Transcript SDK-Kchannel

+
K
Channels
60
40
mV
20
0
-20
-40
-60
-80
0
2
4
6
8
10
msec
PNa or PK
PNa rises quickly then declines
PK rises slowly, declines
with repolarization
0
2
4
6
8
10
4/12/05, MCB610
K Channel Gating
K+
K+
K+
K+
K+
K+
K+
Outside
Inside
K+
K+
K+
K+
K+
K+
K+
-15
-60mV
mV
K+
K+
K+
K+
Electrophysiology
– extracellular recording
– intracellular recording
– whole-cell recording
– single channel recording
How to Study?
“Patch Clamp”
Nobel Prize in Physiology &
Medicine -1991
Extracellular
Inside Cell
Patch Clamp Recording
Technique
A
B
C
D
E
el ect r ode
channel
cel l
cel l- at t ached pat ch
whol e- cel l
out si de- out pat ch
Types of K+ Channels
Voltage-gated
 Inward Rectifying
 Ca2+ sensitive
 ATP-sensitive
 Mechano-sensitive
 Type A
 Receptor-coupled

Classification of K+ Channels
1. Voltage-gated
6 transmembrane domains
 4 subunits surround central pore (S5 & S6
regions of each subunit
 Selectivity filter (P region)

– Hydrophobic sequence between last 2 TMD; contains Gly-Tyr-Gly

Voltage sensor (S4) has multiple positively
charged amino acids
Voltage-gated
con’t
Activated by depolarization
 Present in both excitable and nonexcitable
cells
 Functions

– Regulate resting membrane potential
– Control of the shape and frequency of action
potentials
Voltage Dependent Gating
Outside
S1
S2
S3
S4
S5
S6
Inside
HO2C
H2N
+ + + + + +
LRVIRLVRVFRIFKLSRHS
1. Three Types Ca2+ Sensitive K+ Channels

High conductance (BK) channels (Slo)
– Gated by internal Ca2+ and membrane potential
– Conductance = 100 to 220 picoSiemens (pS)
– Blocked by charybdotoxin and iberiotoxin

Intermediate conductance (IK) channels (SK4)
–
–
–
–

Gated only by internal Ca2+
More sensitive than BK channels
Conductance = 20 to 85 pS
Blocked by charybdotoxin
Small conductance (SK) channels (SK1-3)
–
–
–
–
Gated only by internal Ca2+
More sensitive than BK channels
Conductance = 2 to 20 pS
Blocked by apamin
BK channel
2. KATP channel

KATP
ATP increase-decrease channel
opening
Pancreatic type or cardiac type

KNDP
NDP increase-increase channel
opening in the presence of Mg2+
smooth muscle type
KATP characteristics




Octameric
four a-subunit (KIR6.1 or KIR6.2)
four b-subunit (SUR1, SUR2A, SUR2B)
Smooth muscle type
KIR6.2/SUR2B
Sulfonylurea agents-glibenclamide, tolbutamide inhibit channel
activity
Pharmacological KATP activator
pinacidil, cromakalim, lemakalim, diazoxide, minoxidil, nicorandil
(induce hyperpolarization)
Endocrine Reviews
20 (2): 101-135
Molecular Biology
of Adenosine
TriphosphateSensitive Potassium
Channels.
Lydia AguilarBryan and Joseph
Bryan
KATP channel
3. Inwardly Rectifying K+ Channel
(KIR)

2 transmembrane regions (M1 & M2)
– Corresponds to S5 & S6 in Kv channel






4 subunits surround central pore
P region separates M1 and M2
Non-conducting at positive membrane potentials
Maintains resting membrane potential near Ek
Blocked by external Ba++
Mainly Kir2x
Increasing extracellular K+
induced shortening
of cardiac action potential.
Mg, PA
4. K2P CHANNELS
TWIK: Tandem pore domain Weak
Inwardly rectifying K+ channel
 TREK: TWIK-RElated K+ channel
 TRAAK:TWIK-Related Arachidonic acidActivated K+ channel
 TALK: TWIK-related ALkaline-activated
K+ channel
 TASK: TWIK-related Acid-Sensitive K+
channel

