NE203 Lecture 4 REAL

Patch Clamp Method

Measures current flowing through a single ion channel using a highly sensitive amplifier connected to a recording pipette

4 Patch Clamp Configurations

Cell-attached recording, whole-cell recording, inside-out recording, outside-out recording

Cell-attached recording

Pipette forms tight seal with membrane using mild suction; channel stays intact in cell, tight contact between pipette + membrane

Whole-cell recording

Strong suction pulse ruptures membrane patch so cytoplasm is continuous with pipette interior

Inside-out recording

Pipette pulled away and patch exposed to air; cytoplasmic domain becomes accessible and allows manipulation inside + out

Outside-out recording

Pipette retracted so membrane ends anneal into a vesicle; extracellular domain becomes accessible

Microscopic currents

Currents flowing through individual/single ion channels

Macroscopic currents

Currents through many channels, representing a large surface of membrane

Na+ channel behavior

Most Na+ channels open within the initial 1-2 ms after depolarization, then probability of opening diminishes due to channel inactivation → closes after

K+ channel behavior

K+ channels open with a delay but remain open for the duration of the depolarization

Tetrodotoxin, saxitoxin, mu-Conotoxin

Toxins (pufferfish, dinoflagellates/cyanobacteria, cone snail venom) that block Na+ channels and inhibit depolarization

alpha-Toxins

Poisonous chemicals from Aspergillus molds (corn, peanuts, tree nuts) that prolong action potentials, scrambling information flow

beta-Toxins

From scorpion venom and staph; cause Na+ channels to open at lower-than-normal potentials, causing uncontrolled AP firing

Batrachotoxin

Potent cardiotoxic/neurotoxic alkaloid from frogs, birds, beetles; removes and shifts activation of Na+ channels

Dendrotoxin, apamin, charybdotoxin

Toxins from mamba snake, honeybee venom, and deathstalker scorpion venom that block K+ channels

SCN genes

Encode voltage-gated Na+ channels

KCN genes

Encode voltage-gated K+ channels

CACNA genes

Encode voltage-gated Ca2+ channels

CLCN genes

Encode voltage-gated Cl- channels

5 types of channel gating stimuli

Voltage-gated, neurotransmitter-gated, cyclic nucleotide-gated, thermosensitive, mechanosensitive

Selectivity filter criteria in ion channel pores

Mouth of the pore is made of amino acids that are opposite charge to that of the ion being taken in.

Pore diameter, atomic radius of ion (naked), atomic radius of ion (hydrated), and electronegativity of the ion

Voltage sensor gating (V-gated)

Positively charged amino acids in voltage sensors are pushed outward by depolarization and pulled inward by hyperpolarization, pulling helical linkers to open/close the pore

Ligand gating (AMPA receptor)

Extracellular ligand (e.g., glutamate) binding shuts the clam-shell shaped ligand-binding domain (LBD), moving gate helices of the transmembrane domain to open the pore

Intracellular ligands

Cyclic nucleotide-gated channel is structurally similar to voltage-gated K+ channel; example operates in response to light

TRPV1 channel gating via heat

Heat or capsaicin displaces membrane lipids near the helical linker connecting sensor domains to the pore, causing a conformational change that opens the channel gates

gating of channels via mechanical displacement

Sensitive to light touch and allodynia; extremely large protein (38 transmembrane helices) with 3 'blades' that act as levers sensing membrane curvature; mechanical flattening builds tension and opens the channel

Na+/K+ ATPase pump

Uses ATP hydrolysis to remove 3 Na+ (rate ~1/3 faster, efflux depends on extracellular K+) and take in 2 K+ per cycle; maintains Na+/K+ gradients

PMCA pump

Plasma membrane Ca2+ ATPase; extrudes Ca2+ from the cell

SERCA pump

ER membrane Ca2+ ATPase; extrudes Ca2+ from the cytoplasm into the ER

Ion exchangers

Use electrochemical gradient of other ions as energy source; carry one ion up its gradient while taking another down its gradient

Antiporters

Exchange intracellular and extracellular ions in opposite directions (e.g., Na+/Ca2+ exchanger, Na+/H+ exchanger to maintain pH)

Co-transporters

Carry multiple ions in the same direction (e.g., Na+/K+/Cl- co-transporter, which regulates intracellular Cl-)

Na+/Ca2+ exchanger structure

10 transmembrane helices: 8 form the core domain (ion binding/translocation, 3 negatively charged binding sites), 2 form the gating bundle that slides to switch conformation

Electrical synapses

Use gap junctions as ion channels; bidirectional; faster; synaptic delay less than 0.1 ms

Chemical synapses

Use neurotransmitters and receptors; Ca2+-dependent release; unidirectional; slower

Connexon

Hexamer of 6 connexin subunits; each cell contributes one connexon (half the gap junction channel) providing electrical and cytoplasmic continuity

Hippocampal interneurons

One of the few CNS locations that use electrical synapses; allow synchronized firing across adjacent neurons

Ionotropic vs metabotropic receptors

Two mechanisms by which chemical synapse signaling ultimately opens ion channels to change postsynaptic membrane potential

Presynaptic active zone

Site where vesicle pools are ready for exocytosis; filamentous structures guide vesicles here

Postsynaptic Density (PSD)

Anchors postsynaptic receptors, prevents their lateral diffusion; contains proteins involved in plasticity, learning, memory, health and disease

Chemical synapse signaling steps

AP invades presynaptic terminal -> depolarization opens voltage-gated Ca2+ channels -> Ca2+ influx -> vesicles fuse with membrane -> neurotransmitter released via exocytosis -> ntm binds to postsynaptic receptors -> opens/closes postsynaptic channels -> generates postsynaptic potential -> neurotransmitter removed by glial uptake or enzymatic degradation -> vesicular membrane retrieved

Ion channel pores

composed from arrangement of loops + helices of subunits; large enough for only the ion for which it’s meant to pass through → membrane is permeable to that ion

Active transport

essential to maintain a conc gradient for all physiologically relevant ions