Angiotensin II Type 1 Receptor Mediates Angiotensin II Inhibition on BKCa Channels
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analyzed by LC/MS/MS. A specific MaxiKa immunoprecipitation is supported by the identification of peptides covering 27.1% of MaxiKa protein in samples from wild-type animals and using anti-MaxiKa antibodies but not in samples of immunoprecipitates using IgG or brains of Kcnma1 -/- animals (n=3). MaxiKa splice variants, STREX (in between exon 18 and 19) and SV27 (in between exon 23 and 24) as well as two residue variants A632R and F633L in exon 19 were identified (n=3) . In addition to the 21 proteins immunopreciptated earlier from murine cortical membranes (Gorini et al, FEBS Lett. 2010; 584:845-51), 254 additional proteins were obtained (n=3). Along with the Max- iKb4 subunit (Kcnmb4), Teneurin 2, Insulin receptor-related protein, Kinesin 21A, Ubiquitin-conjugating enzyme 2N, GABA transporter 3&4 (S6A11), SERCA 2, Coronin 1A, Citrate synthase, Ssbp2, and Rl19 were identified as the most prominent proteins interacting with the brain MaxiKa. Several nuclear and mitochondrial proteins were also detected. These results strongly suggest that MaxiKa interacts with a variety of proteins in the brain, and further inves- tigation of functional consequences of these interactions will enhance our un- derstanding of MaxiK and its role in neuronal function. Supported by NIH and AHA. 3501-Pos Board B362 Angiotensin II Type 1 Receptor Mediates Angiotensin II Inhibition on BK Ca Channels Zhu Zhang, Rong Lu, Enrico Stefani, Ligia Toro. UCLA, Department of Anesthesiology, Los Angeles, CA, USA. The renin-angiotensin system (RAS) is a critical regulator of sodium balance, extracellular fluid volume, vascular resistance, and, ultimately, arterial blood pressure. The key RAS molecule angiotensin II type 1 receptor (AT1R), and another major determinant of vascular tone, the large conductance calcium- activated potassium (BK Ca ) channel, are both highly expressed in renal arterial smooth muscle cells (SMCs). Our previous studies in expression systems re- vealed a physical association between AT1R and BK Ca , and that AT1R associ- ation modified BK Ca channel voltage sensitivity. However, the effect of Ang II on BK Ca channels in renal arterial SMCs needs to be defined. Furthermore, whether AT1R association is critical for the alteration of channel activity in re- sponse to Ang II, and whether the coupling changes BK Ca reactivity to specific inhibitors also remain unknown. Our present studies in rat renal arterial SMCs show that application of 100 nM Iberiotoxin (IbTx, a specific BK Ca channel blocker) inhibits whole-cell BK Ca currents by 63.0512.5% (n=5). IbTx- sensitive BK Ca currents were reduced by 44.458.7% (n=3) in response to 1 mM Ang II treatment. In AT1R-IRES-BK Ca -transfected HEK293T cells, ex- tracellular application of 1 mM Ang II also suppressed whole-cell BK Ca cur- rents by 19.750.3% (n=3), while this treatment made no significant changes in cells expressing only BK Ca (n=6). IbTx reduced the whole-cell currents by 37.251.9% (n=4) in BK Ca -transfected HEK293T cells and 37.7512.5% (n=5) in AT1R-IRES-BK Ca -transfected cells. Paxilline treatment (100 nM) produced a 66.858.0% (n=3) reduction of BK Ca whole-cell currents in BK Ca -transfected HEK293T cells and 65.859.2% (n=3) reduction in AT1R- IRES-BK Ca -transfected cells. These results demonstrate that Ang II inhibits BK Ca channel activity in renal arterial SMCs and suggest that AT1R serves as the mediator of the effect. However, the interaction between AT1R and BK Ca does not change channel reactivity towards specific channel inhibitors. Supported by NIH. 3502-Pos Board B363 Kcnma1 Gene Expression Improves Mitochondrial Function in NS1619 Preconditioned Hearts after Ischemia/Reperfusion Injury Jean C. Bopassa 1 , Harpreet Singh 1 , Shahab M. Danesh 1 , Andrea L. Meredith 2 , Ligia Toro 1 , Enrico Stefani 1 . 