Ns101B Module 2: Lecture 1

Front 1

Function of Na/K channel during AP
*4 steps*

Back 1

1. Na channel opens depolarizing mmb potential away from resting potential towards E_na
2. FAST: depolarization opens more Na+ channels ( + feedback)
3. SLOW: Delay; K+ channels open causing hyperpolarization of mmb potential to E_k
4. Delay; Na+ channels inactivate helping K+ channels hyerpolarize mmb potential

Front 2

Important properties of voltage-gated ion channels
1. Pore

Back 2

pore controls ion permeation
-selectivity: which ions go through the channels
-channel conductance: how rapidly ions pass
-channel blockers: bind w/i the pore

Front 3

Important properties of voltage-gated ion channels
1. Pore
2. Gates

Back 3

gates control the gating of the channel
-acitivation --> channel opening
-inactivation/deactivation --> channel closing

Front 4

Molecular identification of voltage gated K+ channels involved a

Back 4

mutant fly (shaker) w/ defective muscle K+ channel

Front 5

1. Fly with shaker mutation are ..
2. Treat WT flies with drug that blocks K+ channel causes... Suggests that.....
3. Gene identified using...

Back 5

1. hyperexcitable Ishake their legs uner anesthesia)
2. causes similar phenotype
Suggests that --> K+ channel is defective
3. positional cloning

Front 6

Expression of gene for functional analysis
Step 1

Back 6

1. synthesize mRNA corresponding to the gene of interest

Front 7

Expression of gene for functional analysis
Step 2

Back 7

express the mRNA by injecting into into Xenopus (frog) oocyte

Front 8

Expression of gene for functional analysis
Step 3

Back 8

measure the channels function by voltage clamp

Front 9

Expression of gene for functional analysis
*picture of process

Back 9

Front 10

Voltage clamp recordings of K+ channel expressed in Xenus oocytes
*Conclusions from experiment*

Back 10

1. expressed protein conducts K+ ions
2. opens among mmb depolarization and closes on repolarization
3. particular K+ channel investigated does not inactivate (others do) --> as long as you depolarize it stays open

Front 11

From learning the sequence of the proteins, the scientists were able to do 3 things.

Back 11

1. functional expression of mRNA
2. predict the structure of the channel (look for long stretches of hydrophobic a.a. for transmmb domains)
3. identify homologous proteins

Front 12

Predicting mmb topology (structure) of voltage gated K+ channel

Back 12

1) Voltage gated K+ channel subunits contain: 6 alpha helical segments termed S1-S6
2) a re-entrant loop between S5-S6
3) large N terminal segment
4) Functional channels are tetramers
a) 4 subunites are designated I-IV

Front 13

From genome, found a large number of voltage gated- channel genes which are called...
-Sequence homology among which channels?
- Why so many?

Back 13

Protein Superfamily
-Na+, Ca+, K+ channels (Cl- unrelated)
-different neurons respond differently to certain excitation (release more or less ions, time open)

Front 14

Different K+ channel genes encode channels w/ functional differences.
-two different ones

Back 14

Normal channels open for depol. and inactivate when depol. remains
-Inward rectifier & 2-pore

Front 15

K+ channel: Inward Rectifier
-----> Function & structure

Back 15

Function: K+ channel that opens in response to hyperpolarization (negative current, -120 mV)
Structure: only has re-entrant loop (S5-S6) and lacks the N terminal 4 transmmb domains
-has 4 subunits

Front 16

K+ channel: 2-pore
-----> Function & structure

Back 16

Function: K+ channel modulated by pH --> responds to basic pH conditions
-not gated by voltage
Structure: only 2 subunits (min. req to be a fxn-al channel)
-two S5-S6 subunits on either side

Front 17

Inward Rectifier and 2-pore similarities

-I.R. looks like it is gated by voltage but it is actually...

Back 17

Both lack the 4th N terminal transmmb domain so both are not gated by voltage

-has something in its structure that is sensitive to changes in mmb potential

Front 18

Diverse channel types underlie...
-voltage clamp recording involves
-Look at AP fired what can be seen?

Back 18

different firing properties
-intracellular recordings: inject current --> initial depol. --> reach threshold --> neuron fires AP
-some fire fast and inactivate quickly & vice versa because
*** expression of different types of voltage gated potassium channels causes differences in the electrical properties of the channel ***

Front 19

-Structure of Voltage Gated Na+ and Ca+ channels (2)
-Each domain has?
-They are...

Back 19

1) 4 main domains, each domain subunit contains 6 transmmb domains
2) each domain is homologous to a K+ channel subunit
-they are monomers

1) 6 alpha helical segments termed S1-S6
2) a non-helical re-entrant loop (P-Loop) between S5-S6

-monomers

Front 20

-Many K+, Na+, and Ca+ channels also have a ___ subunit
-What does this subunit do?
-Subunits that form the actual channel are?

Back 20

Beta subunit

-not req. to make the channel work, they MODULATE (regulate) the FUNCTION of the ion channel
----> Regulate by: making the channel more or less sensitive, incr/decr conduction, incr/decr closure time

-the ALPHA subunits form the channel