K + Channels 4/12/05, MCB610 K Channel Gating K+K+ K+K+ K+K+ K+K+ K+K+ K+K+ K+K+ K+K+ K+K+ K+K+ K+K+...
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Transcript of K + Channels 4/12/05, MCB610 K Channel Gating K+K+ K+K+ K+K+ K+K+ K+K+ K+K+ K+K+ K+K+ K+K+ K+K+ K+K+...
KK++ Channels Channels
4/12/05, MCB610
K Channel GatingK Channel Gating
K+
K+
K+
K+
K+
K+
K+
K+
K+
K+K+
K+
K+
K+ K+
K+
K+
K+
Outside
Inside
-60 mV-15 mV
Electrophysiology
– extracellular recording
– intracellular recording
– whole-cell recording
– single channel recording
Inside Cell
Extracellular
“Patch Clamp”Nobel Prize in Physiology & Medicine -1991
How to Study?How to Study?
Patch Clamp Recording Patch Clamp Recording TechniqueTechnique
A B C D E
electrode
cellchannel
cell- attached patch whole- cell outside- out patch
Types of KTypes of K++ Channels Channels
Voltage-gated Inward Rectifying Ca2+ sensitive ATP-sensitive Mechano-sensitive Type A Receptor-coupled
Classification of Classification of KK++ Channels Channels
1. 1. Voltage-gatedVoltage-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 Voltage-gated con’tcon’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 GatingVoltage Dependent Gating
Outside
Inside
S1 S2 S3 S4 S5 S6
HO2C
H2N
LRVIRLVRVFRIFKLSRHS+ + + + + +
1. Three1. Three Types Ca Types Ca2+2+ Sensitive K Sensitive K++ Channels 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. K2. KATPATP channel 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
KKATPATP characteristics characteristics
Octameric
four -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): 101-135Molecular Biology of Adenosine Triphosphate-Sensitive Potassium Channels.Lydia Aguilar-Bryan and Joseph Bryan
KKATPATP channel channel
3. 3. Inwardly Rectifying KInwardly Rectifying K++ Channel Channel (K(KIRIR))
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 shorteningof cardiac action potential.
Mg, PA
4. 4. K2P K2P CHANNELSCHANNELS TWIK: Tandem pore domain Weak
Inwardly rectifying K+ channel TREK: TWIK-RElated K+ channel TRAAK:TWIK-Related Arachidonic acid- Activated K+ channel TALK: TWIK-related ALkaline-activated
K+ channel TASK: TWIK-related Acid-Sensitive K+
channel
TREK channels
A. on-cell, 0mV, asymmetrical K+
NNEGATIVE PRESSURE ACTIVATES SDK CHANNELEGATIVE PRESSURE ACTIVATES SDK CHANNEL (murine colonic myocyte)(murine colonic myocyte)
B. Pr. and Po relation
-20cmH2O
-40cmH2O
-20cmH2O
-40cmH2O
I-O
-60cmH2O -60cmH2O
-80cmH2O
10 pA
10 sec
0.8
0.6
0.4
0.2
-80
1
-60 -40 -20 00
cmH2O
Pro
bab
ilit
y d
ensi
ty
Should be K+conductance
SDK CHANNEL ACTIVATED BY INCREASE CELL LENGTHSDK CHANNEL ACTIVATED BY INCREASE CELL LENGTH
10 µM
BA
-60 cm H2OC
2sec
10 pA
Cell Elongation
Cells were actually elongated and activated K+ channels with the same properties as those activated by negative pressure.
Stimulus of negative pressure does not necessarily stimulate the effects of cell stretch.
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.
5. A-TYPE CURRENTS IN SMOOTH MUSCLE
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 Kv4.1 Kv4.3
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
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
Voltage dependence of inactivation and activation of delayed rectifier K+ currents
Determination of the reversal potential
The recovery from inactivation of delayed rectifier K+ current
Effect of 5 mM 4-AP on delayed rectifier K+ current
Inhibition of delayed rectifier K+ currents by 10 mM TEA
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 voltage-
dependent 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