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Modulation of Neuronal Kir3 Channels by Cholesterol
608a
Wednesday, March 2, 2016
by 2.450.4, 1.650.1 and 1.650.2 fold respectively; these effects were completely reversed by zinc chelator TPEN (20mM) added in the presence of ZnPy. Fluorescent zinc imaging with FluoZin3TM in HEK293 cells and DRG neurons demonstrated that ZnPy induced robust intracellular zinc rises which were completely reversed by TPEN. Four other chemically unrelated zinc ionophores PDTC, DEDTC, DIQ and Zinc ionophore I had similar to ZnPy effects: all four compounds augmented KCNQ4 and KCNQ2/3 currents and induced intracellular zinc rises; TPEN completely abolished these effects. Using voltage sensitive phosphatase CiVSP we found that the Ci-VSPdependent PIP2 hydrolysis strongly inhibited recombinant KCNQ4 and KCNQ2/3, however, ZnPy and PDTC completely abolished these effect rendering channels insensitive to Ci-VSP activation. TIRF imaging using optical PIP2 reporter PLCdPH-GFP confirmed that neither ZnPy nor PDTC affected the ability of CiVSP to hydrolyze PIP2, as determined by the depolarization-induced PLCdPH-GFP translocation. In sum, our data suggest that intracellular zinc potently augment KCNQ channel activity by reducing channel requirement for PIP2. 3001-Pos Board B378 Modulation of Neuronal Kir3 Channels by Cholesterol Anna N. Bukiya1, Avia Rosenhouse-Dantsker2. 1 Pharmacology, The University of Tennessee Health Science Center, Memphis, TN, USA, 2Pharmacology, University of Illinois at Chicago, Chicago, IL, USA. In recent years, cholesterol emerged as a major regulator of ion channel function. The most common effect of cholesterol on ion channels is a decrease in channel activity. We have recently shown that unexpectedly cholesterol enrichment up-regulates G-protein gated inwardly rectifying potassium (GIRK or Kir3) activity in atrial myocytes. Here we focus on neuronal Kir3 channels, and determine the effect of cholesterol on their function. Several Kir3 subunits are expressed in the hippocampus: Kir3.1, Kir3.2a, Kir3.2c and Kir3.3. Among these, Kir3.2 may be expressed as a homomer or as a heteromer with Kir3.1 and/or Kir3.3, both of which do not express as homomers in the plasma membrane. We first studied the effect of cholesterol on a pore mutant of Kir3.2 that increases its membrane expression and activity, Kir3.2^ (Kir3.2_E152D). We show that when Kir3.2^ was expressed in Xenopus oocytes, the resulting current was up-regulated by in vitro cholesterol enrichment of the oocyte membrane. Next, using planar lipid bilayers we demonstrate that cholesterol significantly increased the open probability of the Kir3.2^ channels. No change was observed in the unitary conductance. Most importantly, we show that also in vitro cholesterol enrichment of freshly isolated hippocampal pyramidal neurons resulted in a strong increase in physiological Kir3 currents. These findings indicate that cholesterol plays a critical role in modulating neuronal Kir3 channels. 3002-Pos Board B379 Cross-Talk between Cholesterol, PIP2 and Caveolin in Regulating Kir2 Channels Huazhi Han1, Avia Rosenhouse-Dantsker1, Radhakrishnan Gnanasambandam2, Frederick Sachs2, Irena Levitan1. 1 Medicine, University of Illinois at Chicago, Chicago, IL, USA, 2Physiology and Biophysics, University at Buffalo, SUNY, Buffalo, NY, USA. Multiple types of ion channels are regulated by two lipid modulators: phosphatidylinositol-4,5-bisphosphate (PIP2) and cholesterol, both modulators being enriched in caveolae. Our earlier studies identified cholesterol as a major negative regulator of Kir2 channels that are also well-known to be critically dependent on PIP2. Furthermore, it was proposed that cholesterol regulation of ion channels might be attributed to the disruption of their association with caveolin-1 and/or PIP2. In this study, we investigated the cross-talk between cholesterol, PIP2 and caveolin in the regulation of Kir2.1 channels. We show that cholesterol depletion strengthens the interaction between Kir2 channels and PIP2 and that a single-point mutation of Kir2.1 L222I that renders the channels cholesterol insensitive abrogates this effect. Furthermore, we present direct evidence that Kir2.1 is negatively regulated by Cav-1 and that Kir2.1-L222I mutation also abrogates the sensitivity of the channels to caveolin. Kinetic analysis of single-channel events also points to a common mechanism for cholesterol and Cav-1 regulation of the channels. However, neither Cav-1, nor intact caveolae are required for Kir2.1 channels to be cholesterol sensitive, thus challenging a general notion that cholesterol regulates ion channels by disruption of caveolae. Furthermore, we present first insights into the structural determinants of the cross-talk between the sensitivity of Kir2 channels to cholesterol, PIP2 and to caveolin: based on recent studies, we suggest that cholesterol, PIP2 and caveolin regulate the channels through distinct binding sites but that the signals generated by the binding of these modulators converge in regulating Kir2.1 gating.
