Sodium Channels and Nonselective Cation Channels

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Sodium Channels and Nonselective Cation Channels

• An Introduction

Corthell, 2007
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Outline

• Sodium Channels
• Types
• Regulatory mechanisms (a few)
• Pharmacology (and what it shows us)
• Structure
• Paper-Role of hydrophobic residues
• Nonselective Cation Channels
• Where they are
• What they are
• TRP channels-well-characterized
• Paper-TRPC3 Channels Are Necessary

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Sodium (Na) Channel Types

• Voltage-gated Na Channels
• Include voltage sensor on protein
• Crucial to establish an action potential (AP)
• Found in various systems with variant effects and
  operating voltages
• Ligand-gated Na Channels
• Bind to specific ligand and generate electrical
  response

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Voltage-Gated
http//www.mun.ca/biology/desmid/brian/BIOL2060/BI
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Ligand-Gated
http//www.mun.ca/biology/desmid/brian/BIOL2060/BI
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Regulation and Modulation in Na Channels

• Phosphorylation effects
• Mutations in ball-and-chain affect inactivation
  speed
• Cleavage of any part of Na channel protein
• Drugs can be used as modulators

• NO modulates Na currents (Ribeiro et al., 2007)
• NO donors reduce peak Na current
• ENaC modulated by accessory proteins (Gormley et
  al., 2003)

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Pharmacology (i.e. drugs of choice)

• Saxitoxin (STX), from red tide, used to count Na
  channels (Ritchie et al. 1976)
• Tetrodotoxin (TTX), from fugu puffer fish, local
  anesthetics also block Na channel flux
• Local anesthetic channels open at once

Saxitoxin
www.chemfinder.com
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• Drugs bind to receptors
• Can be used to count receptors, block channels
  (ex identify which current is responsible for
  some spiking)
• Na channel is not perfectly selective
• Also permeable to K ions, though much less than
  Na (Chandler and Meves, 1965)
• Therefore, drug application may not necessarily
  block one ion completely
• Drug responses are variable
• Cardiac cells respond less to TTX than skeletal
  muscle cells (Ritchie and Rogart, 1977 Cohen et
  al., 1981)

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Structural Drug Use

• TTX and STX used to identify Na channel proteins
  (Henderson and Wang, 1972)
• Irradiated TTX and STX used as markers for bound
  portions of protein

• Other drugs used to identify other channel
  proteins as well as their receptor sites

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Na Channel Structure

• 6 transmembrane domains (S1-S6)
• 4 repeats (Domain 1-4)
• Has ?, ?, and ? subunits
• ? subunit responsible for pore
• P-loop as selectivity filter

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• Single linked protein makes up ion channel
• P-loop reflects speed of inactivation
• ?, ? subunits modify channel function but are not
  essential to create the pore

• Ligand-gated channels do not have voltage sensor,
  but ligand binding site
• Voltage gated channels have voltage sensor on S4
  in each domain
• Speculation domain sensors have special
  functions (Kuhn and Greef, 1999)

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Epithelial Na Channel (ENaC)
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ENaC in kidney, colon, and lungs

• Kidney ENaC aids in NaCl reabsorption
• Maintains body NaCl balance and blood pressure
  (Garty and Benos, 1988)
• Lungs aids in fluid clearance from alveolar
  space
• Maintains normal gas exchange in lungs (Matalon
  and OBrodovich, 1999)

• Affected by aldosterone and vasopressin
• Alter rate of insertion, degradation, recycling
  of channels
• Helped identify channel recycling by
  clathrin-mediated endocytosis (Shimkets et al.,
  1997)

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Nicotinic Acetylcholine Receptor (nAchR)
Model of the ligand-binding domain
Mature muscle expresses different subunits than
fetal muscle
http//s12-ap550.biop.ox.ac.uk8078/dynamite_html/
gallery_files/nAChR_covariance_lines_small.png
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Paper Role of hydrophobic residues in the
voltage sensors of the voltage-gated sodium
channel Bendahhou et al., 2007)

• S4 of each domain is considered the voltage
  sensor
• Major players include Arg and Lys residues
  occurring every 3 a.a.s and separated by 2
  neutral residues
• Mutate nonpolar Phe and Leu to Ala
• Eliminate steric hindrance
• Follow up with patch-clamp recording

