1. Second-Messenger Gated Ion Channels Membrane Biophysics, 2014 Ion Channel Presentation Vehpi Yildirim and Joe McKenna

2. Overview ● Stimulus triggers iIntracelluar signal that modulates channel activity ● Examples – G-protein coupled channels – IP 3 -regulated channels – Adenine nucleotide-sensitive channels

3. Examples ● G-protein coupled inward rectifying K + channel ● IP 3 -regulated Ca 2+ release from ER

4. Review Article

5. ATP-sensitive K + Channels ● Link cellular energetics and excitability ● Gate efflux of K + – Inward rectifier – Shallow voltage-dependence ● Inhibited by ATP, activated by Mg 2+

6. K ATP Architecture ● Functional Octamer ● Kir 6.2: 4 sub-units – Channel pore – Site of ATP inhibition ● Sulphonylurea Receptor (SUR): 4 sub-units – Site of Mg 2+ activation

7. Kir 6.2/SUR Model ● Extrapolated from – Bacteria K + channel crystal – Prokaryotic Kir – Targeted mutation ● ATP binds at interface of SUR NBF1 & 2 (b. green)

8. Mechanism of Gating ● Fast ligand-independent gating by ion selectivity filter ● Ligand-dependent gating by hinged motion of M2 – Inhibited by ATP – Activated by PIP 2, MgADP

9. Gating Kinetic Model ● Fast ligand-independent gating and slow ligand- dependent gating ● One subunit in closed configuration → channel closed ● Two ways to achieve same half-maximal inhibition

10. K ATP -related disease ● Pancreatic beta-cells – Loss of function mutation →Hyperglycemia/diabetes – Gain of function mutation → Hyperinsulinemia ● Coronoary cells – Loss of function mutation → spontaneous contraction, early death

11. Identification and Properties of an ATP-Sensitive K + Current in Rabbit Sino-Atrial Node Pacemaker Cells X. Han, P. E. Light, W. R. Giles and R. J. French Journal of Physiology (1996), 490.2, pp.337-350

12. INTRODUCTION K (ATP) channles have been identified in many cell types. Most studies use myocytes from atrium. Here they use cells from sino-atrium node.

13. Questions to be Answered Are K (ATP) channels present in SA node and, if so, what are their single channel properties? Can physiological, pharmacological and pathological conditions which are known to activate K (ATP) channels alter SA-node activity?

14. METHODS Isolated single cells from SA node of rabbit heart are studied by measuring spontaneous activity. Both whole cell and single channel currents are measured. Pharmacological blockers or openers are used.

15. Ventricular myocytes also isolated to compare results from different regions of heart. Perforated patch technique for Whole cell. Inside-out configuration for single channel.

16. RESULTS

17. Glibenclamide: K (ATP) channel blocker. Acts on SUR subunit. Cromakalim and Pinacidil: K (ATP) channel openers. Act on SUR subunit.

18. Effects of glibenclamide on electrical activity and ion curents.

19. Effects of K ATP channel openers on the Current

20. Effect of metabolic inhibition by NaCN NaCN (Sodium Cyanide) : inhibits ATP production.

21. Properties of Single K ATP Channels

22. Effects of drugs on single channel activity. Effects with high ATP concentration.

23. Open and Close Times

25. Neonatal Diabetes (NDM) Overview ● Presents within first 3 months of life, requires insulin treatment ● Insulin response to sulphonylureas but not glucose or glucagon ● May result from Kir 6.2 gain of function mutations in pancreatic beta-cells

26. K ATP Channels and NDM ● Glucose → ATP → channel closure → Ca 2+ influx → Insulin secretion

27. NDM Patient Screening ● Patients with known diabetes- related mutations excluded ● Physical exam including insulin, sulphonylurea challenges ● Kir 6.2 gene sequenced ● Identified 6 novel mutations ● NDM seen only in patients with Kir6.2 mutations

28. Kir6.2 Affected Residues ● Highly conserved regions → functional role ● Near ATP-binding site or slide helix

29. Patient Response to Secretagogues ● 3 patients with mutations in ATP binding site (ABS) – No secretion from glucose – Secretion from K ATP channel opener

30. K ATP Channels in Oocytes ● Channels with mutated ABS residues – Larger current in steady [ATP] – Current increased by sulphonylurea – Weakly inhibition by ATP

31. K ATP Channels in Oocytes ● NDM pathology more severe in homozygote mutants – Significant difference in half-maximal activation by ATP

32. Conclusion ● Activating mutations in Kir6.2 causes NDM ● Found in 34% of patients with NDM ● Accompanying complications point to vital role of KATP channels in brain and muscle ● Potential therapy: channel blocker acting on SUR receptors

33. Defective Insulin Secretion and Enhanced Insulin Action in K ATP Channel Deficient Mice Takashi Miki, Kazuaki Nagashima, Fumi Tashiro, Kazumi Kotake, Hideyuki Yoshitomi, Atsuko Tamamoto, Tohru Gonoi, Toshihiko Iwanaga, Jun-ichi Miyazaki, And Susumu Seino PNAS Vol. 95, pp. 10402-10406, September 1998, Biochemistry

34. INTRODUCTION K ATP Channels in pancreatic Beta Cells comprise Kir6.2 and SUR1 subunits. K ATP Channels are ATP and ADP sensors and play a very important role in insulin secretion. Mutations in regulatory genes cause hypoglycemia. Here they use Kir6.2 -/- mice to study the role of K ATP channels in insulin secretion.

35. Kir6.2 +/+ and Kir6.2 -/- cells are dialyzed with ATP-free pipette solution.

36. Glucose or Tolbutamide does not effect [Ca] in Kir6.2 -\- cells. AcetylCholine and High K + does effect [Ca] like in wild type cells. Showing voltage gated Ca channels and IP3 sensitive Ca stores are functioning normally in Kir6.2 -\- cells.

37. A rapid rise in Ca concentration is needed for glucose induced insulin secretion, rather than a continuous elevated [Ca]. In Kir6.2 -/- mice, only a small first phase and no second phase secretion observed. (In Vitro)

38. Glucose induced insulin secretion is reduced in knock-out mice. But surprisingly glucose lowering effect of insulin is significantly increased in knock-out mice.

39. Kir6.2 +/+ Kir6.2 -/- BetaAlpha

40. CONCLUSION K ATP channels play a significant role in insulin secretion. Glucose metabolism itself is insufficient for glucose- induced and sulfonylurea-induced insulin secretion, both of which require the rapid rise in [Ca2] caused by closure of the KATP channels.