3003-Pos Board B380 A Shared Mechanism of BK Channel Activation by Mallotoxin and Auxiliary g1 Subunit Xin Guan, Qin Li, Jiusheng Yan. Department of Anesthesiology and Perioperative Medicine, MD Anderson Cancer Center, Houston, TX, USA. The large-conductance, voltage- and calcium-activated Kþ (BK) channels consist of the pore-forming, voltage- and Ca2þ -sensing a-subunits (BKa) and the regulatory b and g subunits. The auxiliary g1 subunit is so far the most potent activator of BK channels which drastically shifts the voltagedependence of channel activation by ~ 140 mV towards the hyperpolarizing potential direction. Mallotoxin, also called rottlerin, is a potent extracellular small-molecule activator of BK channels, which was reported to shift the voltage-dependence of channel activation by ~ 100 mV in whole-cell patchclamp recording condition through a yet largely unknown mechanism. Here, we investigated the effects of mallotoxin on BK channels in excised membrane patches in the absence and presence of the auxiliary g subunit. Mallotoxin exerted significant activating effect on BK channels formed by BK alone in excised membrane patches by a ~ 80 mV shift in voltage-dependence of channel activation. However, the presence of g1 and g3 subunits nearly abolished and g2 subunit greatly attenuated the activating effect of mallotoxin. Most mutations in g1 subunit that caused a loss of its modulatory function on BK channels also resulted in a loss of its influence on mallotoxin. Importantly, we identified a point mutation in the middle of the g1 subunit’s transmembrane domain that had little effect on the g1 subunit’s modulatory function on BK channels in the absence of mallotoxin but caused an irreversible loss of the g1 subunit’s binding and modulatory function on BK channel upon a brief exposure to mallotoxin. Therefore, we conclude that g1 subunit competitively blocks the mallotoxin’s binding or action on BK channel, which can be fully reversed by a point mutation in the middle of the g1 transmembrane region. 3004-Pos Board B381 A Critical Role of the S6 Transmembrane Helix in BK Channel Modulation by Auxiliary g Subunits Qin Li, Jiusheng Yan. Department of Anesthesiology and Perioperative Medicine, MD Anderson Cancer Ctr, Houston, TX, USA. BK channels consist of the pore-forming, voltage- and Ca2þ-sensing a-subunits (BKa or Slo1) and the regulatory auxiliary b or g subunits. BK channels are potently modulated by the auxiliary g subunits, which shift the voltagedependence of channel activation by up to 140 mV in the hyperpolarizing direction. We have recently identified the g subunits’ transmembrane (TM) segment and its neighboring intracellular charged residues as key determinants for their modulatory functions on BK channels. However, the molecular mechanisms underlying BK channel modulation by auxiliary g subunits remain largely unknown. The proton-gated Slo3 channel has a high sequence similarity to Slo1 but showed nearly no sensitivity to the BKg1 (LRRC26) subunit. To identify the key protein regions of Slo1 that are involved in modulation by auxiliary g subunits, we generated a series of Slo1/Slo3 chimeras in the N-terminal TM (S0-S6) domain and analyzed their responses to the auxiliary g1 subunit in voltage-dependence of