TREK channels
NEGATIVE PRESSURE ACTIVATES SDK CHANNEL
(murine colonic myocyte)
A. on-cell, 0mV, asymmetrical K+
B. Pr. and Po relation
-40cmH2O
-60cmH2O
-40cmH2O
-80cmH2O
-60cmH2O
I-O
Probability density
-20cmH2O -20cmH2O
1
0.8
0.6
0.4
0.2
0
-80
-60
-40
cmH2O
10 sec
10 pA
Should be K+conductance
-20
0
SDK CHANNEL ACTIVATED BY INCREASE CELL LENGTH
A
B
Stimulus of
negative pressure
does not necessarily
stimulate the effects
of cell stretch.
C
10 µM
-60 cm H2O
Cell Elongation
2sec
10 pA
Cells were actually elongated and activated K+ channels with
the same properties as those activated by negative pressure.
5. A-TYPE CURRENTS IN SMOOTH MUSCLE
Voltage-dependent, transient outward K+ currents have also been identified in
smooth muscle cells.
The term A-type current to designate rapidly activating, inactivating,
voltage-dependent K+ currents.
In vascular smooth muscle cells of the rabbit (portal vein, pulmonary artery,
aorta), rat (pulmonary artery, renal resistance artery), and human (mesenteric
artery).
In genitourinary (GU) smooth muscle cells of the guinea pig (ureter, seminal
vesicles, and vas deferens), rabbit (vas deferens), rat (myometrium), and
human (myometrium).
In gastrointestinal (GI) smooth muscle cells of the mouse (fundus, antrum,
jejunum, and colon), rat (ileum), guinea pig (colon), and opossum (esophagus
General properties of A-type K+ currents. A: whole cell A-type currents from
holding potentials of -80 (a) and 40 mV (b) recorded from mouse antral
myocytes. B: steady-state inactivation shown as a plot of normalized peak
current (I/Imax) as a function of conditioning potential and fit with a
Boltzmann function.
Table 3. A-type channel and accessory subunit expression in smooth muscle
Smooth Muscle
Transcript
Protein
Rat mesenteric artery
Kv1.4, Kv3.3, Kv3.4, Kv4.2, Kv4.3, Kv 1- 3
Rat tail artery
Kv1.4, Kv3.3, Kv3.4, Kv4.2, Kv4.3, Kv 1- 3
Rat pulmonary artery
Kv1.4, Kv4.1-4.3
Rat aorta
Kv4.3L
Rat vas deferens
Kv4.3L > Kv4.2
Rat urinary bladder
Kv4.3L
Rat myometrium
Kv4.3L > Kv4.2
Rat stomach
Kv4.3L
Rat colon
Kv4.3L
Mouse fundus
Kv4.1, Kv4.2, Kv4.3L, NCS-1, KChIP1, 3, 4
Kv4.2, Kv4.3
Mouse antrum
Kv4.3L > Kv4.2 > Kv4.1, NCS-1, KChIP1, 3, 4
Kv4.2, Kv4.3
Mouse jejunum
Kv4.3L > Kv4.2 > Kv4.1, NCS-1, KChIP1 > KChIP2-4
Kv4.2, Kv4.3
Mouse colon
Kv4.3L > Kv4.2 > Kv4.1, NCS-1, KChIP1 > KChIP2-4
Kv4.3 > Kv4.
2
Kv4.1
Kv4.3
Kv, voltage-gated Ca2+-independent K+ current; NCS, neuronal Ca2+ sensor; KChIP, K+ channel-interacting protein.
Figure 1. Effect of 4-AP on the
electrical activity of intact murine
colonic smooth muscle
Figure 2. Effect of TEA on the
electrical activity of intact
murine colonic smooth muscle
Determination of the reversal
potential
Voltage dependence of inactivation and
activation of delayed rectifier K+
currents
The recovery from inactivation of
delayed rectifier K+ current
Inhibition of delayed rectifier K+
currents by 10 mM TEA
Effect of 5 mM 4-AP on delayed
rectifier K+ current
mRNA expression of Kv1, Kv4 and Kv subunits in
murine proximal colon circular smooth muscle cells
The effect of intracellular Ca2+ buffering on inactivation of
A-type currents
The effect of KN-93 on inactivation time
constants of A-type currents
The effect of KN-93 on the voltage dependence of
inactivation of A-type currents
The effect of KN-93 on recovery from inactivation
of A-type currents
The effect of dialysis with autothiophosphorylated
CaMKII on A-type currents
CaMKII-like immunoreactivity in mouse proximal
colon
Quantification of Kv4 transcripts in colon and jejunum
Inhibition of colonic A-type current by flecainide
Kv4.2- and Kv4.3-like immunoreactivity in the tunica
muscularis of murine colon and jejunum
Quantification of KChIP transcripts in colon and
jejunum
Autothiophosphorylated Ca2+/calmodulin-dependent protein
kinase II (CaMKII) decreases the rate of inactivation of voltagedependent K+ channel 4.3 (Kv4.3) currents.
Autothiophosphorylated CaMKII produced a positive shift in
voltage-dependent activation and inactivation.
Autothiophosphorylated CaMKII accelerates the recovery from
inactivation of Kv4.3 currents.
Effect of mutagenesis on specific CaMKII consensus sequences on
Kv4.3 currents.
Effect of C2 mutagenesis on the rate of recovery from inactivation.
Effect of C2 mutagenesis on Kv4.3 channel inactivation kinetics in
response to application of autothiophosphorylated CaMKII
Effect of C2 mutagenesis on Kv4.3 channel inactivation kinetics
in response to inhibition of CaMKII. A: dialysis with the CaMKII
inhibitory peptide 281–301