3. Membranes and Transport II Flashcards by J L

Lidocaine
• Pain reliever commonly used in dental practice
• Most modern dentistry depends upon lidocaine

• \_\_\_\_ dependent \_\_\_\_ channel blockers
	○ Inhibits transmission of AP

voltage

Na+

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• Nearly all the neural signals emanating from pulp are associated with ____

pain

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Typical ion concentrations across the membrane in mM

These differences matter:
• Small elevations in extracellular K+ can lead to ____ and death
(and malpractice litigations if erroneous infusion)
• Rapid changes form the basis of neuronal signaling and thus thoughts & pain

	• Inside:
		○ High of \_\_\_\_
		○ Low of \_\_\_\_
		○ Low of \_\_\_\_
		○ Very low of \_\_\_\_
		○ Moderate Mg
	• Outside:
		○ Low of \_\_\_\_
		○ High of \_\_\_\_
		○ High \_\_\_\_
		○ Several orders of magnitude of \_\_\_\_ compared to intracellular
	• Differences in ion concentrations are the fundamental basis of signaling 
	• How are gradients established?
		○ NaKATPase
		○ Energy requirement to maintain differences

cardiac arrest
K
Na+
Cl-
Ca++

K
Na+
Cl-
Ca++

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Chemical driving force
Glucose crossing a permeable membrane

Chemical driving force proportional to difference in ____
Chemical driving force = -RT ln([G]i/[G]o)
Where R = gas constant, T = temperature in Kelvin and [G] = glucose concentration

concentration

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Diffusion
• Chemical potential:
– Molecules diffuse from high to low concentration – Move down their ____ gradient; “Downhill”

• Electric potential:
– Opposites attract
– Cations with + charge move toward ____ charged areas
– Anions with - charge move toward ____ charged areas
– Move down their ____ gradient; “Downhill”.

Ions have both ____ and ____ gradients that must be balanced at equilibrium

+
electrical

electrical
chemical

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Electrochemical driving force 1

• T0, membrane is permeable to K
	○ Cl doesn't move
	○ K diffuses from inside to outside due to \_\_\_\_ driving force
		§ Concentration gradient

chemical

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Electrochemical driving force 2

	• Buildup of K on outside of membrane
	• Electrical force
		○ Net positive charge on the outside
		○ Net negative charge on inside
		○ Tries to pull positive charge back inside the cell due to \_\_\_\_ gradient, but \_\_\_\_ gradient pushes K back out

electrical

concentration

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Electrochemical driving force 3

• Reach equilibrium:
	○ Driving forces are \_\_\_\_ and \_\_\_\_
		§ Neither are \_\_\_\_

equal
opposite
resolved

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Electrochemical driving force 4

____ energy = -RT ln([K]i/[K]o)

____ energy = zFV

Where z = valence (K+=1),
F = Faraday constant (magnitude of electric charge per mole of electron) V = voltage

* Bigger difference in cxn results in a larger \_\_\_\_ force (the log)…
* F - number applied to the electrical \_\_\_\_…

chemical
electrical

chemical
charges

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Electrochemical driving force 5

At equilibrium, sum of ____ and ____ energy for K inside equals that outside

zFVi + RT ln[Ki] = zFVo + RT ln[Ko]

chemical

electrical

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Electrochemical driving force 6

LOOK AT THESE SLIDES

• \_\_\_\_  is the membrane potential where they are balanced
	○ Nernst equation

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The Nernst equation:
The Nernst equation lets you balance the electrical and chemical driving forces for ____ ions

EK is Vm when K+ is at ____; the potential at which there is no ____ of K+ for the given intracellular and extracellular concentrations

permeant
equilibrium
net movement

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The equilibrium potential for major ions differs greatly

EK very ____
ENa ____
ECl ____
ECa very ____
Ex is potential where ion is at ____; the major ions in a cell can’t all be at ____ at same time

If Vm=-70, which ion moves least? Most? Basis for Calcium signaling

* Real driving force on \_\_\_\_ (most opposite of +125)
* \_\_\_\_ and \_\_\_\_ barely moves
* \_\_\_\_ moves decently

negative
positive
negative
positive

equilibrium
equilibrium

Ca++
K+
Cl-
Na+

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The membrane potential

A typical cell has a net ____ change, of between ____ mV to ____ mV at rest

• Measure membrane potential via \_\_\_\_

negative

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Determining the contribution of
multiple ions

Membrane potential is always with respect to the
cell ____

How do the individual equilibrium potentials combine to determine what the potential will be?

The permeability of the membrane to different ions varies: the more permeable the ion X, the more it contributes to the ____

interior

membrane potential

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What decides the contribution of each ion to the membrane potential? ____

____ equation

Remember: Cl is ____ in the equation (normal in over out)

Relative permeability
Goldman-Hodgkin-Katz (GHK)

inverted

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In resting cell
K+ is the biggest influence: PK large

PNa =0.05 PK =____ PCl =0.1 Because ____>____, ____, Vm close to ____

1
PK
PNa
PCl
EK

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Ca2+ contributes very little to GHK and membrane potential because ____ low and ____ levels low

PCa++

absolute

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What sets resting membrane potential?

