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K + Channels

K + Channels. 4/12/05, MCB610. -15 mV. -60 mV. K Channel Gating. K +. K +. K +. K +. K +. K +. K +. Outside. Inside. K +. K +. K +. K +. K +. K +. K +. K +. K +. K +. K +. Electrophysiology extracellular recording intracellular recording whole-cell recording

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K + Channels

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  1. K+ Channels 4/12/05, MCB610

  2. -15 mV -60 mV K Channel Gating K+ K+ K+ K+ K+ K+ K+ Outside Inside K+ K+ K+ K+ K+ K+ K+ K+ K+ K+ K+

  3. Electrophysiology • extracellular recording • intracellular recording • whole-cell recording • single channel recording

  4. How to Study? “Patch Clamp” Nobel Prize in Physiology & Medicine -1991 Extracellular Inside Cell

  5. Patch Clamp Recording Technique

  6. Types of K+ Channels • Voltage-gated • Inward Rectifying • Ca2+ sensitive • ATP-sensitive • Mechano-sensitive • Type A • Receptor-coupled

  7. Classification of K+ Channels

  8. 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

  9. 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

  10. + + + + + + LRVIRLVRVFRIFKLSRHS Voltage Dependent Gating Outside S1 S2 S3 S4 S5 S6 Inside HO2C H2N

  11. 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

  12. BK channel

  13. 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

  14. 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)

  15. Endocrine Reviews 20 (2): 101-135Molecular Biology of Adenosine Triphosphate-Sensitive Potassium Channels. Lydia Aguilar-Bryan and Joseph Bryan

  16. KATP channel

  17. 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

  18. Increasing extracellular K+ induced shortening of cardiac action potential. Mg, PA

  19. 4. K2P CHANNELS • 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

  20. TREK channels

  21. Should be K+conductance NEGATIVE PRESSURE ACTIVATES SDK CHANNEL(murine colonic myocyte) A. on-cell, 0mV, asymmetrical K+ B. Pr. and Po relation -40cmH2O -20cmH2O -20cmH2O 1 0.8 -60cmH2O -60cmH2O -40cmH2O 0.6 Probability density 0.4 0.2 I-O -80cmH2O 0 -80 -60 -40 -20 0 cmH2O 10 sec 10 pA

  22. Cell Elongation SDK CHANNEL ACTIVATED BY INCREASE CELL LENGTH A B Stimulus of negative pressure does not necessarily stimulate the effects of cell stretch. C -60 cm H2O 2sec 10 pA Cells were actually elongated and activated K+ channels with the same properties as those activated by negative pressure.

  23. 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). Ingastrointestinal (GI) smooth muscle cells of the mouse (fundus, antrum, jejunum, and colon), rat (ileum), guinea pig (colon), and opossum (esophagus

  24. 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.

  25. Figure 2. Effect of TEA on the electrical activity of intact murine colonic smooth muscle Figure 1. Effect of 4-AP on the electrical activity of intact murine colonic smooth muscle

  26. Voltage dependence of inactivation and activation of delayed rectifier K+ currents Determination of the reversal potential

  27. The recovery from inactivation of delayed rectifier K+ current

  28. Inhibition of delayed rectifier K+ currents by 10 mM TEA Effect of 5 mM 4-AP on delayed rectifier K+ current

  29. mRNA expression of Kv1, Kv4 and Kv subunits in murine proximal colon circular smooth muscle cells

  30. The effect of intracellular Ca2+ buffering on inactivation of A-type currents

  31. The effect of KN-93 on inactivation time constants of A-type currents

  32. The effect of KN-93 on the voltage dependence of inactivation of A-type currents

  33. The effect of KN-93 on recovery from inactivation of A-type currents

  34. The effect of dialysis with autothiophosphorylated CaMKII on A-type currents

  35. CaMKII-like immunoreactivity in mouse proximal colon

  36. Quantification of Kv4 transcripts in colon and jejunum

  37. Inhibition of colonic A-type current by flecainide

  38. Kv4.2- and Kv4.3-like immunoreactivity in the tunica muscularis of murine colon and jejunum

  39. Quantification of KChIP transcripts in colon and jejunum

  40. Autothiophosphorylated Ca2+/calmodulin-dependent protein kinase II (CaMKII) decreases the rate of inactivation of voltage-dependent K+ channel 4.3 (Kv4.3) currents.

  41. Autothiophosphorylated CaMKII produced a positive shift in voltage-dependent activation and inactivation.

  42. Autothiophosphorylated CaMKII accelerates the recovery from inactivation of Kv4.3 currents.

  43. Effect of mutagenesis on specific CaMKII consensus sequences on Kv4.3 currents.

  44. Effect of C2 mutagenesis on the rate of recovery from inactivation.

  45. Effect of C2 mutagenesis on Kv4.3 channel inactivation kinetics in response to application of autothiophosphorylated CaMKII

  46. 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

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