Ion Channel Structure and Function

Ion Channel Structure

• Ion channels are transmembrane proteins critical for rapid ion movement down electrochemical gradients.
• They are selective for anions, cations, or specific cations (e.g., K$^+$, Na$^+$).
• Ion channels have gated states, which are activated by voltage, ligand binding, or physical changes.
• Modulation occurs via auxiliary subunits, G proteins, or neuromodulators.

Structure of Ion Channels

• Size: Large transmembrane proteins, ranging from 640 to 2000 amino acid residues.
• Sugars attach to extracellular regions.
• Multimeric Nature:
  • Homomeric multimers consist of multiple copies of the same subunit.
  • Heteromeric multimers are composed of different subunits.
• Amino acids:
  • Hydrophobic amino acids are lipophilic (fat-loving).
  • Hydrophilic amino acids are lipophobic (water-loving).

Components of Ion Channels

• Protein: Amino acid chain encoded by mRNA.
• Domain: Functional unit of a channel.
• Subunit: Part of a channel, a single protein that may contain one or more domains.
• Transmembrane Segment: Section of a domain that spans the membrane.
• N and C Tails: Allow subunits to associate with other proteins or ligands to modulate channel function.

Transmembrane Spanning

• 20 amino acids (in α-helix or β-sheets) are necessary to traverse the membrane.
• A minimum of 8 transmembrane crossings are necessary to form a functional channel.

Voltage-Gated Ion Channels

• Voltage-gated ion channels typically have 24 transmembrane regions.
• The structure often includes repeating domains (e.g., I, II, III, IV) each with S1-S6 segments.

Deducing Ion Channel Structure

• Sequence a long string of amino acids (~2,000 aa, encoded by ~7,000 bp).
• Identify hydrophobic (uncharged) and hydrophilic (charged) amino acids based on their "R" groups.

Hydrophobicity Plots

• Hydrophobicity Plots: Used to predict transmembrane regions.
• Positive values: indicate hydrophobic regions.
• Negative values: indicate hydrophilic regions.
• Each position along the x-axis corresponds to an amino acid in the peptide sequence.

Determining Extracellular vs. Intracellular Regions

1. Putative glycosylation sites mark extracellular domains.
2. Immunostaining with antibodies directed against specific sites, with or without membrane detergent, helps determine location.

Case Study: Novel Channel on Hippocampal Pyramidal Cells

• A novel channel expressed on hippocampal pyramidal cells was identified and cloned by Dr. Leaky.
• Experiments were performed to predict the structure of the channel.
• Antibodies against the C-terminus were used for immunostaining:
  • Without detergent: No staining.
  • With detergent: Staining.
• Putative glycosylation sites (e.g., asparagine–X–serine/threonine) were identified at specific sites (marked by asterisks).

Potassium Channel Sequence (Drosophila Shaker Locus)

• Potassium currents repolarize electrically excitable membranes.
• The Shaker locus in Drosophila encodes a K$^+$ channel.
• The predicted protein contains seven potential membrane-spanning sequences and is homologous to vertebrate sodium channels in regions involved in voltage-dependent activation.

Real-World Examples

• Drosophila shaker K$^+$ channel
• Bacterial K$^+$ channel
• Human beta-2 adrenergic receptor
• Hydrophobicity calculator (set window size to 15)

Imaging Techniques

• Electron microscopy of single channels provides information about physical properties. Example: nACh Receptors from Torpedo electric fish.
• Super-resolution microscopy can localize single ion channel molecules.

Crystal Structure Analysis

• X-ray analysis is used to deduce the fine tertiary structure of the protein.
• The hardest part is crystallizing the protein.

Ion Selective Channel Crystal Structure

• Rod MacKinnon (1998) performed X-ray crystallography on a bacterial K channel.
• It is similar to the inward rectifying K channel.
• It has 4 subunits with only 2 S domains (S5 and S6).

Cryo-EM

• Cryo-EM (Cryo-Electron Microscopy) is used for 3D reconstruction of channels, like TRPV1.

Four-Fold Symmetry

• TRPV1 and VGICs (Voltage-Gated Ion Channels) share a similar four-fold symmetric architecture, including:
  • S4-S5 linker
  • Pore helix
  • S6
  • Pore module
  • S1-S4
  • TRP domain

Electron Crystallography

• Electron Crystallography allows crystallization in native state and can image multiple conformations.

Diversity of Channels

• Voltage-gated channels
• Ca-activated K channels
• Cyclic nucleotide-gated channels
• Ligand-gated channels (nAChR, GluR, GABA A)
• TRP channels
• Gap Junctions
• Selectivity:
  • Most selective
  • Less selective (cations/anions)
  • Least selective

Structure-Function Relationship

• How does the structure of the ion channel determine its functional properties?

Linking Structure to Function

• Expression Systems:
  • Xenopus oocytes
  • Human cell lines (HEK 293)
• Reductionist Approach:
  • DNA manipulation
  • cDNA clones for single channel type

Voltage-Gated Channels

• Four domains (I-IV), each consisting of 6 transmembrane segments (S1-S6).
• Na and Ca channels: all four domains are on a single subunit.
• K channel has 4 separate subunits; one for each domain.

Ligand-Gated Channels

• Large extracellular tail
  • ATD (Amino-Terminal Domain)
  • LBD (Ligand-Binding Domain)
• M2-pore forming region
  • TMD (Transmembrane Domain)

Pore-Forming Segments

• S5-S6 segments are pore-forming.
• Inwardly rectifying K channel has 4 subunits with only 2 transmembrane segments.

Selectivity Filter

• The selectivity filter is along the channel mouth.
• It must have an exact fit to create an energetically favorable environment to remove water molecules.

K$^+$ Channel

• K$^+$ channel is occupied by 4 K$^+$ ions.
• Electrostatic repulsion ensures high throughput.
• The selectivity filter is composed of a specific sequence of charged amino acids (TVGYG) that act as K$^+$-binding sites (but not Na$^+$-binding sites).
• Movement of ions through a channel is passive.
• Rate: 10 million to 100 million ions per second.

Voltage Sensor (S4 Domain)

• Positive charge every 3 aa.
• Conservation in sequence between many different species.
• Similar in other voltage-gated channels.
• Is absent from non-voltage dependent K channel.

Voltage Sensor Summary

• Located in S4 domain.
• Rich in charged/basic amino acids (Arginines and Lysines).
• Changes in Vm produce movement of S4 (→ gating current).
• Conformational changes in the voltage sensor lead to the opening or closing of the channel’s gate.
• The gate and voltage sensors are separate structures.
  {V_m}

Inactivation Mechanism (Ball and Chain Model)

1. Closed - hyperpolarization.
2. Open - depolarization.
3. Open/inactivated - depolarization.
4. De-inactivated - hyperpolarization.

Modulation

• When a channel is modulated, the same stimulus will give a different response.
• Many different mechanisms and time scales of modulation:
  • Second messengers, phosphorylation (rapid, reversible).
  • Changes in subunit composition – receptor trafficking (slower, longer lasting).
  • Changes in gene expression (slower, long lasting).

Auxiliary Subunits

• Auxiliary subunits modulate channel function.
• β subunits link channels to complex signaling cascades.

Sodium Current

• Classic sodium current (hippocampal pyramidal neuron)
• Resurgent sodium current (cerebellar Purkinje neuron)
• Na channel inactivation recovery is faster in the presence of aB subunit, creating a “resurgent current.”