1 UCLA, Department of Anesthesiology, Los Angeles, CA, USA, 2 University of Maryland, Baltimore, MD, USA. Our previous immunochemical and transcript analyses indicate that the large conductance calcium-activated potassium channel (BK Ca ) is present in cardiac mitochondria and encoded by the Kcnma1 gene. In addition, studies using a BK Ca opener, NS1619, point to the view that opening BK Ca expressed in mi- tochondria (mitoBK Ca ) contributes to cardioprotection after ischemic insult by- modulating mitochondrial function. However, whether the beneficial effect of NS1619 depends on the presence of Kcnma1 has not been studied. To address this question, we used mice expressing (wild type, wt) or not (Kcnma1 -/- ) Kcnma1 gene product and the Langendorff model of global ischemia and reper- fusion.Mitochondrial functions affecting the opening of the mitochondrial per- meability transition pore (mPTP) and cell death were assessed: calcium retention capacity (CRC) and reactive oxygen species (ROS). Mitochondria were isolated after 10 min of reperfusion from hearts preconditioned or not (control) for 10 min before ischemia with 10 mM NS1619. Mitochondria from hearts preconditioned with NS1619 showed a significant increase in CRC from 140þ 11 to 280þ 23 nmoles/mg (n=3) in wt mice but no significant effect was observed in Kcnma1 -/- mice (120þ 23 and 100þ 11 nmoles/mg, n=3) showing that after ischemic insult the protective effect of NS1619 requires ex- pression of Kcnma1 gene. Likewise, mitochondrial ROS production stimulat- ing complex I with 3 mM glutamate/malate was reduced by NS1619 preconditioning in the wt mice from 159þ 13 to 113þ 7 pmoles/min/mg but not in the Kcnma1 -/- mice (n=3). Our results indicate that Kcnma1 gene expres- sion is required for the improvement of mitochondrial function by NS1619 pre- conditioning, which involves: 1) increased CRC, and 2) decreased ROS production of complex I, both events preventing mPTP opening. Further, the results also confirm that mitoBK Ca is encoded by Kcnma1 gene. Supported by NIH and AHA. 3503-Pos Board B364 Genetic Activation of Bk Currents using a Gain-Of-Function Subunit Alters Neuronal Activity Andrea L. Meredith, Jenna R. Montgomery. Univ. of Maryland School of Medicine, Baltimore, MD, USA. K þ channels play key roles in dampening excitability in many types of tissues. In neurons, the large conductance Ca 2þ -activated K þ (BK) channel inhibits ex- citability by participating in the fast afterhyperpolarization and limiting neuro- transmitter release. Paradoxically, BK currents have also been recently linked to enhancing excitability and seizures. Using a transgenic approach, the dual nature of BK’s influence on excitability was investigated. In vivo expression of a gain-of function BK channel alpha subunit (R207Q) recapitulates aspects of both the excitatory and inhibitory influence of BK currents. The direction of influence is dependent upon the relative expression level of wildtype and mu- tant subunits, the resulting BK current activation kinetics, and interaction with other fast-gated K þ currents. 