Sites of Action for Abused Inhalants

1
Sites of Action for Abused Inhalants

• John J Woodward
• Professor, Dept. of Neurosciences Center for
  Drug and Alcohol Programs
• Medical University of South Carolina
• Charleston, SC

2
Abused Inhalants
-Represent a diverse set of compounds found in a
variety of products Volatile alkyl
nitrites-amyl, butyl nitrite Gases-butane,propan
e,fluorohydrocarbons Volatile solvents-paint
thinner,gasoline,glues, correction fluids
-Neuronal sites of action largely unknown
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Lipid versus Protein Sites of Action

• Meyer-Overton
• Anesthetic potency positively correlated with
  lipid solubility membrane fluidity
• -Challenges to Lipid Theory
• Greater effects on synaptic versus axonal
  transmission
• Development of compounds with high lipid
  solubility that are behaviorally inert
• Meyer-Overton dynamics preserved in luciferase a
  lipid-free protein
• Suggests that brain proteins involved in synaptic
  transmission are potential targets for volatile
  CNS depressants

4
Gap Junctions-Connexins

• Assembled from Connexin proteins (Cx 32, 45,
  etc.)
• Allow intercellular passage of ions, metabolites
  and 2nd messengers lt 1 kDa
• Electrical coupling (epithelium, cardiac
  syncitium, neurons)
• Developmental role (neuronal migration,
  differentiation, deafness, cataracts, skin
  disorders)

5
Toluene Inhibits Gap Junctions Expressed in HEK
Cells
Del Re and Woodward, 2005
6
Voltage-Sensitive Calcium Channels Are Both
Potentiated and Inhibited by Toluene and Related
Solvents
Shafer et al., 2005
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Calcium-Activated Potassium Channels (BK)

• Alpha subunit-pore-forming
• Voltage dependent
• Calcium activated (calcium bowl / RCK)
• Located pre-synaptically and post-synaptically
  and are responsible for fAHP
• Potentiated by ethanol

8
BK Channels Expressed in Oocytes
Del Re and Woodward
9
Toluene and Etoh Have Opposing Actions on BK
Channels Expressed in Oocytes
E
Del Re and Woodward
10
G-protein Inwardly Rectifying K Channels (GirK)

• GirK 1-4 subunits
• 2 TM regions
• 4 subunits comprise a functional channel
• GirK 2 and 1/2 are neuronal GirK 1/4 cardiac
• Multiple ?? binding domains
• PIP2 binding domain
• Potentiated by ethanol
• Inhibited by volatile anesthetics - halothane
  isoflurane

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G-protein Inwardly Rectifying K Channels (GirK)
Expressed in Oocytes
GirK 2-brain GirK 1/4-cardiac
12
Toluene Inhibits G-protein Inwardly Rectifying K
Channels (GirK)
D
30

3 mM Toluene
1 mM Toluene
20
Inhibition by Toluene

10
0
1
GIRK 2
GIRK 1/2
GIRK 1/4

Receptor Combination
Del Re and Woodward
13
G-protein Inwardly Rectifying K Channels (GirK)
Are Activated by a Variety of G-protein Linked
Receptors (GPCR)
A
B
hK
hK
Glu
Glu
Tol
Tol
I
Glu


Del Re and Woodward
14
Toluene Inhibition of GirK Channels is Not
Altered by GPCR Activation
C
40
3 mM Toluene
35
1 mM Toluene
30
25
Inhibition by Toluene
20
15
10
5
0
1
GirK 2 /mGluR1?
GirK 2

Receptor Combination
Del Re and Woodward
15
Summary of the Effects of Toluene on Ion Channel
Function
Inhibition
No Effect
Potentiation
GABA ???? Glycine ?? Non-NMDA (10 mM) P2X2, 4
AMPA (GluR1-4) GirK 1/4
VSCC L-type Non L-type
from other investigators
Supported by NIDA DA13951