1. Eaton Lab Innovation & Conventional patch clamping cell

2. Methods: Voltage clamp (single channel patch clamp analysis) The patch clamp set up Pipet holder micromanipulator Inverted microscope Images taken from http://www.sciencedisplay.com/patchclamp.html

3. Pipet is approaching cell of interest

4. Forth coming contact of the pipet tip with the cell surface can be monitored with the oscilloscope MONITOR current time Send out a test (+20mV) current pulse

5. To get a seal, tight contact of the pipet with plasma membrane, a soft suction has to be applied MONITOR current time

6. Suction of the patch pipet causes deformation of the cell membrane and the building of a seal. MONITOR current time

7. Ion channels in the cell membrane can be investigated. MONITOR current time All proteins are in their natural environment. The patch is so small a single channel can be investigated. C C Na+ 1 2 1 2 N

8. N- terminus C C Na+ 1 2 1 2 N N- terminus N The Eaton Lab investigates: Primary cells:Cell lines: Human lung Rat lung Murine lung Xenopus kidney, murine kidney, chinese hampster ovary, murine alveolar lung…. Ion channels: ENaC (sodium) CFTR (chloride) -Highly selective -Non-selective

9. 1.Anesthetize and sacrifice animals in accordance with Institutional Animal Care and Use Committee. 2. Perfuse lungs to remove intravascular blood elements. 3. Agarose fill lungs to stabilize delicate tissue. 4. Section 250  m slices; horizontal plane across the middle lobes. Eaton Lab Innovation 1. Novel In situ patch clamp

10. -V P (mV) G Na =4.4pS I (pA) Figure2. I (pA) G Na =13.5pS -V P (mV) Highly Na + Selective Channel (HSC) Nonselective Cation Channel (NSC) Helms et al A. B. C. +20mV -20mV 0mV 5s 1pA -60mV -80mV -40mV

11. Eaton Lab Innovation 2. Novel Identification of CFTR chloride channels in lung cells. pipet Single channel recording with 100  M amiloride back-filled in pipet

12. CTR SODi 15  M NONOate 2mM NONOate +O 2 Eaton Lab Innovation 3. Complex drug delivery protocols. control Continual patch clamp recording in one A6 cell with multiple drug treatments. + superoxide dismustase inhibitor + low concentration nitric oxide + high concentration nitric oxide increase O 2 -