3504-Pos Board B365 Dystrobrevin Controls Neurotransmitter Release and Muscle Ca 2D Tran- sients by Localizing BK Channels in C. Elegans Bojun Chen, Ping Liu, Haiying Zhan, Zhao-Wen Wang. University of Connecticut Health Center, Farmington, CT, USA. Dystrobrevin is a major component of a dystrophin-associated protein com- plex (DAPC). It is widely expressed in mammalian tissues including the ner- vous system, where it is localized to the presynaptic nerve terminal with unknown function. In a genetic screen for suppressors of a lethargic phenotype caused by a gain-of-function (gf) isoform of SLO-1 in C. elegans, we isolated multiple loss-of-function (lf) mutants of the dystrobrevin gene dyb-1. dyb-1(lf) phenocopied slo-1(lf), causing increased neurotransmitter release at the neuro- muscular junction, increased frequency of Ca 2þ transients in body-wall mus- cle, and abnormal locomotion behavior. Neuron- and muscle-specific rescue experiments suggest that DYB-1 is required for SLO-1 function in both neu- rons and muscle cells. DYB-1 colocalized with SLO-1 at presynaptic sites in neurons and dense body regions in muscle cells, and dyb-1(lf) caused SLO-1 mislocalization in both types of cells without altering SLO-1 protein level. The neuronal phenotypes of dyb-1(lf) were partially rescued by mouse a-dystrobrevin-1 (aDB1). These observations revealed novel functions of the BK channel in regulating muscle Ca 2þ transients, and of dystrobrevin in controlling neurotransmitter release and muscle Ca 2þ transients by localizing the BK channel. 3505-Pos Board B366 Dominant-Negative, Spliced Variant of the Intermediate-Conductance Ca 2D -Activated K D Channel, K Ca 3.1 in Lymphoid Cells Susumu Ohya, Satomi Niwa, Ayano Yanagi, Yuka Fukuyo, Hisao Yamamura, Yuji Imaizumi. Nagoya City University, Nagoya, Japan. Intermediate-conductance Ca 2þ -activated K þ channel (IK Ca channel) encoded by K Ca 3.1 is responsible for the control of proliferation and differentiation in various types of cells. We identified novel spliced variants of K Ca 3.1 (hK Ca 3.1b) from the human thymus, which were lacking the N-terminal do- mains of the original hK Ca 3.1a as a result of alternative splicing. hK Ca 3.1b was significantly expressed in human lymphoid tissues. Western blot analysis showed that hK Ca 3.1a proteins were mainly expressed in plasma membrane fraction, whereas hK Ca 3.1b was in the cytoplasmic fraction. We also identi- fied a similar N-terminus lacking K Ca 3.1 spliced variants from mice and rat lymphoid tissues (mK Ca 3.1b, rK Ca 3.1b). In the HEK293 heterologous Webnesday, February 29, 2012 691a
Transcript of Angiotensin II Type 1 Receptor Mediates Angiotensin II Inhibition on BKCa Channels
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analyzed by LC/MS/MS. A specific MaxiKa immunoprecipitation is supportedby the identification of peptides covering 27.1% of MaxiKa protein in samplesfrom wild-type animals and using anti-MaxiKa antibodies but not in samples ofimmunoprecipitates using IgG or brains of Kcnma1-/- animals (n=3). MaxiKasplice variants, STREX (in between exon 18 and 19) and SV27 (in betweenexon 23 and 24) as well as two residue variants A632R and F633L in exon19 were identified (n=3) . In addition to the 21 proteins immunopreciptatedearlier from murine cortical membranes (Gorini et al, FEBS Lett. 2010;584:845-51), 254 additional proteins were obtained (n=3). Along with theMax-iKb4 subunit (Kcnmb4), Teneurin 2, Insulin receptor-related protein, Kinesin21A, Ubiquitin-conjugating enzyme 2N, GABA transporter 3&4 (S6A11),SERCA 2, Coronin 1A, Citrate synthase, Ssbp2, and Rl19 were identified asthe most prominent proteins interacting with the brain MaxiKa. Several nuclearand mitochondrial proteins were also detected. These results strongly suggestthat MaxiKa interacts with a variety of proteins in the brain, and further inves-tigation of functional consequences of these interactions will enhance our un-derstanding of MaxiK and its role in neuronal function. Supported by NIH andAHA.
