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

3. 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+ K+K+ K+K+ K+K+ K+K+ K+K+ K+K+ K+K+ Outside Inside -60 mV -15 mV

4. Electrophysiology –extracellular recording –intracellular recording –whole-cell recording –single channel recording

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

6. Patch Clamp Recording Technique

7. Types of K + Channels l Voltage-gated l Inward Rectifying l Ca 2+ sensitive l ATP-sensitive l Mechano-sensitive l Type A l Receptor-coupled

8. Classification of K + Channels

10. 1. Voltage-gated l 6 transmembrane domains l 4 subunits surround central pore (S5 & S6 regions of each subunit l Selectivity filter (P region) –Hydrophobic sequence between last 2 TMD; contains Gly-Tyr-Gly l Voltage sensor (S4) has multiple positively charged amino acids

11. Voltage-gated con’t l Activated by depolarization l Present in both excitable and nonexcitable cells l Functions –Regulate resting membrane potential –Control of the shape and frequency of action potentials

12. Voltage Dependent Gating Outside Inside S1S2S3S4S5S6 HO 2 C H2NH2N LRVIRLVRVFRIFKLSRHS + + +

13. 1. Three Types Ca 2+ Sensitive K + Channels l High conductance (BK) channels (Slo) –Gated by internal Ca 2+ and membrane potential –Conductance = 100 to 220 picoSiemens (pS) –Blocked by charybdotoxin and iberiotoxin l Intermediate conductance (IK) channels (SK4) –Gated only by internal Ca 2+ –More sensitive than BK channels –Conductance = 20 to 85 pS –Blocked by charybdotoxin l Small conductance (SK) channels (SK1-3) –Gated only by internal Ca 2+ –More sensitive than BK channels –Conductance = 2 to 20 pS –Blocked by apamin

14. BK channel

15. 2. K ATP channel l K ATP ATP increase-decrease channel opening Pancreatic type or cardiac type l K NDP NDP increase-increase channel opening in the presence of Mg 2+ smooth muscle type

16. K ATP characteristics l Octameric four  -subunit (KIR6.1 or KIR6.2) four b-subunit (SUR1, SUR2A, SUR2B) l Smooth muscle type KIR6.2/SUR2B l Sulfonylurea agents-glibenclamide, tolbutamide inhibit channel activity l Pharmacological K ATP activator pinacidil, cromakalim, lemakalim, diazoxide, minoxidil, nicorandil (induce hyperpolarization)

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

19. K ATP channel

20. 3. Inwardly Rectifying K + Channel (K IR ) l 2 transmembrane regions (M1 & M2) –Corresponds to S5 & S6 in Kv channel l 4 subunits surround central pore l P region separates M1 and M2 l Non-conducting at positive membrane potentials l Maintains resting membrane potential near E k l Blocked by external Ba ++ l Mainly Kir2x

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

22. 4. K2P CHANNELS l TWIK: Tandem pore domain Weak Inwardly rectifying K + channel l TREK: TWIK-RElated K + channel l TRAAK:TWIK-Related Arachidonic acid- Activated K + channel l TALK: TWIK-related ALkaline-activated K + channel l TASK: TWIK-related Acid-Sensitive K + channel

23. TREK channels

24. A. on-cell, 0mV, asymmetrical K + NEGATIVE PRESSURE ACTIVATES SDK CHANNEL (murine colonic myocyte) B. Pr. and Po relation -20cmH 2 O -40cmH 2 O -20cmH 2 O -40cmH 2 O I-O -60cmH 2 O -80cmH 2 O 10 pA 10 sec 0.8 0.6 0.4 0.2 -80 1 -60-40-200 0 cmH 2 O Probability density Should be K + conductance

25. SDK CHANNEL ACTIVATED BY INCREASE CELL LENGTH B A -60 cm H 2 O C 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.

26. 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

27. 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.

28. Table 3. A-type channel and accessory subunit expression in smooth muscle Smooth MuscleTranscript Protein Rat mesenteric arteryKv1.4, Kv3.3, Kv3.4, Kv4.2, Kv4.3, Kv 1- 3 Rat tail arteryKv1.4, Kv3.3, Kv3.4, Kv4.2, Kv4.3, Kv 1- 3 Rat pulmonary arteryKv1.4, Kv4.1-4.3 Rat aortaKv4.3L Rat vas deferensKv4.3L > Kv4.2 Rat urinary bladderKv4.3L Rat myometriumKv4.3L > Kv4.2 Kv4.1 Kv4.3 Rat stomachKv4.3L Rat colonKv4.3L Mouse fundusKv4.1, Kv4.2, Kv4.3L, NCS-1, KChIP1, 3, 4 Kv4.2, Kv4.3 Mouse antrumKv4.3L > Kv4.2 > Kv4.1, NCS-1, KChIP1, 3, 4 Kv4.2, Kv4.3 Mouse jejunumKv4.3L > Kv4.2 > Kv4.1, NCS-1, KChIP1 > KChIP2-4 Kv4.2, Kv4.3 Mouse colonKv4.3L > Kv4.2 > Kv4.1, NCS-1, KChIP1 > KChIP2-4 Kv4.3 > Kv4. 2 Kv, voltage-gated Ca 2+ -independent K + current; NCS, neuronal Ca 2+ sensor; KChIP, K + channel-interacting protein.

29. 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

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

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

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

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

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

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

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

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

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

39. CaMKII-like immunoreactivity in mouse proximal colon

41. Quantification of Kv4 transcripts in colon and jejunum

42. Inhibition of colonic A-type current by flecainide

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

44. Quantification of KChIP transcripts in colon and jejunum

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

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

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

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

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

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

51. 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