BIOL 436 Exam 2

Lectures 15-???

What was the method of recording mbP developed in the 1940s?

Using two electrodes, a mbRP of -60 mV is displayed when one electrode is in the cell and one electrode is out.

How does injecting current affect mbP?

Injecting a positive I causes depolarization, whereas reversing the flow causes hyperpolarization.

What about the stimulus affects the AP?

Stimulus intensity determines its frequency, and stimulus duration determines the number of them.

Draw a detailed diagram of the AP. Indicate any time periods, the refractory periods, and each phase of polarization.

AP threshold, absolute and relative RPs, and all three polarizations must be present. Make sure the mbP rests around -65 mV.

What are the six characteristics of the AP?

Unique long-ranged signal.

AP triggered by depolarization of 10-20 mV.

“All or none” event.

Regenerative; propagates without decrement.

Overshoot and undershoot in its shape.

Absolute refractory period.

What causes the AP?

An initial influx of Na⁺ followed by an efflux of K⁺.

Describe the voltage clamp method and what it can achieve.

It allows one to control mbP at any level while measuring the I necessary to maintain it.

It allows us to determine hoe mbP influences ionic I flow across the mb.

How does whole cell patch clamp recording apply to measuring AP and mbP?

V_m is recorded and the amount of I needed to keep V_m = N_c is injected.

What are the currents produced following depolarizaiton to the threshold? Draw the graph.

Initial brief capacitive I followed by a rapidly rising inward ionic I, followed by a slower and delayed outward I.

What are some pieces of evidence for the Na⁺ current?

A 90% reduction in external Na and replacement with choline eliminated the early inward current.

Tetrodoxin (TTX), saxitoxin (STX), and conotoxins block V-gated Na⁺ channels and remove the early inward current.

When the V_m is set to 55 mV, there is no inward current of Na⁺.

What are some pieces of evidence for the K⁺ current?

Replacement of intracellular K⁺ with Cs⁺ eliminates the late outward current.

Tetraethylammonium (TEA) and 4-aminopyridine block the outward current.

What the general process of an AP?

At rest, only leak K⁺ channels open. During the rising phase, more V-gated Na⁺ channels open as the mb is depolarized. Once the overshoot phase hits, the sodium channels close and V-gated K⁺ channels open. During the falling phase, V-gated potassium channels are at their peak activity while sodium channels fully close. During the undershoot phase, the potassium channels begin to close; they finish closing during recovery.

What are three important phenomena of the conductances of K and Na during an AP?

Both of them are V-dependent, as they increase progressively as the neuron is depolarized.

Both change over time; the inward flow of Na (.5 ms) precedes the outward flow o K (1-2 ms).

Activation saturates. The initial depolarization opens Na channels which causes further depolarization. The rate of depolarization decreases because Na’s driving force decreases, Na channels become inactivated, and K channels open to repolarize the mbP.

How does the positive feedback loop in APs work?

Depolarization causes an increase in g_Na, which causes more inward I_Na, leading to more depolarization.

How does the negative feedback loop in APs work?

Depolarization causes an increase in g_K, which leads to an outward K current. This causes repolarization, which leads to a decrease in g_K.

Describe the absolute refractory period.

It occurs after the neuron fires its AP. It’s impossible to make the neuron fire another AP due to the inactivation of Na channels. It usually lasts 1 ms, capping the AP frequency to 1000 Hz.

Describe the relative refractory period.

It’s after ARP; it is unlikely BUT possible to fire another AP due to the opening of K channels and repolarization.

What else does the refractory period contribute to?

It contributes to the AP maintaining this direction.

How does the refractory period affect the direction of propagation?

RP limits the firing frequency while causing the AP to continue propagating in one direction. It causes the membrane downstream to depolarize.

How do the time and length constants affect AP velocity?

A small time constant lets neurons reach the threshold faster.

A large length constant will maintain depolarization over a longer distance.

Why does the AP get regenerated along the axon?

Mb leakiness causes adjacent Na channels to open.

What two factors influence AP velocity?

Axonal diameter (larger axon is better) and membrane resistance (higher is good because there is less signal loss).

How does myelination affect AP velocity?

The membranes of myelinated axons have V-sensitive Na and K channels restricted to nodal regions with a typical distance of 1 mm apart.