3501-Pos Board B362Angiotensin II Type 1 Receptor Mediates Angiotensin II Inhibition onBKCa ChannelsZhu Zhang, Rong Lu, Enrico Stefani, Ligia Toro.UCLA, Department of Anesthesiology, Los Angeles, CA, USA.The renin-angiotensin system (RAS) is a critical regulator of sodium balance,extracellular fluid volume, vascular resistance, and, ultimately, arterial bloodpressure. The key RAS molecule angiotensin II type 1 receptor (AT1R), andanother major determinant of vascular tone, the large conductance calcium-activated potassium (BKCa) channel, are both highly expressed in renal arterialsmooth muscle cells (SMCs). Our previous studies in expression systems re-vealed a physical association between AT1R and BKCa, and that AT1R associ-ation modified BKCa channel voltage sensitivity. However, the effect of Ang IIon BKCa channels in renal arterial SMCs needs to be defined. Furthermore,whether AT1R association is critical for the alteration of channel activity in re-sponse to Ang II, and whether the coupling changes BKCa reactivity to specificinhibitors also remain unknown. Our present studies in rat renal arterial SMCsshow that application of 100 nM Iberiotoxin (IbTx, a specific BKCa channelblocker) inhibits whole-cell BKCa currents by 63.0512.5% (n=5). IbTx-sensitive BKCa currents were reduced by 44.458.7% (n=3) in response to1 mM Ang II treatment. In AT1R-IRES-BKCa-transfected HEK293T cells, ex-tracellular application of 1 mM Ang II also suppressed whole-cell BKCa cur-rents by 19.750.3% (n=3), while this treatment made no significant changesin cells expressing only BKCa (n=6). IbTx reduced the whole-cell currents by37.251.9% (n=4) in BKCa-transfected HEK293T cells and 37.7512.5%(n=5) in AT1R-IRES-BKCa-transfected cells. Paxilline treatment (100 nM)produced a 66.858.0% (n=3) reduction of BKCa whole-cell currents inBKCa-transfected HEK293T cells and 65.859.2% (n=3) reduction in AT1R-IRES-BKCa-transfected cells. These results demonstrate that Ang II inhibitsBKCa channel activity in renal arterial SMCs and suggest that AT1R servesas the mediator of the effect. However, the interaction between AT1R andBKCa does not change channel reactivity towards specific channel inhibitors.Supported by NIH.
3502-Pos Board B363Kcnma1 Gene Expression Improves Mitochondrial Function in NS1619Preconditioned Hearts after Ischemia/Reperfusion InjuryJean C. Bopassa1, Harpreet Singh1, Shahab M. Danesh1,Andrea L. Meredith2, Ligia Toro1, Enrico Stefani1.1UCLA, Department of Anesthesiology, Los Angeles, CA, USA,2University of Maryland, Baltimore, MD, USA.Our previous immunochemical and transcript analyses indicate that the largeconductance calcium-activated potassium channel (BKCa) is present in cardiacmitochondria and encoded by the Kcnma1 gene. In addition, studies usinga BKCa opener, NS1619, point to the view that opening BKCa expressed in mi-tochondria (mitoBKCa) contributes to cardioprotection after ischemic insult by-modulating mitochondrial function. However, whether the beneficial effect ofNS1619 depends on the presence of Kcnma1 has not been studied. To addressthis question, we used mice expressing (wild type, wt) or not (Kcnma1-/-)Kcnma1 gene product and the Langendorff model of global ischemia and reper-fusion.Mitochondrial functions affecting the opening of the mitochondrial per-meability transition pore (mPTP) and cell death were assessed: calciumretention capacity (CRC) and reactive oxygen species (ROS). Mitochondriawere isolated after 10 min of reperfusion from hearts preconditioned or not
(control) for 10 min before ischemia with 10 mM NS1619. Mitochondriafrom hearts preconditioned with NS1619 showed a significant increase inCRC from 140þ11 to 280þ23 nmoles/mg (n=3) in wt mice but no significanteffect was observed in Kcnma1-/- mice (120þ23 and 100þ11 nmoles/mg, n=3)showing that after ischemic insult the protective effect of NS1619 requires ex-pression of Kcnma1 gene. Likewise, mitochondrial ROS production stimulat-ing complex I with 3 mM glutamate/malate was reduced by NS1619preconditioning in the wt mice from 159þ13 to 113þ7 pmoles/min/mg butnot in the Kcnma1-/- mice (n=3). Our results indicate that Kcnma1 gene expres-sion is required for the improvement of mitochondrial function by NS1619 pre-conditioning, which involves: 1) increased CRC, and 2) decreased ROSproduction of complex I, both events preventing mPTP opening. Further, theresults also confirm that mitoBKCa is encoded by Kcnma1 gene. Supportedby NIH and AHA.
