4.2.4  Patch clamping: a window on ion regulation

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Figure 4.13 illustrates a patch clamp experiment. It summarises a study of an ion channel which transports K+ ions across the tonoplast separating the vacuole from the cytoplasm of beetroot. Figure 4.13(a) is a photomicrograph of the whole vacuole configuration, where the vacuole is attached to the end of a microcapillary and the bit of membrane covering the orifice at the tip has been ruptured. The microcapillary is filled with 50 mM potassium orthophosphate (KH2PO4) and so the vacuole also contains this solution. The pattern of current traces obtained during a sequence of pulses at voltages ranging from –111 to +129 mV is shown in Figure 4.13(b). When the cytoplasmic side (i.e. bathing solution) is positive relative to the vacuole (+49 to +129 mV), a flow of current develops slowly over one or two seconds in response to the square wave pulse. At negative voltages (–111 mV) there is very little current. This is a case of voltage gating (Section 4.1.3(a)).

Single channels can be studied too using small patches of membrane. Figure 4.13(c) shows data from four channels in an ‘outside-out’ patch of membrane isolated from a beetroot vacuole. Opening and closing of individual channels are seen as abrupt step-like changes in current. Traces for a range of voltages show that as the voltage gets more positive the height of the current steps increases. A plot of the height of current steps against voltage (Figure 4.13d) can be extrapolated back to the x-axis, showing the point where the diffusion gradient for the ions moving through these channels is balanced by the voltage applied (reversal potential). Calculation of the Nernst potentials (Equation 4.7) shows that this voltage is close to the diffusion potential for orthophosphate but at least 50 mV too negative for K+ ions. Figure 4.13(c) also shows that as voltage increases, the channel is open more frequently and for longer. A careful analysis of each transition permits us to calculate the probability of a channel being open (Popen) and to plot it against voltage. Figure 4.13(d) shows that at voltages of less than +100 mV the chances of a channel being open are at most 20%. Consequently, these experiments have not only identified a channel in the tonoplast from beetroot which allows orthophosphate to cross from vacuole to cytoplasm but also indicate how flow is achieved. Voltage is critical. When the energy gradient for orthophosphate flow out of the vacuole is favourable (cytoplasm positive relative to vacuole), orthophosphate channels are most likely to open and let this flux proceed. While strongly negative voltages could drive orthophosphate the other way, the channels close and consequently it is effectively a one-way transport. We also know that the channel’s
initial response to voltage is rather sluggish.