Myelination increases r_m, and there is saltatory conduction between the Nodes of Ranvier.

How are Na and K channels distributed in a myelinated neuron?

Na channels are at the nodes, and K channels are at the paranodes.

Where can APs occur?

APs usually happen where the highest density of Na channels is, which is usually the axon hillock.

There is emerging evidence that AP can occur in dendrites with elaborate branches, such as Purkinje cells and cortical pyramidal cells. At branch points, the AP may fail to go both ways!

What effect does lidocaine have on AP?

It acts as a reversible inhibitor of Na channels to temporarily block AP propagation.

The “selectivity filter” in an ion channel is most accurately described as a part of the pore that…

…contains a ring of charged residues that interact with specific ions.

What is membrane permeability?

The ease with which ions can move across the membrane.

How are ions and mbP related?

Changes in mbP are due to the movement of ions through the Pmb.

What are the typical flows of some common ions, and what are their effects?

Na in, depol.

K out, hyperpol.

Cl in hyperpol.

What are ion channels?

They are integral mbProteins found in the Pmb and ICmbs. They allow the diffusion of ions DOWN their electrochemical gradient through the membrane.

How do ion channels differ from pumps and active transporters?

Ion channels move DOWN gradients and don’t need energy; pumps and ATs do the opposite.

Why are ion channels important? Name three reasons.

2% of the genome encodes for ion channels!

Many biological processes, such as muscle contraction, cell proliferation/death, and neuronal signaling all rely on ion channels.

Several nerve and muscle diseases, such as Long QT syndrome, are due to defects in ion channels.

Describe Long QT syndrome.

It is a rare congenital heart disease characterized by arrhythmia in cardiac muscle. The prolonged QT interval is due to mutations in K_v channels, leading to delayed repolarization after an AP.

What are the four important ion channel features?

They’re found in all excitable cells.

They are either multiple proteins/SUs/polypeps. OR a single polypep. that spans the mb multiple times.

Can be highly selective or not selective.

Either regulated (gated) or not.

What is an example of an ungated ion channel?

K⁺ leak channels, which are vital for mbRP.

Describe the basic structure of the Nicotinic Acetylcholine Receptor (nAChR).

It has a pentameric channel structure (two alpha, one of each subsequent Greek letter). Each SU contains 4 mb-spanning regions (M1-M4) and contributes to a hydrophilic central pore. Each alpha SU binds the ligand (ACh).

How can ion selectivity differ between channels?

Some channels only allow a certain ion, whereas others allow those with a specific charge to pass.

Channels can be classified by the ions they’re permeable to.

What are the three modes of activation for ion channels? What is an example of each?

Voltage: Na_v

Ligand: nAChR (EC ligands) [can be EC or IC ligands]

Mechanical: TMC1 and TMC2

What are TMC1 and TMC2?

Stretch-activated ion channels in hair cells of the inner ear.

Describe V-Activated Ion Channels.

They undergo conformational changes in response to mbP.

They also exhibit high ion selectivity (K_v channels are 10x more selective to K than Na).

Some examples include Na_v and K_v channels, as well as Ca_v channels responsible for NT release.

They are categorized into structural families on the basis of ion selectivity.

Describe the Na_v channel.

It is a large, 200 kD, single polypep. with 4 domains that are architecturally equivalent to the AChR SUs. Each domain has 6 mb-spanning segments. Between S5 and S6, there is a P region that embeds into the mb WITHOUT spanning it that controls ion selectivity.

Describe ligand-activated ion channels.

Their main role is to turn chemical into electrical signals.

They’re activated by NT binding; they have a low degree of selectivity, are relatively slow, and are categorized by the ligands they respond to.

What are the four main NTs, and which receptors do they bind to?

GABA: GABA_A

Glycine: Glycine

Glutamate: Kainate, AMPA, and NMDA (all glutamate analogs, glutamate binds as well!)

Acetylcholine: nAChR

Which NTs are inhibitory, and which ones are excitatory?

Inhibitory: GABA and Glycine

Excitatory: Glutamate and ACh

Describe glutamate receptors.

They are the most important excitatory NT in the CNS. They can be ionotropic or metabotropic.

The three different ionotropic glutR can be distinguished by their sensitivity to the glutamate analogs.

