Bernard Ribalet

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.

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.

Characterization of the G protein coupling of a somatostatin receptor to the K+ATP channel in insulin‐secreting mammalian HIT and RIN cell lines.

1. The G protein‐mediated coupling of a somatostatin (somatotropin‐releasing inhibitory factor; SRIF) receptor to the ATP‐dependent K+ channel (K+ATP channel) has been studied in insulin‐secreting cells using the patch clamp technique. 2. In excised outside‐out patches, the concentration‐dependent stimulation of the K+ATP channel by SRIF was biphasic. Stimulation reached a maximum at 15 nM (EC50 = 5.5 nM), then decayed to a minimum at 50 nM and returned to maximum stimulation at 500 nM. 3. In cell‐attached patches, bath‐applied SRIF caused K+ATP channel stimulation in most experiments. In a few cases, however, SRIF suppressed channel activity, a response that was reversed by addition of dibutyryl cyclic AMP (DBcAMP). Channel stimulation by SRIF or by DBcAMP did not occur in the presence of glucose. 4. In excised inside‐out patches, the alpha‐subunits of Gi or G(o)‐type G proteins stimulated the K+ATP channel (EC50 = 29 and 42 pM, respectively). The K+ATP channel stimulation by alpha i‐ or alpha o‐subunits had no effect on the concentration‐dependent inhibition by ATP. 5. In excised inside‐out patches, K+ATP channel activity was reduced by inhibitors of protein kinase C (PKC) and stimulated by a PKC activator. The stimulatory effect of PKC was unaffected by the presence of pertussis toxin, but stimulation by exogenous alpha‐subunits of the G protein Gi or G(o) was prevented by PKC inhibitors. 6. From these data we deduce that SRIF can affect K+ATP channel activity directly via a membrane‐delimited pathway or indirectly via a pathway requiring diffusible messengers. In the former case, alpha i/alpha o may either enhance PLC activity, stimulating PKC and thus inducing K+ATP channel phosphorylation with consequent increase of activity, or channel phosphorylation by PKC may facilitate a direct stimulation of the channel by alpha i/alpha o. In the latter case, an alpha i/alpha o‐induced fall in cAMP contributes to reduced PKA‐mediated phosphorylation and suppression of channel activity.

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.

The Intracellular Loop of Orai1 Plays a Central Role in Fast Inactivation of Ca2+ Release-activated Ca2+ Channels

Store-operated Ca2+ entry (SOCE) due to activation of Ca2+ release-activated Ca2+ (CRAC) channels leads to sustained elevation of cytoplasmic Ca2+ and activation of lymphocytes. CRAC channels consisting of four pore-forming Orai1 subunits are activated by STIM1, an endoplasmic reticulum Ca2+ sensor that senses intracellular store depletion and migrates to plasma membrane proximal regions to mediate SOCE. One of the fundamental properties of CRAC channels is their Ca2+-dependent fast inactivation. To identify the domains of Orai1 involved in fast inactivation, we have mutated residues in the Orai1 intracellular loop linking transmembrane segment II to III. Mutation of four residues, V151SNV154, at the center of the loop (MutA) abrogated fast inactivation, leading to increased SOCE as well as higher CRAC currents. Point mutation analysis identified five key amino acids, N153VHNL157, that increased SOCE in Orai1 null murine embryonic fibroblasts. Expression or direct application of a peptide comprising the entire intracellular loop or the sequence N153VHNL157 blocked CRAC currents from both wild type (WT) and MutA Orai1. A peptide incorporating the MutA mutations had no blocking effect. Concatenated Orai1 constructs with four MutA monomers exhibited high CRAC currents lacking fast inactivation. Reintroduction of a single WT monomer (MutA-MutA-MutA-WT) was sufficient to fully restore fast inactivation, suggesting that only a single intracellular loop can block the channel. These data suggest that the intracellular loop of Orai1 acts as an inactivation particle, which is stabilized in the ion permeation pathway by the N153VHNL157 residues. These results along with recent reports support a model in which the N terminus and the selectivity filter of Orai1 as well as STIM1 act in concert to regulate the movement of the intracellular loop and evoke fast inactivation.

Calcium Signaling via Orai1 Is Essential for Induction of the Nuclear Orphan Receptor Pathway To Drive Th17 Differentiation

Orai1 is the pore subunit of Ca2+ release–activated Ca2+ (CRAC) channels that stimulate downstream signaling pathways crucial for T cell activation. CRAC channels are an attractive therapeutic target for alleviation of autoimmune diseases. Using high-throughput chemical library screening targeting Orai1, we identified a novel class of small molecules that inhibit CRAC channel activity. One of these molecules, compound 5D, inhibited CRAC channel activity by blocking ion permeation. When included during differentiation, Th17 cells showed higher sensitivity to compound 5D than Th1 and Th2 cells. The selectivity was attributable to high dependence of promoters of retinoic-acid-receptor-related orphan receptors on the Ca2+-NFAT pathway. Blocking of CRAC channels drastically decreased recruitment of NFAT and histone modifications within key gene loci involved in Th17 differentiation. The impairment in Th17 differentiation by treatment with CRAC channel blocker was recapitulated in Orai1-deficient T cells, which could be rescued by exogenous expression of retinoic-acid-receptor-related orphan receptors or a constitutive active mutant of NFAT. In vivo administration of CRAC channel blockers effectively reduced the severity of experimental autoimmune encephalomyelitis by suppression of differentiation of inflammatory T cells. These results suggest that CRAC channel blockers can be considered as chemical templates for the development of therapeutic agents to suppress inflammatory responses.

The third transmembrane segment of orai1 protein modulates Ca2+ release-activated Ca2+ (CRAC) channel gating and permeation properties.

Orai1, the pore subunit of Ca2+ release-activated Ca2+ channels, has four transmembrane segments (TMs). The first segment, TMI, lines the pore and plays an important role in channel activation and ion permeation. TMIII, on the other hand, does not line the pore but still regulates channel gating and permeation properties. To understand the role of TMIII, we have mutated and characterized several residues in this domain. Mutation of Trp-176 to Cys (W176C) and Gly-183 to Ala (G183A) had dramatic effects. Unlike wild-type channels, which exhibit little outward current and are activated by STIM1, W176C mutant channels exhibited a large outward current at positive potentials and were constitutively active in the absence of STIM1. G183A mutant channels also exhibited substantial outward currents but were active only in the presence of 2-aminoethoxydiphenyl borate (2-APB), irrespective of STIM1. With W176C mutant channels inward, monovalent currents were blocked by Ca2+ with a high affinity similar to the wild type, but the Ca2+-dependent blocking of outward currents differed in the two cases. Although a 50% block of the WT outward current required 250 μm Ca2+, more than 6 mm was necessary to have the same effect on W176C mutant channels. In the presence of extracellular Ca2+, W176C and G183A outward currents developed slowly in a voltage-dependent manner, whereas they developed almost instantaneously in the absence of Ca2+. These changes in permeation and gating properties mimic the changes induced by mutations of Glu-190 in TMIII and Asp-110/Asp-112 in the TMI/TMII loop. On the basis of these data, we propose that TMIII maintains negatively charged residues at or near the selectivity filter in a conformation that facilitates Ca2+ inward currents and prevents outward currents of monovalent cations. In addition, to controlling selectivity, TMIII may also stabilize channel gating in a closed state in the absence of STIM1 in a Trp-176-dependent manner.