Negatively charged proteins
- ____
____
- maintains gradients
Large ____ conductance
- Vm near Ek

Together these enable the intracellular concentration of ions to be different from extracellular and provide a ____ membrane potential. This results in large ____ gradients that can be used to signal quickly

impermeable
Na+-K+ pump
K+

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Many charges are impermeant

The presence of negative ions “stuck” ____ the cell creates a permanent force that influences the distribution of ions and water across membrane
based upon rules of electrochemical gradients and osmolarity

inside

Impermant anions - equilibrium
without equality

____ a key reason that ions can be at equilibrium but concentrations differ across membrane

Ionic balance means osmotic forces would swell cells:
in reality Na/K pump also keep cells from bursting as it forces out ____ and ____ follows – net loss of 1 ion per cycle

• NAKATPase
	○ Keeps cells from \_\_\_\_ from influx of H2O
	○ Forces out Na+ in conjunction with one H2O

impermeant intracellular anionic proteins

Na+
water

bursting

Primary active transport Na-K-ATPase

Transport of ions against gradient
Uses 33-70% of cell ATP - critical to function
Found in all animal cells
Electrogenic – 3 Na+/2 K+ drives a net charge across membrane but only makes membrane ____ mV more negative
Main role is to set up gradients
Also stops bursting

If block with ____, takes a while for the gradients to dissipate

* \_\_\_\_, a net negative movement across membrane
* Mainly used to set up cxn gradients

2-4
oubain
electrogenic

The number of ions needed for membrane potential is relatively ____

For typical 10 μM diameter cell, excess charge for a -90 mV potential

small

0.002%

The proportion of ions ____ as the diameter ____

In small axon, consistent ____ can add up

The number of charges in setting the membrane
potentials and APs is a ____ number of ions in the cell

increases
decreases
changes
small

Leak K+ currents add to negative membrane potential * Gradients drive ____ out of cell when leak channel opens * (If increase ____ to an ion, Vm moves toward ____ for that ion) * ____ pore channels * Molecular identity: two-pore channels * Maintains ____

``` K+ permeability Ex two equilibrium ```

Pump, K+ channels and thus membrane potential key to salivary gland function • Salivary glands Different combo's of ion channels and transporters that are involved in secreting solution (Na, Cl, followed by ____) The contribution of ion channels and transporters lead to a coordinated effect There are many ____ channels of various types that allow us to get the membrane potential; especially the two pore channels Other channels: • ____ exchange • ____ • ____ exchangers

H2O Na-anion Cl-bicarbonate Na-H

Summary of equilibrium and membrane potential * The Nernst equation tells the potential at which ions are in ____ for a given concentration – no net ____ * The GHK equation sums the contribution of multiple ions based upon their ____ and ____ * Membrane potential determined by ____, ____, ____ currents.

``` equilibirum movement concentration relative permeability impermeant anions Na/K ATPase leak ```

Key ideas on equilibrium and membrane potential * A small change in ion concentration makes a big change in ____ * Osmotic pressures are ____ if water is permeable * If an ion X is at equilibrium, ____ = Vm * If Ex is not = Vm at steady state, ions are pumped against the ____ of ____ movement * The greater the ____ of a membrane to an ion X, the closer Vm moves to Ex

``` membrane voltage equal Ex direction passive relative permeability ```

Ion channels • The region through the middle contains a pore, and a response to ____ change leads to ____ changes which will gate it open.

voltage | allosteric

Facilitated diffusion: Transport across membranes with a little help from the ____ * Many large and/or charged molecules cannot diffuse across the bilipid membrane * Specialized proteins aid in the crossing * Transport called facilitated diffusion if proteins provide a route through membrane to avoid lipid barrier •____ transport - ____ - ____

proteins passive carriers channels

Ion channels are protein holes in lipid membranes ____ center lets ions dissolved in an aqueous solution through the pore ____ periphery keeps channel stable in lipid membrane

hydrophilic | hydrophobic

Ion channel structure Multiple segments span the membrane ____ AAs inside lipid membrane ____ AAs on extracellular face, cytoplasmic face and make pore Voltage gated ion channels: ____ subunits create a pore * ____ TM for Na+ and K+ domains with ball and chain * No connection in ____ channels

``` hydrophobic hydrophilic 4 6 K ```

Ion channel structure ____ resolution- can actually “see” an ion channel Starting to correlate structure and function identify regions involved in gating, selectivity, binding etc.

xray defraction

CryoEM of TRPM1 ion channel ____ > ____ crystallography in terms of resolution CryoEM takes these ion channels, grows lots of them, purify them, freeze them, and can begin to see orientation through ____ and through ____ power. • You begin to orient them and see how they go together. You also get to see the different portions.

