Figure 8
Experimental configuration. (a) Top view of bath setup for loose patch clamp. Scheme showing the bath obturator insert with pinned muscle preparation. Holes allow insertion of the bath sense and ground electrodes, used to actively clamp the bath potential, and tubes for solution exchange. They are continuous with the bath through cut-outs below the surface, demarcated by the dashed lines. (b) Side view of loose patch clamp setup showing micromanipulator-mounted headstage with recording pipette, holder, suction syringe and solution exchanger. (c) Equivalent circuit for loose patch clamp electrode on muscle membrane. The solution in the pipette, of resistance Rpip, is clamped at pipette voltage, Vpip. The voltage error arising from current flow through the patch, represented by patch resistance Rpatch and the cell membrane resistance Rcell, along with the pipette resistance (Rpip) is corrected by bridge circuit in the loose patch clamp amplifier9,10. The cell is at its resting membrane potential, RMP, represented by the resting voltage Vrest during establishment of the patch. (d) Voltage clamp step protocol consisting of hyperpolarizing pre-pulse to (RMP − 40) mV followed by a variable test pulse starting at − 40 mV altered in 10 mV increments with successive pulse cycles to a test voltage of (RMP + 120) mV, followed by test steps in the reverse order from (RMP + 120) to (RMP − 40) mV.
