Biol-K 416 Lec 11

Front 1

voltage clamp method

Back 1

1. capable of resolving the aggregate current resulting from flow of ions through thousands of channels

2. does not have sensitivity to measure flow of current via a single channel

Front 2

voltage clamp method mechansim

Back 2

1. voltage clamp amplifier (from command)

2. measure current

3. current passes through electrode

4. compared to reference electrode

5. measures Vm

Front 3

what have we learned from voltage-clamp experiments?

Back 3

1. membrane channels have to be able to allow ions to move across the membrane at high rates

2. currents depend on electrochemical gradient across membrane

3. Na and K ions flow across membrane independently of each, different channel types have to be able to discriminate between the ions and allow only one type to pass

4. ionic conductances are voltage-dependent, channels have to sense voltage changes and open only at appropriate levels

Front 4

types of patch clamp recordings

Back 4

1. whole cell recordings

2. cell-attached recordings

3. inside-out recordigns

Front 5

whole cell recording

Back 5

1. patch clamp method

2. membrane patch disrupted briefly by applying strong suction

3. interior or pipet is continuous with cytoplasm

4. allows measurements of electrical potentials and currents from the whole cell

Front 6

patch clamp method

Back 6

1. glass pipet makes tight contact with a small patch of the neuronal membrane

2. suction applied to seal pipette and membrane together

3. disallows the flow of ions in this space

4. all the current flows when a single ion channel opens must flow into the pipette and recorded

Front 7

Back 7

whole cell recording

Front 8

Back 8

cell attached recording

Front 9

cell attached recording

Back 9

1. small piece of the membrane can be pulled away without disrupting seal

2. allows study of single channel without complications due to the rest of the cell

3. possible to change the medium to which intracellular surface is exposed

Front 10

Back 10

inside-out recording

Front 11

what does patch clamp measure?

Back 11

measurements of single Na channels

Front 12

microscopic currents

Back 12

1. small inward currents

2. caused by depolarization

3. much smaller than macroscopic currents

4. result of the flow of thousands of ions per milisecond

Front 13

macroscopic currents

Back 13

obtained by voltage clamping an entire axon

Front 14

Back 14

microscopic Na+ channels

Front 15

Back 15

macroscopic Na+ channels

Front 16

Back 16

macroscopic K+ channels

Front 17

Back 17

microscopic K+ channels

Front 18

ion channel selectivity

Back 18

1. detect differences between Na+ and K+

2. voltage-gated

3. contain voltage sensors

4. depolarization increases opening

5. hyperpolarization closes channel

Front 19

voltage sensor

Back 19

1. present in both Na and K channels

2. detects membrane potential

3. formed by 4 transmembrane structures

4. found in each voltage-gated K+ cahnnel

5. separate domains that extend into the membrane

6. positively charged - allow movement IN membrane

7. depolarization pushes sensors outwards

8. hyperpolarization pushes sensors inwards

9. paddle like movement (open v close)

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Back 20

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Back 21

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Back 22

Front 23

Back 23

Front 24

ADD TTX

Back 24

Front 25

SCN genes

Back 25

1. 10 known genes

2. encode for different types of Na channesl

Front 26

Ca++ channesl

Back 26

1. more than 10 different genes encode for Ca channels

2. varied functions

3. release of NT at synapse

Front 27

K+ channels

Back 27

1. over 100 known genes encode for K channels

2. differ inactivation, gating, and inactivation

Front 28

Ligand-gated ion channels

Back 28

1. respond to chemical signals (ligands) instead of changes in membrane potential

2. less selective than voltage-gated channels

3. allow more than one type of ion to flow

Front 29

Back 29

cyclic nucleotide gated ligand-gated channel

Front 30

Back 30

Ca+2 activated K+ ligand-gated channel

Front 31

Back 31

NT receptors

Front 32

structure of channels

Back 32

1. four subunits are assembled together to form a channel

2. channel pore located in the center of the channel

3. narrowest part is near the outside of the channel

4. constricted so that only non hydrated K+ can pass

5. selectivity filter

Front 33

channel pore

Back 33

1. located in the center of the channel

2. narrow tunnel that allows K+ to flow through

3. formed by poor loops of each subunit

Front 34

selectivity filter

Back 34

1. found in channels

2. filter ions based on charge and size

Front 35

depolarization pushes voltage sensors ______

Back 35

outwards

Front 36

hyperpolarization pushes voltage sensors ______

Back 36

inwards

Front 37

Tarantula toxin: strong binding

Back 37

resting voltage sensors

Front 38

Tarantula toxin: weak binding

Back 38

active voltage sensors

Front 39

active transporters

Back 39

1. function to maintain concentration gradients

2. facilitate ion translocation

3. ex: ATPases, ion exchanges, co-transporters

Front 40

ion translocation

Back 40

1. via active transporters

2. takes several milliseconds

3. much slower than channels

Front 41

Back 41

co-transporters

Front 42

Back 42

ion exchangers

Front 43

Back 43

ATPase pumps

Front 44

Na+/K+ ATPase pump

Back 44

1. 2 K+ into cell for 3 Na+ out (must bind both)

2. net charge produced (+1 out) = electrogenic

3. charge produced is small relative to voltage-gated channels because transporters are slow

4. must have sites to bind ATP and receive phosphate

5. must bind ouabain

Front 45

Na+/K+ ATPase pump mechanism

Back 45

1. Na+ binding

ATP --> ADP

2. phosphorylation

3. conformational change: Na release and K binding

dephosphorylation

4. conformational change: K+ release

Front 46

ouabin

Back 46

toxin that pumps Na/K ATPase pump