Pardo LA et al. (1998), Cell cycle-related changes in the conducting pr...

XB-ART-13978

J Cell Biol 1998 Nov 02;1433:767-75.

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Cell cycle-related changes in the conducting properties of r-eag K+ channels.

Pardo LA , Brüggemann A , Camacho J , Stühmer W .

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Release from arrest in G2 phase of the cell cycle causes profound changes in rat ether-à-go-go (r-eag) K+ channels heterologously expressed in Xenopus oocytes. The most evident consequence of the onset of maturation is the appearance of rectification in the r-eag current. The trigger for these changes is located downstream of the activation of mitosis-promoting factor (MPF). We demonstrate here that the rectification is due to a voltage-dependent block by intracellular Na+ ions. Manipulation of the intracellular Na+ concentration indicates that the site of Na+ block is located approximately 45% into the electrical distance of the pore and is only present in oocytes undergoing maturation. Since the currents through excised patches from immature oocytes exhibited a fast rundown, we studied CHO-K1 cells permanently transfected with r-eag. These cells displayed currents with a variable degree of block by Na+ and variable permeability to Cs+. Partial synchronization of the cultures in G0/G1 or M phases of the cell cycle greatly reduced the variability. The combined data obtained from mammalian cells and oocytes strongly suggest that the permeability properties of r-eag K+ channels are modulated during cell cycle-related processes.

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Species referenced: Xenopus
Genes referenced: cdk1 kcnh1

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	Figure 2. Raw current traces from the same patch in the absence (A) or in the presence of 20 mM NaCl in the internal solution (B). C shows the currentâvoltage relationships under both conditions. The voltage protocol consisted of a 200-ms depolarization from a holding potential of â80 mV. The amplitude was determined as mean current at the end of the test pulse. The scale bars correspond to 25 pA and 50 ms.
	Figure 3. (A) Normalized currentâvoltage relationships in an inside-out patch from an oocyte expressing rectifying currents in the presence of different internal Na+ concentrations. The stimulation protocol was as the one in Fig. 2. (B) Doseâresponse plots for effects of Na+. The solid lines are fits to the Hill equation. (C) IC50 plotted versus voltage. The exponential fit to Eq. 1 gave a value for Î´ of 0.446.
	Figure 4. Sample traces and amplitude histograms obtained from the same patch without (A and B) or with (C and D) 5 mM NaCl added to the intracellular side, at either 0 (A and C) or +80 mV (B and D). The single channel currents are obtained from the fit to a Gaussian distribution. The smaller amplitude in the histograms at +80 mV corresponds to the amplitude of the flickering of the channel, and remains constant in the absence (B) or in the presence (D) of Na+.
	Figure 5. Currents recorded from a whole oocyte before (A) and after (B) the injection of 50 nl 2 M NaCl. (C) Currentâvoltage plots for steady-state currents from A and B. (Inset) The normalized currents. (D) Lack of correlation between the internal Na+ concentration (calculated from the reversal potential of Na+ currents) and the degree of rectification of r-eag currents. The correlation coefficient is â0.1.
	Figure 6. Before maturation, patches expressing r-eag currents show only slight rectification with 20 mM Na+ (A, control traces; B, 20 mM Na+), as can be seen in the I-V plot (C; open circles, control; solid circles, 20 mM NaCl) and more clearly after normalization of the current amplitude (inset). After MPF injection, patches from the same oocyte show rectification comparable to mature oocytes (D; solid squares); for comparison, the normalized current amplitudes from A and B have been included (symbols as in C).
	Figure 7. Variability in the degree of blockade by internal Na+ in CHO-K1 cells. (A and B) Raw currents traces obtained from two apparently identical cells (500-ms depolarizations to voltages between â60 and +100 mV, in 20-mV increments). (C and D) Corresponding I-V relationships. The internal solution contained 140 mM KCl and 10 mM NaCl.
	Figure 8. (A) Outward currents in a CHO-K1 cell expressing r-eag exposed to an internal solution containing Cs+ as the sole charge carrier; the same internal solution was used in BâD. (B) Tail currents in a cell exposed to an external solution containing (mM) 140 CsCl, 2 CaCl2, 2 MgCl2, 10 Hepes/CsOH, pH 7.2. The reversal potential is â¼0 mV. (C) Instantaneous I-V plots in cells with Cs+ internal solution and varying extracellular K+ and Cs+ concentrations. (D) Anomalous mole fraction effect obtained from tail currents in a cell perfused with different K+-Cs+ mixtures. The tail current at â100 mV (extrapolation to t = 0 of the exponential decay of the amplitude) was normalized to the outward current at +60 mV (mean amplitude at the final 10% of the pulse).
	Figure 9. Normalized currents obtained during voltage ramps in CHO-K1 cells expressing r-eag currents. The internal solution contained Cs+ as charge carrier, and the external solution contained K+. From a holding potential of â100 mV, the cell was progressively depolarized to +75 mV during 1 s. Notice the strong variability in the reversal potential, while the shape of the current is similar in all cases.

References [+] :

Baukrowitz, Modulation of K+ current by frequency and external [K+]: a tale of two inactivation mechanisms. 1995, Pubmed