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