ca-1 Data

Experimental data obtained using a CA-1 by Dr. Enrico Stefani in collaboration with Dr. Francisco Bezanilla, both with the UCLA School of Medicine

Cut Open Oocyte Vaseline Gap Technique

Xenopus oocytes are widely used to express ionic channels from MRNA and to record the resulting ionic currents. Two main electrophysiological techniques are presently used: a) the two microelectrode voltage clamp for the recording of whole oocyte currents, and b) the patch clamp for the study of single channels or multichannel currents. We describe the development of a new cut-open oocyte method that allows a fast clamp with low noise and the possibility to control the intracellular components. In addition, with this technique stable recordings (0.5 to 2 hrs) could be obtained. The following are examples of different recordings which can be obtained from expressed ion channels. The technique is particularly suitable to record fast ionic currents and gating currents.

FIGURE 1 SCHEMATIC DIAGRAM OF THE EXPERIMENTAL CHAMBER AND ELECTRONICS.

A: An oocyte is shown in contact with the internal, external and guard compartments. The internal solution can passivity diffuse or can be directly pumped into the oocvte through a metal perfusion cannula. The system consists in three voltage clamps. Amplifiers 1 and 3 clamp the external and the guard compartment to the command pulse. Amplifier 5 clamps the oocyte interior facing the external pool to ground. Amplifier 4 records the tansmembrane potential while amplifier 2 records the transmembrane currents. Since the guard and external compartment are actively clamped to the same command potential, no current can flow between these two compartments through the vaseline seals.

FIGURE 2 PERFOFIMANCE OF THE INTERNAL PERFUSION SYSTEM

Values of peak K currents (ShakerB IR) are plotted as function of time. The curves show the time course of replacement of internal K by N-methylglucamine.

FIGURE 3 EXAMPLES OF EXPRESSED K IONIC CURRENTS FROM DRK1 CLONES.

A: Traces show the speed of the capacity transient for a 20 mV pulse. Note the virtual absence of leak currents.

B: Outward K currents from -70 mV in 10 mV increments to +50 mV. Unsubtracted records. The initial upward points correspond to the capacity transient that is followed by the ionic current.

C: Instantaneous I-V relationship to illustrate the instantaneous current jump during a constant 20 mV pulse after first stimulating pulses to various potentials. The size of this jump is proportional to the conductance.

D: Instantaneous I-V relationship with a constant first pulse to 0 mV followed by pulses to different potentials. Note the instantaneous current jump during the post-pulse and the well defined K reversal potential.

FIGURE 4 K GATING CURRENTS IN SINGLE TRACES AND WITHOUT SUBTRACTION PROTOCOLS.

The clamp is fast enough (20-80 us settling time) that allows the recording of gating currents after charging the linear capacity. The figure shows that gating currents obtained with the P/-4 subtracting method (B,D) (from -1 20 mV) had similar properties to unsubtracted gating currents (A,C). This indicates the adequacy of the subtracting procedure and the lack of non-linear charge movement in the subtracting pulses. Gating currents had no rising phase for small depolarizations (-70 mV), and a prominent rising phase for larger depolarizations (-30 to 10 mV). For different duration pulses (C and D) the rising phase in the OFF became noticeable with pulses of longer durations.

FIGURE 5 SHAKERB GATING CURRENTS SHOW A RISING PHASE.

Unsubtracted current traces to different depolarizing potentials show the initial capacity transients followed by the K gating currents. Gating currents had a rising phase for larger depolarizations.

CONCLUSIONS

The new system "cut-open oocyte vaseline-gap voltage clamp' can perform as a fast clamp (10-50 Khz) and allows the perfusion of intracellular solutions. The main features of this technique are: 1) low current noise (1nA at 3 Khz), 2) control of the ionic composition of both the internal and external media, 3) fast time resolution (20 to 1 00 lisec time constant of decay of the capacity transient) and 4) stable recordings for several hours.1 ILA These features allow reliable measurements of gating currents and fast ionic currents. Furthermore, this system can be used with high level of channel expression to record gating currents in single and unsubtracted traces. This method should be valuable to study structure-function relationships of cloned ionic channels and as a read-out system for expression-cloning.