Alter D1-D3, as D4 S4 has been studied extensively
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• D1 and D2 voltage sensor mutations did not result
  in significantly altered activation/inactivation
  kinetics

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• but did alter the activation curve. L224A is
  shifted to a hyperpolarized voltage, enhancing
  the open state, while L227A is shifted to a
  depolarized voltage (favors closed)

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• D3 mutations led to altered fast inactivation and
  a voltage shift in inactivation to
  hyperpolarization

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Paper Summary

• Hydrophobic residues are also important to the
  voltage sensor
• Need correct shape
• Altering the voltage sensor on D1 and D2 alters
  inactivation/activation kinetics
• Mutations on D3 S4 alter kinetics and voltage
  dependence
• Leads to idea perhaps each S4 responsible for
  different aspects of channel gating? Do they
  function independently?

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Nonselective Cation Channels

• Where?
• Across most sensory systems as transduction
  channels
• Examples retinal rods, hair cells, Pacinian
  corpuscle, spindle organs, taste cells (amino
  acid taste), nociception
• TRP channels extensively studied
• Broad family of nonselective cation channels
• In brain, aiding in spontaneous firing (Kim et
  al., 2007)

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Stretch Receptors
www.unm.edu/toolson/ pacinian_corpuscle.gif
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What are nonselective cation channels?

• Obvious answer
• However, most NCCs are known for fluxing Ca2
• Mostly due to chemical gradient of Ca outside of
  cell
• Still flux Na, K

• Not necessarily a universal structure like Na
  or K channels
• Depends on sequence homology, location of channel

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Transient Receptor Potential (TRP) Channels

• Very large gene family-many divisions
• TRPM, TRPC, TRPV
• Widely expressed in brain (including hippocampus)
• Structural similarity, but still many differences
  between channel structures and functions

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Structure

• TRP channels have 6 transmembrane segments
  (similar to Kv channels)
• Between S5 and S6 is believed to be pore
• TRP domain highly conserved 25 a.a.s C-terminal
  to S6
• Include 6 invariant a.a.s , called TRP box

• Different subunits made up of homo- and
  heterotetramers
• Ankyrin repeats (33 a.a.s) crucial for some
  subunits to assemble

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TRPC3 structure (proposed)
Mio et al., 2007
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• TRP channels are known to have many different
  ligands (capsaicin-TRP relative VR1 Cesare and
  McNaughton, 1996, 1997, PIP2-TRPV Nilius et
  al., 2007)

• Many of these channels are also activated by Ca2
  binding (Amaral and Pozzo-Miller, 2007)

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Paper-TRPC3 Channels Are Necessary for
Brain-Derived Neurotrophic Factor to Activate a
Nonselective Cationic Current and to Induce
Dendritic Spine Formation Amaral and
Pozzo-Miller, 2007.

• BDNF elicits a current that is not blocked by
  tetrodotoxin or saxitoxin but is blocked by
  interfering RNA-mediated knockdown of TRPC3
• BDNF application also increases surface TRPC3 in
  cultured hippocampal neurons

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• Long-term BDNF exposure leads to various effects
  on hippocampal neurons
• Can modulate synaptic transmission
• Can change structure of dendrites, spines, and
  presynaptic terminals

• Kept in serum-free media to avoid effects of
  serum nutrients
• Slowly activating, sustained current
• Different than other Trk receptor cation fluxes

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• In voltage clamp. K-252a is a tyrosine kinase
  inhibitor, showing that the BDNF response
  requires one

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• Current is not blocked by saxitoxin
• TRPC currents expressed in hippocampal neurons

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• BDNF application alters amount of TRPC3 on surface

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• Spines affected by different drugs, including
  TRPC inhibitors

• Spines counted

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Paper Summary

• BDNF increases density of dendritic spines on
  hippocampal neurons (CA1)
• Works via a TRPC3 conductance
• Uses TrkB receptors, phospholipase C, others

• Therefore, TRPC3 channels are mediators of
  BDNF-mediated dendritic remodeling

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Summation

• Na channels have multiple locations, uses,
  responses
• Well-studied
• Structure still not elucidated
• Isoforms part of historical work

• Nonselective cation channels are found in most
  sensory systems
• Transduction channels or TRP channels
• Many different purposes, depending on host cell

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