CryoEM x-ray EM computational

Key characteristics of ion channels 1. ____: What opens and closes pore? 2. ____: What ions go through the pore? How quickly do they ions pass through? 3. ____: Non-conducting state after activity 4. ____: Preventing flow of ions through pore

gating permeation/selectivity inactivation block

Can measure activity from a single channel with a ____ Can see activity of an individual protein When depolarize, channels open, Na+ ions flow into cells, inward current We can measure the opening and closing of ____ ion channels through the patch clamp. • You press an ____ against the cell membrane. You can get a very tight seal against the membrane which allows you to measure ion ____ flowing in and out of an ion channel. • You can look at a ____ ion channel here which is unique. • If you depolarize, you can see the channels open. • Important to understand that we can ____ different ion channels in this regard.

patch clamp individual electrode current single correlate

Gating of ion channels: how do they open and close ``` Channels can be gated open using: ____ messengers (phosphorylation) ____ binding - tooth pain ____ change ```

secondary neurotransmitter voltage

Channel gating – there really is an opening Opening of connexin 26 Addition of extracellular ____ closes channel (can see visually)

Ca++

Selectivity of ion channels: how to discriminate Not just dependent on ____; K+ vs. Na+ ____ interactions with channel wall – sometimes ____ ion gives closer fit • Selectivity comes down to ____ of fit with the ion channel ○ Tighter fit releases more ____ and allows it to go through

``` size molecular bigger tightness free energy ```

Ion channel selectivity obeys Nernst equation Single channel steps – (Channel as enzyme) Can determine which ion flows through channel by seeing the voltage at which no ____ flows (equilibrium) and using intracellular and extracellular ion concentrations to compare to EK, ENa etc.. • Change voltage via ____ • Change ____ of current steps • More depolarized, they get larger • Plot relationship between current and voltage = ____ ○ How rapidly do we get more current through ion channel based on voltage

current patch clamp magnitude conductance

Conductance of ion channels: how ____ can ions pass through • Different channels let more ions through per second – bigger ____ • Rate (current jump) ____ for given channel type at given gradient • To increase total current across membrane increase number of channels ____ or increase the amount of ____ each channel is open - ____ • Conductance = how rapidly ions pass through via charge/second ○ ____ mechanism! • Larger pore, but sometime its about ____ balance = higher conductance • Put more in membrane, or keep them open longer ○ Increase current

``` quickly current constant open time gating ``` time-dependent energetic

Channel inactivation Ball and chain model of inactivation Protein loop moves into region exposed when voltage gates channel open * ____ charged ball is tethered to membrane, binds the pocket, thereby inactivating * Controlling conductance in a very short ____ * Can be open but ____

positively timeframe inactivated

Channel block ____ block movement of ions through pore ____ is very effective blocker of Na+ channels. Beware of fugu (puffer fish) Channel closed more of the time with ____ • Inhibited ____ dependent ____ channels • Can no longer generate AP • ____ - relative of lidocaine ○ More often the channel is closed, and is open infrequently

local anesthetics tetrodotoxin (TTX) lidocaine derivative voltage Na+ QX-222

Lidocaine block of Na+ channel * Na channel has ____ subunits. * Lidocaine binds very ____ within the pocket. * Lidocaine fits very snugly in the pocket. Lots of this is dependent on the interactions between the ____ on the side chains of these ____ and the ____ itself.

``` 4 deeply atoms proteins Lidocaine ```

Schematic map of ion channels and transporters related to physiological and biological activities in tooth development. • Ion channel mutations contributing to dental issues • Tooth eruption: Cl, KIR, TRP • Dentin development: Cl, TRP • Calcium homeostasis: Ca, Cx43 (gap junction) • pH regulation: development of enamel - dentin • Coordinated action of ____ in order to develop teeth • ____ in ion channels that contribute to tooth development ○ Not just ____ factors * Ca homeostasis: there are Ca channels here. * pH regulation is critical to the development of the enamel. Its located in the area of the dentin.

ion channels polymorphisms behavioral

Figure 1. Schematic image of ion channels and transporters in ameloblasts (A) and odontoblasts (B). • Heres ameloblasts and odontoblasts. • You can see the balance of ____. • Odontoblasts have more complex regulation with different ____ channels. ◦ ____ • As you look at the production of the enamel and others, this ____-dependent regulation leads to the formation. • Ion channels on ____

``` ion channels Cl TRP-V3 pH lysosomes ```

TRP channels let + ions into odontoblasts when stretched • TRP - ____ in odontoblast • Characterize the type of ____ via the drugs and channel blockers ○ In combination with ____

polarized ion channel genetics

Summary - Membranes 2 * The ____ movement of an ion across the membrane is determined by balance between ____ charges and ____ forces and calculated using the Nernst equation * The combined influence of individual ions is determined by their ____ and calculated using the Goldman equation * The membrane potential is determined by ____, the ____ and ____ channels * The various characteristics of ion channels determine when and how much current flows through * In dental structures, ____ are key in development and for responding to stimuli

``` passive electrical osmotic relative permeability impermeant anions NaK ATPase leak K ion channels ```