channel activation. We found that the Slo3 channel’s lack of sensitivity to BKg1 is largely caused by its S6 TM segment, whose inclusion in the Slo1/Slo3 chimera greatly attenuated the channel’s response to BKg1. Our further mutational analyses of the differential residues on the S6 TM segment identified several key residues that are largely responsible for the difference between Slo1 and Slo3 channels in their responses to BKg1. Therefore, we concluded that the S6 TM helix is critical in BK channel modulation by auxiliary g subunit. 3005-Pos Board B382 The Stretch-Activated BK (SAKca) Channel in Chick Heart is Inhibited by the Spider Peptide GsMTx-4 Qiong-Yao Tang1, Xiao-Dong Tang1, Yan-Jun Feng1, Hui Li1, Fei-Fei Zhang1, Zhe Zhang1, Masahiro Sokabe2. 1 Jiangsu Province Key Laboratory of Anesthesiology, Xuzhou Medical College, Xuzhou, China, 2Mechanobiology Laboratory, Nagoya University. Graduate School of Medicine, Nagoya, Japan. GsMTx4 is a 34-residue peptide isolated from the tarantula Grammostola spatulata and inhibits atrial fibrillation potentiated by dilatation in heart by blocking the stretch-activated (SA) ion channels. However, the mechanism by which GsMTx4 inhibits SA channels remains unknown. Here we report that the extracellularly applied nM concentrations of GsMTx4 inhibited SAKca channels in excised inside-out patch membranes of isolated chick heart cells. We found that GsMTx4 inhibited the stretch-induced SAKca channel opening in a dose-dependent manner without altering the single-channel conductance. Membrane stretch
Wednesday, March 2, 2016
by 2.450.4, 1.650.1 and 1.650.2 fold respectively; these effects were completely reversed by zinc chelator TPEN (20mM) added in the presence of ZnPy. Fluorescent zinc imaging with FluoZin3TM in HEK293 cells and DRG neurons demonstrated that ZnPy induced robust intracellular zinc rises which were completely reversed by TPEN. Four other chemically unrelated zinc ionophores PDTC, DEDTC, DIQ and Zinc ionophore I had similar to ZnPy effects: all four compounds augmented KCNQ4 and KCNQ2/3 currents and induced intracellular zinc rises; TPEN completely abolished these effects. Using voltage sensitive phosphatase CiVSP we found that the Ci-VSPdependent PIP2 hydrolysis strongly inhibited recombinant KCNQ4 and KCNQ2/3, however, ZnPy and PDTC completely abolished these effect rendering channels insensitive to Ci-VSP activation. TIRF imaging using optical PIP2 reporter PLCdPH-GFP confirmed that neither ZnPy nor PDTC affected the ability of CiVSP to hydrolyze PIP2, as determined by the depolarization-induced PLCdPH-GFP translocation. In sum, our data suggest that intracellular zinc potently augment KCNQ channel activity by reducing channel requirement for PIP2. 3001-Pos Board B378 Modulation of Neuronal Kir3 Channels by Cholesterol Anna N. Bukiya1, Avia Rosenhouse-Dantsker2. 