의과학과, 생리학교실 호원경 Excitation-Secretion Coupling: role of ion channels in insulin secretion Metabolism-Excitation Coupling Closure of

The stimulus-secretion coupling pathway of glucose-stimulated insulin secretion (GSIS) (1) Glucose enters the cells via the GLUT2 transporter (2) and undergoes glycolytic and mitochondrial metabolism, (3) which ultimately has the effect of increasing the ATP:ADP ratio. (4) An increased ATP:ADP ratio leads to the closure of ATP-sensitive KATP channels (5) and to membrane depolarization, (6) which triggers the opening of voltage-dependent Ca2+ channels (VDCCs). (7) The influx of Ca2+ (8) induces the fusion of insulin granules with the plasma membrane and insulin release Excitation-Secretion Coupling (Exocytosis of vesicles) Pancreatic  -cell Metabolism-Excitation Coupling

Single channel recording Whole-cell recording a b ab ATP wash-out Cell-attached Inside-out From Rorsman and Trube (1985) ATP removal ATP-free Discovery of K ATP channels in pancreatic  -cells

K ATP channels: Molecular sensors of cellular metabolism Mg2+-nucleotide binding site ATP inhibition site

KATP conductance high P O low P O ATP ATP removal K ATP channels: inhibited by ATP and activated by ATP removal

Factors that decrease ATP sensitivity high P O low P O ATP PIP2, ADP KATP conductance ATP sensitivity 감소 KATP current 증가  -cell excitability 감소

K ATP channels: targets for diseases and anti-diabetic therapy 1.K ATP channels are target for anti-diabetic therapy. 2.Molecular defects cause neonatal diabetes or hyperinsulinemia.

KATP channel mutations the most common cause of neonatal diabetes mellitus (NDM) in humans Small decrease in ATP sensitivity causes severe DM Rip-Cre/Rosa26-Kir6.2[K185Q,  N30] double-transgenic (Rip-DTG) or Pdx-Cre/Rosa26-Kir6.2[K185Q,  N30] double-transgenic (Pdx-DTG) mice

Reduced excitability/Ca 2+ Insulin secretion 저하 Failure of M-E coupling -ATP 생성저하 (ex, GCK knock/out) -KATP 의 gain of function mutation -calmodulin over-expression (excessive Ca buffering) Closure of

Current (I) is determined by Open probability (P O ) and Number of channels (N) I=NxPo However, signaling mechanisms involved in regulation of channel trafficking are poorly understood. high Nlow N Trafficking mechanism Gating mechanism high P O low P O

Gating mechanism Trafficking mechanism

Pretreatment in low glucose conditions increases K ATP currents A B C Rat pancreatic  -cells or INS-1 cells

Question 1 What happens during glucose deprivation? -signaling mechanism? -channel gating vs channel density?

From Current Opinion in Nephrology and Hypertension 2005 AMPK, AMP-activated protein kinase, a sensor of cellular energy status Roles of AMPK AMPK

AMPK is involved in increases in K ATP conductance induced by glucose deprivation CC : compound C, AMPK inhibitor, 10  M AICAR : AMPK activator, 0.5 mM Total Total-AMPK

I= N X P O high Nlow N high P O low P O ATP AMPK?

K ATP channel surface density was increased by AMPK activation, and inhibited by AMPK inhibitor (Compound C) Kir2.1

AMPK increases surface localization of K ATP channel * Scale bar = 2  m 11 mM Glucose GD: 2 hr in Glucose-free GD with AMPK inhibitor AICAR in 11G

trafficking

Further Questions Molecular mechanisms? Are there endogenous regulators other than glucose? Does K ATP channel trafficking regulation occur in-vivo in physiological conditions?

WT-feeding 1 hr in 8G WT islets 11G GD

Leptin a pivotal regulator of energy homeostasis Leptin Deficiency After leptin tx leptin deficiency leptin insulin Leptin deficiency is associated with diabetes. Dysregulated adipo-insular feedback loop may underlie type 2 diabetes.

