Planar patch clamp (Mergler)

Question 1

Galvani experiments

Answer 1

Studied contraction of frog legs without metals
Did experiments with metallic arcs
1st demonstrated propagation of an AP

Question 2

Bernstein experiment/finding

Answer 2

1st recording of an AP in the nerve

“Negative schwankung”

Question 3

Hodgkin & Huxley findings

Answer 3

1st intracellular recordings of AP in squid axon

Question 4

What can voltage clamp be used for?

Answer 4

Intracellular recordings from individual cells

Extracellular recordings of currents generated by collective activity of many cells

Question 5

Dimensions of voltage clamp glass micropipette

Answer 5

Tip should be 20 um (relatively wide center/no sharp inside of tip, not thick walled)
2-2.5 MOhm resistance
1 um^2 tip opening area
Width of rim approximately 0.2 um
24º

Question 6

Thick-walled pipettes

Answer 6

8-10 MOhm resistance
Walls are not actually that much thicker than soft glass, but have extra inwand curvature, allowing for larger seal area in experiments
Angle 8-12º

Question 7

Soft glass pipettes

Answer 7

2-2.5 MOhm resistance
2-2.5 MOhm resistance
1 um^2 tip opening area
Width of rim approximately 0.2 um
24º

Question 8

What type of clamp is planar patch in?

Answer 8

Voltage clamp

Question 9

Patch clamp set up

Answer 9

Patch pipette connected to electrode
Other side has bath electrode (connected to earth)

Crucial part of set up = patch tip touching cell surface

Question 10

Automated patch clamp systems

Answer 10

Ionflux system

• Has microfluidic network connecting cells (cathode plate)
• Cells are trapped in an ensemble array with a main flow channel, compound introduction channels, and cell trapping zones

Patchliner → fully automated patch-clamp platform

Cytopatch

*Are all high throughput

Question 11

Step-by-step procedure for conventional, whole-cell patch configuration

Answer 11

1. Tip of glass pipette brought into contact with the adherent cell, applying positive pressure, allowing the intracellular solution to touch the cell
   → membrane under the pipette becomes imprinted with a concave curvature
2. Slight negative pressure/suction applied, pulling the membrane into the tip opening → gigaseal formation
3. Apply more negative pressure or short voltage pulses, rupturing the membrane patch under the tip of the pipette
   (→4. Intracellular access for clamping and detecting the current flowing through ion channels)

Question 12

Step-by-step procedure for planar whole-cell patch

Answer 12

1. Suction on microopening, attracting suspended cell (micro-opening both serves for suction application to attract suspended cell and for sealing/recording)
2. Sealing by cell adhesion in the planar contact area surrounding the opening and along the wall of the channel penetrating the substrate
3. More suction makes membrane rupture
4. Recording

Question 13

Step-by-step procedure for cytocentering

Answer 13

1. Move suspended cells by fluid flow to a suction opening
2. Suction opening brings membrane patch into contact with micro-machined patch contact inside of the opening
3. Placement of contact tip → imprinting w/ concave curvature on membrane under tip
4. Pressure applied → gigaseal formation
5. Suction to break membrane
6. Recording

Question 14

What are the different conventional patch clamp configurations and what can we record with them?

Answer 14

1. On-cell
2. Whole-cell → can record currents through multiple channels simultaneously
3. Inside-out → Can change chemical composition of inside surface of membrane
4. Outside out→ Can change chemical composition of outside surface

Question 15

What configurations can we have with planar patch clamp and what kinds of cells can we use?

Answer 15

Only use cells in suspension

Can have on-cell and whole-cell configuration

Question 16

Which patch method requires the most skill level?

Answer 16

Conventional requires greater skill level

Question 17

Benefits of planar over conventional

Answer 17

• Easier to seal for whole-cell
• Less skill required
• Not vulnerable to unwanted electrical noise
• Greater throughput
• Less vulnerable to physical disturbances
• Faster extracellular solution exchange
• Less/not vulnerable to contamination between extracellular and intracellular solutions

Question 18

TRPV1 role in dry eyes syndrome study findings

Answer 18

Calcium imaging in corneal cells + planar patch showed CAP increased whole cell currents → inward currents blocked by L-Carnitine

Hypertonic solution led to an increase in intracellular [Ca2+]

CAP induced IL-6 and IL-8 secretion

Treating TRPV1 w/ L-Carnitine reduced dry eye syndrome

Question 19

Pathomechanism of dry eye syndrome

Answer 19

disordered tear film productions

Upregulated cytokines, ILs, and immune cell reactivity