Shih TM and Goldin AL (1997), Topology of the Shaker potassium channel probed...

XB-ART-16773

J Cell Biol 1997 Mar 10;1365:1037-45.

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Topology of the Shaker potassium channel probed with hydrophilic epitope insertions.

Shih TM , Goldin AL .

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The structure of the Shaker potassium channel has been modeled as passing through the cellular membrane eight times with both the NH2 and COOH termini on the cytoplasmic side (Durrell, S.R., and H.R. Guy. 1992. Biophys. J. 62:238-250). To test the validity of this model, we have inserted an epitope consisting of eight hydrophilic amino acids (DYKDDDDK) in predicted extracellular and intracellular loops throughout the channel. The channels containing the synthetic epitope were expressed in Xenopus oocytes, and function was examined by two-electrode voltage clamping. All of the mutants containing insertions in putative extracellular regions and the NH2 and COOH termini expressed functional channels, and most of their electrophysiological properties were similar to those of the wild-type channel. Immunofluorescent staining with a monoclonal antibody against the epitope was used to determine the membrane localization of the insert in the channels. The data confirm and constrain the model for the transmembrane topology of the voltage-gated potassium channel.

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	Figure 2. Current recordings from oocytes expressing the Flag insertion mutants. (a) 200 pg of RNA encoding the wild-type Shaker and insertion mutants were injected into oocytes, which were incubated at 20Â°C for 24â48 h before recording currents using a two-electrode voltage clamp, as described in Materials and Methods. The currents were elicited by depolarizations from a holding potential of â80 mV to between â30 mV and +30 mV in 10 mV increments. The slowly activating outward currents for pk91, pk333, and pk418 result from calcium activated chloride channels that are endogenously present in oocytes from some frogs. (b) 50 ng of RNA encoding insertion mutant pk300 was injected into oocytes. Incubation and recording conditions were as described in a.
	Figure 3. Electrophysiological properties of the Flag insertion mutants. (a) Conductance voltage relationships were determined by depolarizations from a holding potential of â80 mV to between â80 mV and +150 mV in 10 mV increments. Currents were converted to conductance, as described in Materials and Methods. Conductance values were normalized to the maximum conductance, and the average value is plotted against the depolarizing potential. The curve represents the best fit of a two state Boltzmann equation, as described in Materials and Methods. Symbols represent the means, and error bars indicate the standard deviations for data from four to eight oocytes. (b) Time constants for inactivation were determined by fitting the current traces obtained as in a with a single exponential function, as described in Materials and Methods. The average time constants are plotted against the depolarizing potential. Symbols represent the means, and error bars indicate the standard deviations for data from four to six oocytes. (c) Recovery from inactivation was determined by a 50 mV inactivating pulse for 50 ms, a variable recovery interval from 10 to 250 ms, and a test pulse to +50 mV for 40 ms to assess recovery, as described in Materials and Methods. The maximum current during each test pulse was normalized to the maximum current during the inactivating pulse, and the average values are plotted against the recovery interval. The smooth curve represents a fit of the average data to a double exponential equation, as described in Materials and Methods. Symbols represent the means, and error bars indicate the standard deviations for data from four to eight oocytes.
	Figure 4. Immunofluorescent staining of control, wild-type, pk91, pk252, and pk276. Schematic diagrams to the left of each row of microscopy images indicate the region in which each Flag insertion is located. Photomicrographs of oocytes labeled on the extracellular side of the membrane are shown in columns a (fluorescence) and b (phase contrast). Photomicrographs of oocytes labeled on the intracellular side of the membrane are shown in columns c (fluorescence) and d (phase contrast).
	Figure 5. Immunofluorescent staining of pk333, pk348, pk356, pk418, and pk488. Schematic diagrams to the left of each row of microscopy images indicate the region in which each Flag insertion is located. Photomicrographs of oocytes labeled on the extracellular side of the membrane are shown in columns a (fluorescence) and b (phase contrast). Photomicrographs of oocytes labeled on the intracellular side of the membrane are shown in columns c (fluorescence) and d (phase contrast).

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