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1. Potassium channel
Function:1.shape action potentials and set
the resting membrane potential 2. maintaining vascular tone 3. secretion of hormones 4. ion transport
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Types
Voltage-gated potassium channel Inwardly rectifying potassium channel resting potassium channels" or "leak channels Calcium-activated potassium channel
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6TM: The voltage-gated K+ channels
Kv, KCNQ, Kca2+
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A-type currrent
M-current
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2-TM. The inward rectifier K+
KIR1.x, KIR2.x, KIR3.x, KIR4.x,
KIR5.x, KIR6.x and KIR7.x
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4-TM. Sensitive to halothane,insensitive to TEA and 4-APRegulate cell volume
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2. Cell recording solutions and Protocol for Ca2+ 、 Na+ 、 K+
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(1) Calcium channels and Protocol
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激活曲线 Protocol
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失活曲线稳态失活曲线
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失活后再激活曲线
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( 2 ) Sodium channel
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Protocol
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( 3 ) Potassium channel
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① Inwardly rectifying potassium channel(KIR)
激活方案
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②The delayed rectifier current (IK)
largely responsible for repolarization and consists of two components namely IKs and IKr.
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Voltage-Time dependent activation
IKtail
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Time dependent activation
IKtail
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ms
0 200 400 600 800 1000 1200 1400 1600 1800 2000 2200
mV
-100
-80
-60
-40
-20
0
20
40
60
controldrugwash out
豚鼠心室肌细胞
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③ KATP
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KATP
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βcellSmooth muscle cells
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inside-out patch. The pipette potential is -100 mV
Whole cell recording,With ATP and GDP in the pipette solution, Vh-60
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inside-out patch
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Inside out-recording(-100mv)Bath solution (for the intracellular side of the membrane) included (mM): KCl 120, KOH 20, MgCl2 1, EGTA 5, Hepes 10, glucose 5, Na2ATP 0.3, and MgADP 0.5 (pH=7.2); while pipette solution (for the extracellular side of the membrane) contained (mM): KCl 140, MgCl22, EGTA 2, glucose 10, and Hepes 10 (pH=7.4).
Why Vh-60?Answer:minimize Kca
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Whole cell-recordingThe bath solution contained (mM):NaCl 140, KCl 5.4, MgCl2 1.2, HEPES 10, EGTA 2, glucose 10 (pH adjusted to 7.4 with NaOH). The pipette solution comprised (mM): KCl 140, MgCl2 1, EGTA 10, HEPES 10, glucose 5, Na2ATP 0.3, and MgADP 0.5 (pH=7.2)
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The membrane potential recordingThe bath solution contained (mM):NaCl 140, KCl 5.4, MgCl2 1.2, HEPES 10, EGTA 2, glucose 10 (pH adjusted to 7.4with NaOH). The pipette solution comprised (mM): KCl 140, MgCl2 1, EGTA 10,HEPES 10, glucose 5, nystatin 250 μg/ml
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④ IK,M------(KCNQ2-KCNQ3)
the M-current characterized as a low-threshold, voltage-gated K+ current that is active around the resting membrane-potential and constitutes an important regulator of neuronal excitability
a voltage-and time-dependent, low-threshold, slowly activating current.
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Traditional M-currentvoltage-clamp protocol: holding potential was -30 mV; step potentials were -40 to -80 mV
Activation from a holding potential of -60 mV in 5 mV increments.
IKM tail
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0.3 nA, 200-ms current-pulse injection
M- current
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Predominant K+ current in many mature neurons that is initially activated at the subthreshold range of membrane potential and deactivated during depolarizing pulses of duration.
⑤Transient inactivating A-type current (IA)
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50ms
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14 (A1 ), 10 (A2 ), or 30 (A3 ) pA for 800 ms
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⑥ Kca
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BKSKIK
voltage gated and calcium modulated
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NATURE 2004,427(26)
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voltage-clamped at-30mV and pulsed for 400 ms from-100mV to +140mV For the perforated-patch experiments, a stock solution of 100 mg/ ml amphotericin B and final concentration of 200 mg/ml amphotericin The pipette solution contained (in mM): 140 KCl, 10 NaCl, 2 MgCl2, 0.7 CaCl2,1 EGTA and 10 HEPES (made to pH 7.3 with KOH). Cells were perfused with anextracellular buffer containing (in mM): 140 NaCl, 2.5 KCl, 0.5 MgCl2, 1.2 CaCl2,101 HEPES and 5 glucose (made to pH 7.4 with NaOH).
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Journal of physiology
a holding potential of –70 mV to test potentials between –50 and +80 mV in 10-mV increments.
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To obtain steady-state KCa channel measurements in perforated patch-clamp experiments, Ca2+ sparks and thus transient KCa currents were abolished using thapsigargin (100 nM), an inhibitor of sarcoplasmic reticulum Ca2+-ATPase
the bathing solution contained (in mM) 134 NaCl, 6 KCl, 2 CaCl2, 1 MgCl2, 10 HEPES, and 10 glucose (pH 7.4, NaOH). The perforated-patch pipette solution contained (in mM) 110 potassium aspartate, 30 KCl, 10 NaCl, 1 MgCl2, 10 HEPES, and 0.05 EGTA (pH 7.2 with KOH). Amphotericin B (1 μg/ml)
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For inside-out recordings, the pipette solution contained (in mM) 140 NaCl, 6 KCl, 10 HEPES, 1 CaCl2, and 2 MgCl2. The inside-out bath solution contained (in mM) 140 KCl, 10 HEPES, 2 MgCl2, 5 or 1 EGTA, 1.6 HEDTA, and appropriate amounts of CaCl2 to obtain free Ca2+ concentrations of 0.1, 0.3, 1, 3, 10, 30, or 100 μM.