1 / 40

Regulation of ion channels by lipid signaling

Regulation of ion channels by lipid signaling. Hana Cho Department of Physiology, Sungkyunkwan University School of Medicine. acetylcholine. GIRK. OUT. M 2. M 2. bg. a. bg. IN. a. GTP. K +. GDP. GIRK channels in atrial myocytes. acetylcholine. ACh. ACh.

tad
Download Presentation

Regulation of ion channels by lipid signaling

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. Regulation of ion channels by lipid signaling Hana Cho Department of Physiology, Sungkyunkwan University School of Medicine

  2. acetylcholine GIRK OUT M2 M2 bg a bg IN a GTP K+ GDP GIRK channels in atrial myocytes acetylcholine

  3. ACh ACh a b c d GIRK channels are regulated by membrane phospholipid, PIP2 500 pA 2 min Logothetis & Zhang. J. Physiol. 520:630, 1999

  4. PIP2: a substrate of phospholipase C DAG PIP2 IP3

  5. a DAG PLC Gq IP3 Is PIP2 a signaling molecule? GIRK PIP2

  6. Cardiac GIRK channels: Inhibited by GqPCRs via PIP2 depletion J Biol Chem 276:159 in a receptor-specific manner ; J Biol Chem 277: 27742; PNAS 102: 4643 ACh ACh ACh ET-1 BK PE

  7. Excitable cells endocrine cells neurons myocytes Electrical signals underly functions of excitable cells

  8. Ion channels/transporters are basis of electrical signals Ion current through ion channels electrical signal

  9. Electrical signals are measured by patch clamp technique and microfluorometry (Fura-2)

  10. Nobel Prize in Chemistry, 2008 The Nobel Prize in Chemistry in 2008 awarded Osamu Shimomura, Martin Chalfie and Roger Y. Tsien "for the discovery and development of the green fluorescent protein, GFP". Osamu Shimomura Martin Chalfie Fura-2 Roger Tsien

  11. Ca2+ imaging by Fura-2

  12. objective 505 nm Ca2+ signals can be studied using Fura-2 Ca2+ 340 & 380 nm PC AD/DA converter Xe & Monochromator Photodiode

  13. Ca2+ is a common second messenger for cellular output Ca2+ Shaping Ca2+ signals Cellular outputs contraction secretion

  14. Patch clamp? Patch; a small piece of cell membrane Patch Clamp; imposing on a membrane patch a defined voltage (" volt age-clamp") with the purpose to measure the resulting current for the calculation of the patch conductance  voltage- clamp of a membrane patch. Neher and Sakmann in 1976 Nobel prize; 1992

  15. Voltageclamp technique Current to voltage converter

  16. When the pipette touches the cell membrane and forms a high resistance seal (~1GOhm), you are in the "cell attached" recording configuration. 

  17. Whole Cell Recording

  18. Equipment

  19. Recording equipment • Patch-clamp set의 일반적 구성은 다음과 같다 • Optics; Microscope, TV camera & monitor • 2. Mechanics; Vibration isolation table, Micromanipulator • 3. Electronics; Patch-amplifier,, ADDA converter Computer • 4. Environment; bath • 5. 기타; Feraday cage, Photometry system

  20. Overview of the recording set-up: microscope, Micromanipulator, patch clamp amplifier, computer.

  21. A close-up view of the recording scope. The recording electrode is attached to the blue headstage.

  22. A close-up view of the microscope stage. The cells are plated on a glass coverslip that is adhered to the bottom of the plastic dish. The electrode holder does not have a pipette on it in this picture.

  23. Patch clamper is using the computer to run the patch clamp amplifier and collect data. 

  24. The top trace is the current response of the electrode in the bath. The bottom trace shows the small voltage step that is being applied repeatedly.

  25. As the seal begins to form, the horizontal lines of the current trace move closer together (compare to previous photo).

  26. Patch clamper is gently sucking on the back of the syringe that is connected to  the back of the pipette. This suction will help seal the tip of the pipette onto the cell.

  27. The amplifier is used to compensate, i.e. to electronically remove, the pipette capacitance from the current recording.

  28. After electrically removing the pipette capacitance, the vertical spikes are gone.

  29. To break into the whole cell recording mode, additional suction is applied to the syringe. As shown here, you'll know when a whole cell recording in acheived because the capacitative transients (the vertical "spikes" due to the cell capacitance) are large and have a slow decay. 

  30. Effect of phenylephrine on GIRK channels

  31. GIRK a M2 DAG PLC PIP2 Gq Gi/o IP3 Cardiac GIRK channels: Inhibited by GqPCRs via PIP2 depletion J Biol Chem 276:159 in a receptor-specific manner ; J Biol Chem 277: 27742; PNAS 102: 4643 ACh ACh ACh ET-1 BK PE

  32. Finally we can propose comprehensive model for GIRK channel regulation by Gq-coupled receptors

  33. ET-1 ACh ACh ET-1 5 pA 20 sec 2 pA 20 sec Recording GIRK current in cell-attached patches

  34. Fluorescence Recovery After Bleaching (FRAB) to measure diffusion of second messenger (PIP2) confocal image Slope = R0 [R 02 + 8Dt]-1/2 where R0 is the half-width (1/e2) of the bleach D=0.000390.000038 mm2/s (n=5)

  35. Cytoskeleton blocks the diffusion of PIP2

  36. Acknowledgement SKKU, SBRI Jin Young Yoon Sun Ok Choi Slee Lee Hyun Tae Cho SNU Dr. Won-Kyung Ho Dr. Suk-Ho Lee Jong Woo Shon Doyun Lee

More Related