Measurement of Ion Concentration in the Unstirred Boundary Layer with Open Patch-Clamp Pipette: Implications in Control of Ion Channels by Fluid Flow

This submittal can help answer key questions in the electro-physiologic sphere, such as the fluid flow in just the regulations of ion channels. The main advantage of this technique is that it can estimate the actual ion concentration in the unstirred boundary layer of the membrane surface for data interpretation related to fluid flow regulation of ion channels. To begin this procedure, bend the fired-glass capillary tube to form a U shape.

The inner diameter of the capillary should be large enough for reducing series resistance when recording large ion currents. Next, dissolve three grams of agarose in 100 milliliters of three-molar potassium chloride, and place it on a hot plate between 90 and 100 degrees Celsius. Then, load the bridge with the potassium chloride agarose, by immersing the glass bridge in the solution.

Keep it overnight at room temperature for the agarose to set and harden. The next day, carefully dig out the agarose potassium chloride-loaded glass bridge from the hardened agarose salt. Store the bridge in a wide-necked bottle of three-molar potassium chloride in the refrigerator.

In this procedure, place a container loaded with bathing solution, above the patch-clamp chamber. Next, fill the patch-clamp chamber with the bathing solution by suctioning the tube. To stop the fluid flow, clip the tube at the container's side, to block the fluid flow.

Then clip the tube at the suction side to stop the suction at the same time. This is the stationary control condition. To apply fluid flow shear force, open both tubes on the container and suction sides at the same time.

Before or after applying the fluid flow shear force to the cell, calculate the flow rate in milliliter per minute by measuring the decrease in fluid volume over a given time. To measure changes in liquid-metal junction potential, prepare a normal physiological-salt saline for the bathing chamber, and compare them in the absence and presence of agar potassium chloride bridge. Next, place a patch pipette containing three-molar potassium chloride solution in the chamber, to minimize the junction potential shift between the pipette and bathing solutions.

Then set the voltage clamp amplifier to the current clamp mode. After nullifying the initial offset potential, measure the changes in voltage induced by varying the flow rates. To verify that the changes in voltage are liquid-metal junction potentials, re-examine the effect of fluid flow on the junction potential, using the agarose salt bridge between the bath solution and the reference electrode.

With the results of the changes in liquid-metal junction potential, draw the function potential flow rate relationships, and estimate the saturating value of junction potential shift by the supra-fluid flow rate. Then, change the chloride concentration in the bathing fluid, and draw the junction potential chloride concentration relationship. Note that the fluid rate should be constant, and sufficiently high to prevent the decrease of chloride concentration to that of the adjacent silver-silver chloride reference electrode.

From the two relationship curves, estimate the changes in chloride concentration from the measured junction potential shift. VDCC-L currents were recorded in the enzymatically dispersed rat mesenteric arterial myocytes, with nystatin perforated patch-clamp recording. With agarose potassium chloride bridge, the junction potential between the reference electrode and bathing solutions could be minimized, and the fluid flow increased the VDCC-L current's voltage independently.

However, when the silver-silver chloride reference electrode was directly connected to the bathing fluid, without an agarose potassium chloride bridge, the IV relationship in the presence of fluid flow shifted to the right, compared to that of the VDCC-L currents under a static condition. The fluid flow-induced increase of Kerr 2.1 currents, which were recorded in rat basophilic leukemia cells with the agarose bridge, can be explained by the unstirred layer effect. After its development, this technique paved the way for researchers in the over electro-physiology.

So it's for the regulation of fluid flow in the ion channel currents. In terms of electro-chemical phenomena, in the unstirred boundary layer at cell membrane surface.