1 Pharmacology, The University of Tennessee Health Science Center, Memphis, TN, USA, 2Pharmacology, University of Illinois at Chicago, Chicago, IL, USA. In recent years, cholesterol emerged as a major regulator of ion channel function. The most common effect of cholesterol on ion channels is a decrease in channel activity. We have recently shown that unexpectedly cholesterol enrichment up-regulates G-protein gated inwardly rectifying potassium (GIRK or Kir3) activity in atrial myocytes. Here we focus on neuronal Kir3 channels, and determine the effect of cholesterol on their function. Several Kir3 subunits are expressed in the hippocampus: Kir3.1, Kir3.2a, Kir3.2c and Kir3.3. Among these, Kir3.2 may be expressed as a homomer or as a heteromer with Kir3.1 and/or Kir3.3, both of which do not express as homomers in the plasma membrane. We first studied the effect of cholesterol on a pore mutant of Kir3.2 that increases its membrane expression and activity, Kir3.2^ (Kir3.2_E152D). We show that when Kir3.2^ was expressed in Xenopus oocytes, the resulting current was up-regulated by in vitro cholesterol enrichment of the oocyte membrane. Next, using planar lipid bilayers we demonstrate that cholesterol significantly increased the open probability of the Kir3.2^ channels. No change was observed in the unitary conductance. Most importantly, we show that also in vitro cholesterol enrichment of freshly isolated hippocampal pyramidal neurons resulted in a strong increase in physiological Kir3 currents. These findings indicate that cholesterol plays a critical role in modulating neuronal Kir3 channels. 3002-Pos Board B379 Cross-Talk between Cholesterol, PIP2 and Caveolin in Regulating Kir2 Channels Huazhi Han1, Avia Rosenhouse-Dantsker1, Radhakrishnan Gnanasambandam2, Frederick Sachs2, Irena Levitan1. 1 Medicine, University of Illinois at Chicago, Chicago, IL, USA, 2Physiology and Biophysics, University at Buffalo, SUNY, Buffalo, NY, USA. Multiple types of ion channels are regulated by two lipid modulators: phosphatidylinositol-4,5-bisphosphate (PIP2) and cholesterol, both modulators being enriched in caveolae. Our earlier studies identified cholesterol as a major negative regulator of Kir2 channels that are also well-known to be critically dependent on PIP2. Furthermore, it was proposed that cholesterol regulation of ion channels might be attributed to the disruption of their association with caveolin-1 and/or PIP2. In this study, we investigated the cross-talk between cholesterol, PIP2 and caveolin in the regulation of Kir2.1 channels. We show that cholesterol depletion strengthens the interaction between Kir2 channels and PIP2 and that a single-point mutation of Kir2.1 L222I that renders the channels cholesterol insensitive abrogates this effect. Furthermore, we present direct evidence that Kir2.1 is negatively regulated by Cav-1 and that Kir2.1-L222I mutation also abrogates the sensitivity of the channels to caveolin. Kinetic analysis of single-channel events also points to a common mechanism for cholesterol and Cav-1 regulation of the channels. However, neither Cav-1, nor intact caveolae are required for Kir2.1 channels to be cholesterol sensitive, thus challenging a general notion that cholesterol regulates ion channels by disruption of caveolae. Furthermore, we present first insights into the structural determinants of the cross-talk between the sensitivity of Kir2 channels to cholesterol, PIP2 and to caveolin: based on recent studies, we suggest that cholesterol, PIP2 and caveolin regulate the channels through distinct binding sites but that the signals generated by the binding of these modulators converge in regulating Kir2.1 gating.
