ENDOCYTOSIS and the REGULATION of CELL and COMPARTMENTAL pH

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ENDOCYTOSIS and the REGULATION of CELL and
COMPARTMENTAL pH
Mitochondria - Green Endo/Lysosomes - Red
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(No Transcript)
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Endocytosis and Receptor Recycling
- Fluid Phase Endocytosis Generally to
Lysosomes - Receptor-Mediated (Targeted) may
take several paths Recycled back to the Plasma
Membrane Moved to the Lysosomes Back to the
Golgi Lipids for Membrane Synthesis Also may be
Transcytosed Across an Epithelial Layer
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Fluid Phase Endocytosis A7R5 Cell
NUCLEUS
GFP Targeted Golgi
Lysosomes Texas Red Dextran
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Low Density Lipoprotein
- Phospholipid
- Cholesterol Molecule
- Cholesterol Ester
- - Lipoprotein
LDL Receptor Binding Site
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Endocytosis and Receptor Recycling
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LDL Membrane Mechanisms
Regulation of Receptor Expression vs. Receptor
Down-Regulation
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Hetero-bivalent Ligand (CCK/MC4R 0.8 nM)
Labeling of HEK cells Expressing the MC4 and CCK
Receptors
2 Min
10 Min
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Deltorphin Coated Qdot Labeling of CHO Cells
A
B
C
A. 1 min
(Bar 20 mm)
B C. 20 min
Deltorphin 750 nm 11000 dilution 50
deltorphin per Qdot
From Zhou et al., 07 Neel Ghosh, Chemistry
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Receptor Mediated Endocytosis

• - Regulated Uptake of Metabolites and
  Co-factors LDL (Cholesterol), Transferrin
  (Iron)
• Retrieval and Storage of Surface Receptors and
  Transporters. Hormone Receptors, Glut-4
  Transporters
• Surface Expression Controlled by
• Endosomal Cycling Rate and Gene
  Transcription/Translation

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Neuron Synaptic Vesicle Recycling
Trans-Golgi

• Axonal Transport. 2. Pinocytosis.
• E/L Incorporation of NT Metabolism. 4. Filling
  with NT.
• 5. Entering the Secretory Pathway

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Acidification Regulates Trafficking
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Calcium as a Second Messenger
Extracellular Ca2 is 1 2 mM (10-3 M) _at_ a
membrane potential of -60 mV intracellular
Ca2 should be 200 mM Intracellular Ca2 is
100 nM (10-7 M) Active Processes Must
Regulate Ca2I Open a Channel, and Ca2
will Flow
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ER
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Ca2 Entry Channels.
Voltage Operated (VOC) and Ligand Operated (LOC)
Channels Voltage Operated (VOCs) L-type
Channels Dihydropyridine (DHP) Receptors.
Inhibited by DHP Antagonists Nifedipine and
Nitredipine. Regulated by cAMP-dependent
Phosphorylation. N-type Channels
Neuron-specific, blocked by a-conotoxin,
responsible for high voltage activated Ca2
currents. T-type Channels Found in excitable
and non-excitable cells low-threshold channels,
activated 30-40 mV more negative than are L
channels.
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Ca2 Channels.
Ligand Operated (LOCs) - Receptor Operated
(ROCs) Directly Activated by Agonists
Nicotinic Acid Receptor (Na),
Extracellular ATP Channel (Cation)
N-methyl-D-aspartate (Glutamate) Channel
(Ca2). - Second Messenger Operated (SMOC)
Activated by Soluble IP3 -Sensitive Channel
Second Messengers. Ca2 Induced Ca2
release G-protein Operated (GOCs).
Insulin-like Growth Factor 2 (IGF-2)
(Ca2) N-methyl-D-aspartate (NMDA)
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Ca2 Removal - Ca2 ATPases
PM-ATPase and Sarco-Endoplasmic Reticulum
Ca2ATPase PM-ATPase Km 0.8 mM Regulated
by Calmodulin, Inhibited by Vanadate.
Non-Electrogenic Antiport of 2 H per
Ca2. SERCA Km 0.3 mM Inhibited by
Thapsigargin or cyclopiazonic acid Regulated by
Phospholamban Also Mitochondrial Ca2
Uniporter Km 5 mM Inhibited by ruthenium red
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Na/ Ca2 exchange
The Km of the exchanger for Ca2 is relatively
high (0.5-2 mM) Transports Na and Ca2
with a stoichiometry of 31 so its
Electrogenic and VoltageSensitive Involved in
Gross Movement, but not Steady State
Maintenance of Cell Ca2
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Effects of Calcium Removal the Steady State
Remove Extra-cellular Ca2
Add Ca2 Back
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Calcium Binding Proteins
Signaling Calmodulin wide range of activity,
contraction in smooth muscle via MLCK Troponin
C - Contraction in Striated Muscle Protein
Kinase C - wide range of activities Ryanodine
Receptors Ca2 activated Ca2 release Channels
(K) - membrane hyperpolarization Sequestration
Calsequestrin, Calreticulin Parvalbumen
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Ca2 Stores
Ca2 Stores and Ca2 Induced Ca2 Release SERCA
Comprises 90 of the Protein in Striated Muscle
SR. 100-fold Less in Non-Muscle Cells-
Calciosomes. Role for PLB? Calciosomes -
Contain Ca2 Binding Proteins that Buffer Ca2
in Stores (calreticulin, calbindin,
calsequestrin) Kds gt 0.5 mM, High Capacity
8-12 Sites/Molecule Ca2 Release From Stores
Muscle Ryanodine Receptor (Ca2 induced Ca2
release) Non-Muscle Inositol Trisphosphate
(IP3) Receptor