3503-Pos Board B364Genetic Activation of Bk Currents using a Gain-Of-Function SubunitAlters Neuronal ActivityAndrea L. Meredith, Jenna R. Montgomery.Univ. of Maryland School of Medicine, Baltimore, MD, USA.Kþ channels play key roles in dampening excitability in many types of tissues.In neurons, the large conductance Ca2þ-activated Kþ (BK) channel inhibits ex-citability by participating in the fast afterhyperpolarization and limiting neuro-transmitter release. Paradoxically, BK currents have also been recently linkedto enhancing excitability and seizures. Using a transgenic approach, the dualnature of BK’s influence on excitability was investigated. In vivo expressionof a gain-of function BK channel alpha subunit (R207Q) recapitulates aspectsof both the excitatory and inhibitory influence of BK currents. The direction ofinfluence is dependent upon the relative expression level of wildtype and mu-tant subunits, the resulting BK current activation kinetics, and interaction withother fast-gated Kþ currents.
3504-Pos Board B365Dystrobrevin Controls Neurotransmitter Release and Muscle Ca2D Tran-sients by Localizing BK Channels in C. ElegansBojun Chen, Ping Liu, Haiying Zhan, Zhao-Wen Wang.University of Connecticut Health Center, Farmington, CT, USA.Dystrobrevin is a major component of a dystrophin-associated protein com-plex (DAPC). It is widely expressed in mammalian tissues including the ner-vous system, where it is localized to the presynaptic nerve terminal withunknown function. In a genetic screen for suppressors of a lethargic phenotypecaused by a gain-of-function (gf) isoform of SLO-1 in C. elegans, we isolatedmultiple loss-of-function (lf) mutants of the dystrobrevin gene dyb-1. dyb-1(lf)phenocopied slo-1(lf), causing increased neurotransmitter release at the neuro-muscular junction, increased frequency of Ca2þ transients in body-wall mus-cle, and abnormal locomotion behavior. Neuron- and muscle-specific rescueexperiments suggest that DYB-1 is required for SLO-1 function in both neu-rons and muscle cells. DYB-1 colocalized with SLO-1 at presynaptic sitesin neurons and dense body regions in muscle cells, and dyb-1(lf) causedSLO-1 mislocalization in both types of cells without altering SLO-1 proteinlevel. The neuronal phenotypes of dyb-1(lf) were partially rescued by mousea-dystrobrevin-1 (aDB1). These observations revealed novel functions ofthe BK channel in regulating muscle Ca2þ transients, and of dystrobrevin incontrolling neurotransmitter release and muscle Ca2þ transients by localizingthe BK channel.
3505-Pos Board B366Dominant-Negative, Spliced Variant of the Intermediate-ConductanceCa2D-Activated KD Channel, KCa3.1 in Lymphoid CellsSusumu Ohya, Satomi Niwa, Ayano Yanagi, Yuka Fukuyo,Hisao Yamamura, Yuji Imaizumi.Nagoya City University, Nagoya, Japan.Intermediate-conductance Ca2þ-activated Kþ channel (IKCa channel) encodedby KCa3.1 is responsible for the control of proliferation and differentiation invarious types of cells. We identified novel spliced variants of KCa3.1(hKCa3.1b) from the human thymus, which were lacking the N-terminal do-mains of the original hKCa3.1a as a result of alternative splicing. hKCa3.1bwas significantly expressed in human lymphoid tissues. Western blot analysisshowed that hKCa3.1a proteins were mainly expressed in plasma membranefraction, whereas hKCa3.1b was in the cytoplasmic fraction. We also identi-fied a similar N-terminus lacking KCa3.1 spliced variants from mice and ratlymphoid tissues (mKCa3.1b, rKCa3.1b). In the HEK293 heterologous
