Whole-cell configuration

The whole-cell configuration is achieved by breaking the cell membrane the buffer in the recording pipette is now in free exchange with the cytosol of the cells. 

The patch clamp technique suddenly became the workhorse of modern electrophysiology, and the whole-cell configuration became one of the most crucial and popular techniques for the biophysical and pharmacological study of ion channels. 

In an alternative, whole-cell configuration, the small patch of membrane is ruptured by suction or brief high voltage, resulting in continuity between the patch pipette solution and the cytosol. Diffusional equilibration occurs between the large volume of the pipette solution and the cytoplasm, so that within a few minutes the cytosolic ion composition is essentially that provided in the pipette solution, with the possible exception of ions for which the cell has a large buffering capacity, e.g. and Ca. In the whole cell... 

The inside-out patch technique is to retract the pipette that is in the cell-attached configuration so that a small vesicle of membrane remains attached. By exposing the tip of the pipette to an external electrolyte, it is possible to control the medium to which the intracellular surface of the membrane is exposed. Alternatively, if the pipette is retracted while it is in the whole-cell configuration, the membrane ruptured ends at the tip of the... 

As the direct communication is established between the pipette and the intracellular compartment in whole-cell configuration, the intracellular solution is rapidly dialyzed by the pipette solution, giving a control of the internal composition of the cell [29]. Nevertheless, as during the cell dialysis the intracellular material is extensively diluted this can be reflected by a run-down of the activity of some channels, whose normal function depends on the presence of some intracellular components, as for example, the calcium channel. The membrane potential of the cell in the whole-cell configuration is the same as that applied to the pipette, and negative currents are interpreted as cationic currents flowing into the cell, while positive currents represent outward cationic currents. 

With whole-cell configuration, the series resistance plays an important role in the control of the voltage of the cell membrane. The series resistance problem has been described for the first time by Hodgkin, Huxley and Katz [31]. It produces a double effect on the voltage-clamp A passive filtering of the voltage pulses applied to the membrane, and a drop of the applied membrane potential when a current is flowing... 

Fig. 8. Whole-cell configuration. After obtaining the cell attached configuration (A), giving a negative pressure or potential pulse, the membrane under the pipette tip can be broken, and a direct access of the pipette to the cell interior is obtained (B). This process can be monitored by the change of the current recorded as response of small voltage pulse, where a significant increase of the capacitative component is observed. In whole-cell configuration, macroscopic currents can be observed, for example, as a response to membrane potential.

The whole-cell configuration has been extensively used to measure macroscopic membrane currents, either voltage-activated (see for example [23, 31]) or receptor-activated (see for example [32, 33]), being an excellent method to study the electrophysiology of small cells that cannot be efficiently voltage clamped using classical intracellular electrodes. This configuration has also been extensively used to study exocytosis, since with appropriate measurement protocols it is possible to monitor continuously the whole-cell membrane capacity [34, 35]. 

Outside-out configuration From the whole-cell configuration it is possible to achieve a new excised-patch. Withdrawing the patch pipette very slowly (Fig. 9A), a small membrane vesicle can be formed on the tip of the pipette (Fig. 9B). This is the outside-out configuration, where the outer surface of the membrane is exposed to the bath solution, and the internal surface of the patch to the pipette solution. In this configuration the pipette potential is equal to that of the membrane, and the recorded current convention is the same as that in whole-cell configuration. 

Fig. 9. Outside-out configuration. After obtaining the whole-cell configuration (A), the pipette is withdrawn, and an excised membrane forms an outside-out patch. When the outside-out patch is obtained, the capacity current observed in response to a small voltage pulse changes significantly, since a reduction of the capacity occurs (from whole-cell to a patch of membrane) (B). At this configuration, single-channel currents are recorded.

Macroscopic sodium currents can be recorded from mouse neuroblastoma neurones [23]. Currents were recorded from whole-cell configurations, using an external solution containing (in mM) 160 NaCl, 2 CaCh, 1 MgCl2, 10 HEPES. The pipette solution, dialyzing the intracellular compartment was 110 CsF, 10 NaCl, 11 EGTA, 10 HEPES. Potassium channels were blocked by the presence of Cs in the intracellular solution, and calcium current were blocked with 1 mM Co in the external solution. 

Fig. 10. Sodium macroscopic current obtained from a neuroblastoma in whole-cell configuration. A Ionic current recorded as a response of a test pulse of —70 mV to - 60mV. Each trace is an average of four records. B Peak current vs. voltage relationship. Peak current is maximal (260 pA) at —7.8 mV, and the reversal potential is 53.5 mV. C Voltage dependence of the activation parameter m. The half activation potential is — 37 mV and the maximum stepness of the voltage dependence yields an e-fold increase of m infinity for 8 mV change in voltage. D Voltage dependence of the inactivation parameter h. The half inactivation potential is -71 mV and an e-fold decrease of h infinity is obtained for a voltage change of 11 mV.

As with extracellular measurements, intracellular measurements can be made by combining electrophysiological and electrochemical techniques (120). For intracellular measurements, the initial patch electrochemical setup is in the cell attached configuration. However, after some time additional suction is applied to the patch pipette, the membrane is ruptured and the whole cell configuration is attained (Figure 17.1.15). Disruption of the membrane allows oxidizable intracellular content to diffuse to the UME where it is detected in either the amperometric or voltammetric mode. 

If the pipette is retracted while it is in the whole cell configuration, a membrane patch is produced that has its extracellular surface exposed. This arrangement is called the outside-out recording configuration. 

Figure 4 Dual effects of ACh on K" " current. Glomus cells were cultured as described above. Cells were continuously perfused with Krebs equilibrated with 5% C02/air. A conventional whole-cell configuration was achieved. K" " current was induced by applying step pulses from —60mV to - -50mV with -1-lOmV increments for 40msec (top). ACh was applied topically via a puff pipette. Tyrode was used as control for ACh application. Low doses of ACh (100 nM-1 pM) increased the Kv current, and high doses of ACh (100 pM-1 mM) decreased the current. The effects of ACh were reversible (data not shown). Current-voltage ciuves (bottom) were constructed with values at 35 msec from the start of voltage pulses.

Calcium current (I ) CsCl is substituted for KCl in the patch pipette. Intracellular K+ is replaced with Cs+ via perfusion in whole-cell configuration for 5 minutes. In this configuration, no outward K+ currents should be present. Thus, the inward I can be recorded in the absence of all four outward potassium currents. 

---