What do ionotropic and metabotropic mean in relation to GlutR?

Ionotropic: Is the ion channel

Metabotropic: G protein-coupled receptors that activate/inhibit other channels via signaling.

What are the three glutamate analogs?

NMDA, AMPA, and Kainate.

Describe mechanosensitive ion channels.

These respond to stretching pressure, and shear stress.

Their functions include touch sensation, hearing, pain perception, proprioception, BP regulation, and cell volume regulation.

What is the PIEZO2 Channel?

A large homotrimeric channel in sensory neurons that responds to stretching. It has non-selective cation permeability, but has a notable preference for Ca²⁺, which makes them important for calcium signaling.

What si the TRPV4 Channel?

An osmotic and mechanical sensor in kidneys, cartilage, and lungs.

It has a non-selective cation permeability with a preference for Ca²⁺.

Who won the 2021 Nobel Prize in Physiology or Medicine?

David Julius and Ardem Patapoutian for their discoveries of receptors for temperature and touch.

What are the three electrophysiological recording methods for recording ion channels?

EC recording, IC recoding, and Patch Clamp recording (only PCR allows for measurement of single ion channels).

Why is ion channel structure important to know?

To understand their functions and how they’re regulated, and to develop drugs to manipulate them.

What are some important biochemical and molecular techniques to reveal structural information? (Mainly to extract the protein)

Protein purification methods, such as chromatography, cloning, sequence analysis, expression, and mutagenesis.

What are some important biophysical techniques to reveal structural information? (looking at structure)

Cryo-EM, X-ray crystallography and diffraction, nuclear magnetic resonance (NMR), and spectroscopic methods (CD).

What are some important computational techniques to reveal structural information?

Molecular modeling via computers and software.

Why is it difficult to get macromolecular structural information about ion channels?

It’s harder to get crystals from mbProteins, as they have complex and irregular structures that make diffraction difficult.

What the three structure types for ion channels?

Hetero-oligomer: several different SUs.

Homo-oligomer: several identical SUs.

Single polypep. spanning mb. multiple times.

What are some facts about the nAChR?

It is a ligand-gated ion channel and the first one studied in detail. It is prominent at the neuromuscular junction (NMJ), as well as at synapses between specific neurons.

It has two types: nicotinic (ion channel) and muscarinic (GPCR).

Describe the nAChR.

These are ionotropic AChRs that responds ot nicotine. The receptor has 5 SUs clustered around a central core. Upon opening, Na and Ca enter the cell while K leaves.

Describe the mAChR.

It is the metabotropic AChR the responds to muscarine. It consists of seven GPCRs that starts an intracellular signaling cascade. It is NOT an ion channel.

How was the structure of nAChR determined?

Mbs of the electric organ of the Pacific Electric Ray were used, as they have mNAchRs.

a-bungarotoxin affinity purification revealed 4 glycoprotein SUs with 40, 50, 60, and 65 kD.

Through cDNA cloning, they discovered that each SU has h-phobic region called M1-M4, which are believed to be the mb-spanning portions.

The structure was solved by getting a transverse section of vesicular tube of Torpedo. M2 is in the center and moves upon binding.

What is alpha-bungarotoxin?

A neurotoxin known to bind with ACh with high specificity.

It is a snakevenom from Bungarus multcinctus.

Describe nAChR’s pentameric channel structure.

At the NMJ, there are 2 a SUs, 1 B, 1 g, and 1 d.

In neurons, there are various combos of 12 different nicotinic receptor SUs (a2-a10 and B2-B4).

Each SU contains 4 mb-spanning regions, and each a SU binds ACh.

What are functional domains of the Na_V channel?

S4 segments: Voltage sensors

P regions: For ion selectivity

Extracellular loops: B₁ interaction sites; B₁ SU regulates gating

III-IV loop: PKC phosphorylation; fast inactivation

How does Na_v’s Voltage sensor work?

On the S4 segment, every third position is a (+) charged AA (lys/arg) that faces inward to the pole. Depolarization causes the S4 segment to move electrostatically outward.

What two factors affect ion selectivity?

The different electrical field built by different ions and the water shell that interacts with specific AAs in the P-loop.

What’s the ion selectivity of Na⁺ in terms of the electrical field and water shells?