A Leptin Kir6.2-S Kir6.2-T Kir2.1-S Kir2.1-T Actin (min) SUR1-S pSTAT3 STAT Leptin (  ) Leptin (+) Kir6.2 Kir2.1 B Kir6.2 INS-1 cell  -cell Leptin increases K ATP channel surface trafficking and activity 10 sec CTR (11 mM glucose) Leptin (+) b a 1 min -50 mV -70 mV C

pAMPK AMPK Actin pACC ACC A Kir6.2-T Kir6.2-S Leptin CC AMPK Actin pAMPK  +  +  ++ Leptin (min) siAMPK Kir6.2-T Kir6.2-S Leptin AMPK pAMPK scRNA  +  + B   C Leptin activates AMPK and leptin effects on K ATP channel trafficking is mediated by AMPK Leptin (  ) Leptin (+) CC (  ) CC (+) D

1 hr in 8 mM glucose + leptin WT ob/ob WT-feeding 1 hr in 8 mM glucose

Further Questions Molecular mechanism of trafficking? Is there an endogenous regulator other than glucose? Does K ATP channel trafficking regulation occur in-vivo in physiological conditions? “Yes, it is leptin.” “Yes, it does.”

Myosin II and Myosin regulatory light chain (MRLC) Myosin II motor activity is regulated by phosphorylation of myosin regulatory light chain (MRLC). AMPK regulates cell morphology by phosphorylating MRLC in epithelial cells (Lee et al., 2007).

A B pMLCP pMRLC MLCP MRLC actin Leptin  +  + siAMPK scRNA   pAMPK AMPK scRNA Leptin ++ siAMPK  +   +    ++ pMRLC/MRLC 0 4 * * 2 C siMRLC Leptin Kir6.2-S  ++  +  Kir2.1-S +  Kir6.2-T Kir2.1-T ++  scRNA Leptin ++ siMRLC  +   +    ++ Kir6.2-S 0 3 * * actin MRLC 1 2 pAMPK AMPK Leptin scRNA +  + +  G (nS/pF) siMRLC +    + D 0 6 scRNA siMRLC Leptin (  ) Leptin (+) *** 3 Leptin-induced increase in MRLC phosphorylation mediates K ATP channel trafficking

Myosin II mediates K ATP channel trafficking

0 min Glucose deprivation 120 min 11 mM Glucose C B A 11G GD GD + CC

A GD+NSC23766GD B 11GGD GD+Phal GD+NSC NSC23766: Rac inhibitor

Acknowledgement to Cell Physiology Lab Members ( Ajin Lim Sun Hyun ParkYoung Eun Han

The stimulus-secretion coupling pathway of glucose-stimulated insulin secretion (GSIS) (1) Glucose enters the cells via the GLUT2 transporter (2) and undergoes glycolytic and mitochondrial metabolism, (3) which ultimately has the effect of increasing the ATP:ADP ratio. (4) An increased ATP:ADP ratio leads to the closure of ATP-sensitive KATP channels (5) and to membrane depolarization, (6) which triggers the opening of voltage-dependent Ca2+ channels (VDCCs). (7) The influx of Ca2+ (8) induces the fusion of insulin granules with the plasma membrane and insulin release Metabolism-Excitation Coupling Excitation-Secretion Coupling (Exocytosis of vesicles)

Biphasic insulin release: ‘storage-limited model’. Geographically or functionally distinct pools of insulin secretory granules that differ in release competence: 1)a readily releasable pool (RRP):less than 5% (50 ~ 200/cell). 2)a reserve pool (RP): the vast majority of granules (>10,000/cell) first-phase insulin release M-E coupling Calcium entry Fusion of the RRP by means of a soluble NSF attachment protein receptor (SNARE)2-dependent mechanism. a second and sustained phase of insulin release: attributed to the refilling of the RRP by mobilizing RP

Role of the cytoskeleton in granule mobilization Ultrastructure of b-cells: F-actin organized as a dense web beneath the plasma membrane; this web impedes access of insulin-containing granules to the cell periphery.

Three different modes of insulin exocytosis mode 1: predocked granules are immediately fused to the plasma membrane by stimulation (old face): mode for K-dependent insulin secretion mode 2: granules are newly recruited by stimulation and immediately fused to the plasma membrane (restless newcomer) mode 3: granules are newly recruited by stimulation but are first docked and then fused to the membrane (resting newcomer) J Clin Invest. 2011;121(6):2118–2125.