to K185 induces a motion of the surrounding region, which destabilizes the open state, favouring closure of the M2 gate. Binding of the α phosphate group of ATP to R201 then stabilizes the closed state. R50 on the N‐terminus controls ATP binding by facilitating the interaction of the β phosphate group of ATP with K185 to destabilize the open state.

Molecular Basis for Kir6.2 Channel Inhibition by Adenine Nucleotides

K(ATP) channels are comprised of a pore-forming protein, Kir6.x, and the sulfonylurea receptor, SURx. Interaction of adenine nucleotides with Kir6.2 positively charged amino acids such as K185 and R201 on the C-terminus causes channel closure. Substitution of these amino acids with other positively charged residues had small effects on inhibition by adenine nucleotide, while substitution with neutral or negative residues had major effects, suggesting electrostatic interactions between Kir6.2 positive charges and adenine nucleotide negative phosphate groups. Furthermore, R201 mutation decreased channel sensitivity to ATP, ADP, and AMP to a similar extent, but K185 mutation decreased primarily ATP and ADP sensitivity, leaving the AMP sensitivity relatively unaffected. Thus, channel inhibition by ATP may involve interaction of the alpha-phosphate with R201 and interaction of the beta-phosphate with K185. In addition, decreased open probability due to rundown or sulfonylureas caused an increase in ATP sensitivity in the K185 mutant, but not in the R201 mutant. Thus, the beta-phosphate may bind in a state-independent fashion to K185 to destabilize channel openings, while R201 interacts with the alpha-phosphate to stabilize a channel closed configuration. Substitution of R192 on the C-terminus and R50 on the N-terminus with different charged residues also affected ATP sensitivity. Based on these results a structural scheme is proposed, which includes features of other recently published models.