Smaller ion = stronger EF = more hydration. Can only pass pores with acidic AAs that interact strongly with Na⁺, and the water molecules are partially lost.

What’s the ion selectivity of K⁺ in terms of the EF and water shell?

Larger ion = weaker EF = less hydration. Can pass the K⁺ channel, but not the Na⁺ channel due to the electrostatic interactions not being strong enough to remove water molecules as well as in the case of Na⁺.

Describe the K⁺ channel structure.

Each one is 4 SUs with 2 mb-spanning helices.

It was the first X-ray structure of an ion channel solved; specifically, the K_est channel from Streptomycus lividans at 3.2A resolution.

What are nociceptors?

Sensory receptor cells for noxious stimuli. They are high-threshold, pseudo-unipolar neurons. Their CBs are in the DRG and have two axons: one goes from the periphery to the DRG, and the other goes from the DRG to the SC.

They are the primary sensory afferent neurons and are glutaminergic (excitatory).

What are the two types of nociceptors?

C-fibers and A-fibers.

Describe C-fibers.

They are unmyelinated and have a small diameter, resulting in a slow conduction velocity of <2 m/s.

They are for dull pain and prolonged burning sensations.

There are two types: substance P (thermal) and non-peptidergic (mechanical).

Describe A-fibers.

These are myelinated an have a larger diameter, resulting in a faster conduction velocity (30 m/s).

They are for fast and intense pain; Adelta types detect and encode pain.

Type 1 is primarily mechanical, and Type 2 is primarily thermal.

Which two Na_v channels are most prevalent in nociceptors?

1.7 and 1.8.

What are the two types of synapses?

Electrical and chemical.

How do electrical and chemical synapses differ from one another?

Esyn: Involves direct transmission of ions and other small molecules through gap junctions (GJs).

Csyn: Conversion of electrical signal into chemical one through NT release.

What was the general belief about synaptic transmission in the late 19th century?

Interneuron and neuron-muscular signaling was mostly electrical.

Who gave the first direct demonstration of chemical transmission, and what was their contribution?

In 1921, Otto Loewi showed that vagus nerve stimulation of a frog heart slows the heartbeat. Transferring the fluid from this heart to an unstimulated heart also slowed the heartbeat; this substance was later identified as ACh.

What were some initial points of opposition to chemical transmission?

Transmission is fast and there isn’t sufficient time for molecule release.

Few synaptic preparations are large enough to study in detail.

Very few NTs were known until around 100 years ago.

What are four critical properties of Esyns?

Small molecules move through a pore (connexon) directly from one cell to the other.

Direct electrical coupling.

Fast!

BIDIRECTIONAL; not ion selective and gates are open on both sides.

What makes up GJs?

Connexons.

Describe the connexon.

It’s made up of one type or different connexins, a polypep. SU hemichannel that has 4 transmb. helices and come in 20 different versions.

Its pore size is 1.5 nm. It allows ions, small molecules (cAMP), and nutrients (1 kD) to pass.

In what conditions can gap junctions (and connexons) close?

In response to lowering cytoplasmic pH of Ca²⁺ elevation. Both of these occur when the cell is damaged, and this is done to protect adjacent healthy cells.

What are the two important purposes of Esyns?

Synchronous cell firing (in hearts, etc.) and cell nutrition.

What are two examples of Esyns?

Needed in the crayfish abdominal nerve for fast response in the animal’s escape reflex.

Needed for synchronous ink release from glands in Aplysia Ink Release.

Describe the basics of Csyns.

They the most common syns allowing direction transmission of info via the release of NTs.

Pre- and postsynaptic cells are separated by a synaptic cleft of ~50 nm. Upon depol. vesicles fuse with Pmb and release NTs.

What happens when AP reaches Csyn?

The arrival causes depol. of presynaptic terminal followed by opening of V-gated Ca²⁺ channels. These flood the cell causing vesicular fusion and NT release.

What differs direct and indirect synaptic channels?

Direct (ionotropic) is an ion channel; the NT binds and changes the channel.

Indirect (metabotropic) is a GPCR; the NT binds and starts an IC cascade.

What are the criteria for NTs?

It must be present in the presyn. neuron, it must be released in response to depol. and is dependent on Ca²⁺ influx into the presynaptic terminal, and specific receptors must be present on the postsyn. cell.