3003-Pos Board B380 A Shared Mechanism of BK Channel Activation by Mallotoxin and Auxiliary g1 Subunit Xin Guan, Qin Li, Jiusheng Yan. Department of Anesthesiology and Perioperative Medicine, MD Anderson Cancer Center, Houston, TX, USA. The large-conductance, voltage- and calcium-activated Kþ (BK) channels consist of the pore-forming, voltage- and Ca2þ -sensing a-subunits (BKa) and the regulatory b and g subunits. The auxiliary g1 subunit is so far the most potent activator of BK channels which drastically shifts the voltagedependence of channel activation by ~ 140 mV towards the hyperpolarizing potential direction. Mallotoxin, also called rottlerin, is a potent extracellular small-molecule activator of BK channels, which was reported to shift the voltage-dependence of channel activation by ~ 100 mV in whole-cell patchclamp recording condition through a yet largely unknown mechanism. Here, we investigated the effects of mallotoxin on BK channels in excised membrane patches in the absence and presence of the auxiliary g subunit. Mallotoxin exerted significant activating effect on BK channels formed by BK alone in excised membrane patches by a ~ 80 mV shift in voltage-dependence of channel activation. However, the presence of g1 and g3 subunits nearly abolished and g2 subunit greatly attenuated the activating effect of mallotoxin. Most mutations in g1 subunit that caused a loss of its modulatory function on BK channels also resulted in a loss of its influence on mallotoxin. Importantly, we identified a point mutation in the middle of the g1 subunit’s transmembrane domain that had little effect on the g1 subunit’s modulatory function on BK channels in the absence of mallotoxin but caused an irreversible loss of the g1 subunit’s binding and modulatory function on BK channel upon a brief exposure to mallotoxin. Therefore, we conclude that g1 subunit competitively blocks the mallotoxin’s binding or action on BK channel, which can be fully reversed by a point mutation in the middle of the g1 transmembrane region. 3004-Pos Board B381 A Critical Role of the S6 Transmembrane Helix in BK Channel Modulation by Auxiliary g Subunits Qin Li, Jiusheng Yan. Department of Anesthesiology and Perioperative Medicine, MD Anderson Cancer Ctr, Houston, TX, USA. BK channels consist of the pore-forming, voltage- and Ca2þ-sensing a-subunits (BKa or Slo1) and the regulatory auxiliary b or g subunits. BK channels are potently modulated by the auxiliary g subunits, which shift the voltagedependence of channel activation by up to 140 mV in the hyperpolarizing direction. We have recently identified the g subunits’ transmembrane (TM) segment and its neighboring intracellular charged residues as key determinants for their modulatory functions on BK channels. However, the molecular mechanisms underlying BK channel modulation by auxiliary g subunits remain largely unknown. The proton-gated Slo3 channel has a high sequence similarity to Slo1 but showed nearly no sensitivity to the BKg1 (LRRC26) subunit. To identify the key protein regions of Slo1 that are involved in modulation by auxiliary g subunits, we generated a series of Slo1/Slo3 chimeras in the N-terminal TM (S0-S6) domain and analyzed their responses to the auxiliary g1 subunit in voltage-dependence of channel activation. We found that the Slo3 channel’s lack of sensitivity to BKg1 is largely caused by its S6 TM segment, whose inclusion in the Slo1/Slo3 chimera greatly attenuated the channel’s response to BKg1. Our further mutational analyses of the differential residues on the S6 TM segment identified several key residues that are largely responsible for the difference between Slo1 and Slo3 channels in their responses to BKg1. Therefore, we concluded that the S6 TM helix is critical in BK channel modulation by auxiliary g subunit. 3005-Pos Board B382 The Stretch-Activated BK (SAKca) Channel in Chick Heart is Inhibited by the Spider Peptide GsMTx-4 Qiong-Yao Tang1, Xiao-Dong Tang1, Yan-Jun Feng1, Hui Li1, Fei-Fei Zhang1, Zhe Zhang1, Masahiro Sokabe2. 1 Jiangsu Province Key Laboratory of Anesthesiology, Xuzhou Medical College, Xuzhou, China, 2Mechanobiology Laboratory, Nagoya University. Graduate School of Medicine, Nagoya, Japan. GsMTx4 is a 34-residue peptide isolated from the tarantula Grammostola spatulata and inhibits atrial fibrillation potentiated by dilatation in heart by blocking the stretch-activated (SA) ion channels. However, the mechanism by which GsMTx4 inhibits SA channels remains unknown. Here we report that the extracellularly applied nM concentrations of GsMTx4 inhibited SAKca channels in excised inside-out patch membranes of isolated chick heart cells. We found that GsMTx4 inhibited the stretch-induced SAKca channel opening in a dose-dependent manner without altering the single-channel conductance. Membrane stretch