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Dagan Corporation Minneapolis MN USA 612-827-5959 (FAX 612-827-6535) Email: info@dagan.com 4/4/97

Xenopus oocytes are widely used to express ionic channels from MRNA and to record the resulting ionic currents. Two main electrophysiological techniques are presently used: a) the two microelectrode voltage clamp for the recording of whole oocyte currents, and b) the patch clamp for the study of single channels or multichannel currents. We describe the development of a new cut-open oocyte method that allows a fast clamp with low noise and the possibility to control the intracellular components. In addition, with this technique stable recordings (0.5 to 2 hrs) could be obtained. The following are examples of different recordings which can be obtained from expressed ion channels. The technique is particularly suitable to record fast ionic currents and gating currents.
	FIGURE 1 SCHEMATIC DIAGRAM OF THE EXPERIMENTAL CHAMBER AND ELECTRONICS. A: An oocyte is shown in contact with the internal, external and guard compartments. The internal solution can passivity diffuse or can be directly pumped into the oocvte through a metal perfusion cannula. The system consists in three voltage clamps. Amplifiers 1 and 3 clamp the external and the guard compartment to the command pulse. Amplifier 5 clamps the oocyte interior facing the external pool to ground. Amplifier 4 records the tansmembrane potential while amplifier 2 records the transmembrane currents. Since the guard and external compartment are actively clamped to the same command potential, no current can flow between these two compartments through the vaseline seals.
	FIGURE 2 PERFOFIMANCE OF THE INTERNAL PERFUSION SYSTEM Values of peak K currents (ShakerB IR) are plotted as function of time. The curves show the time course of replacement of internal K by N-methylglucamine.
	FIGURE 3 EXAMPLES OF EXPRESSED K IONIC CURRENTS FROM DRK1 CLONES. A: Traces show the speed of the capacity transient for a 20 mV pulse. Note the virtual absence of leak currents. B: Outward K currents from -70 mV in 10 mV increments to +50 mV. Unsubtracted records. The initial upward points correspond to the capacity transient that is followed by the ionic current. C: Instantaneous I-V relationship to illustrate the instantaneous current jump during a constant 20 mV pulse after first stimulating pulses to various potentials. The size of this jump is proportional to the conductance. D: Instantaneous I-V relationship with a constant first pulse to 0 mV followed by pulses to different potentials. Note the instantaneous current jump during the post-pulse and the well defined K reversal potential.
	FIGURE 4 K GATING CURRENTS IN SINGLE TRACES AND WITHOUT SUBTRACTION PROTOCOLS. The clamp is fast enough (20-80 us settling time) that allows the recording of gating currents after charging the linear capacity. The figure shows that gating currents obtained with the P/-4 subtracting method (B,D) (from -1 20 mV) had similar properties to unsubtracted gating currents (A,C). This indicates the adequacy of the subtracting procedure and the lack of non-linear charge movement in the subtracting pulses. Gating currents had no rising phase for small depolarizations (-70 mV), and a prominent rising phase for larger depolarizations (-30 to 10 mV). For different duration pulses (C and D) the rising phase in the OFF became noticeable with pulses of longer durations.
	FIGURE 5 SHAKERB GATING CURRENTS SHOW A RISING PHASE. Unsubtracted current traces to different depolarizing potentials show the initial capacity transients followed by the K gating currents. Gating currents had a rising phase for larger depolarizations.
CONCLUSIONS The new system "cut-open oocyte vaseline-gap voltage clamp' can perform as a fast clamp (10-50 Khz) and allows the perfusion of intracellular solutions. The main features of this technique are: 1) low current noise (1nA at 3 Khz), 2) control of the ionic composition of both the internal and external media, 3) fast time resolution (20 to 1 00 lisec time constant of decay of the capacity transient) and 4) stable recordings for several hours.1 ILA These features allow reliable measurements of gating currents and fast ionic currents. Furthermore, this system can be used with high level of channel expression to record gating currents in single and unsubtracted traces. This method should be valuable to study structure-function relationships of cloned ionic channels and as a read-out system for expression-cloning.