Correspondence to: José López-Barneo, Departamento de Fisiología Médica y Biofísica, Universidad de Sevilla, Facultad de Medicina, Avenida Sánchez Pizjuán 4, E-41009, Sevilla, Spain. Fax:(34)-954-551769 E-mail:lbarneo{at}cica.es.
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Abstract |
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Voltage-dependent K+ channel gating is influenced by the permeating ions. Extracellular K+ determines the occupation of sites in the channels where the cation interferes with the motion of the gates. When external [K+] decreases, some K+ channels open too briefly to allow the conduction of measurable current. Given that extracellular K+ is normally low, we have studied if negatively charged amino acids in the extracellular loops of Shaker K+ channels contribute to increase the local [K+]. Surprisingly, neutralization of the charge of most acidic residues has minor effects on gating. However, a glutamate residue (E418) located at the external end of the membrane spanning segment S5 is absolutely required for keeping channels active at the normal external [K+]. E418 is conserved in all families of voltage-dependent K+ channels. Although the channel mutant E418Q has kinetic properties resembling those produced by removal of K+ from the pore, it seems that E418 is not simply concentrating cations near the channel mouth, but has a direct and critical role in gating. Our data suggest that E418 contributes to stabilize the S4 voltage sensor in the depolarized position, thus permitting maintenance of the channel open conformation.
Key Words: K+-channel gating, extracellular K+, acidic residues, open-state stabilization, glutamate mutation
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INTRODUCTION |
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Voltage-dependent K+ channels of the plasmalemma participate in fundamental cellular electrical events such as the genesis of the resting membrane potential, action potential repolarization, and repetitive firing (140 mM), and the extracellular milieu with lower [K+] (
2.5 mM in most mammalian tissues). However, the low external [K+] imposes a major challenge to many K+ channels since they do not gate properly, or even become nonfunctional if extracellular K+ is too scant. Although the channels were classically viewed as pores with gates that move independently of the permeating ions, there are several reports indicating that occupation of the channels by the permeating ions modulates their gating properties (for review, see
In K+ channels, both the intra- and extracellular entryways contain negatively charged amino acids that presumably contribute to increase the local concentration of cations while lowering the concentration of anions (see
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MATERIALS AND METHODS |
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Molecular Biology and Expression of K+ Channels in Chinese Hamster Ovary Cells
We used as wild-type construct the Shaker B6-46 (Shaker B
) channel, which has a deletion in the amino terminus that completely removes N-type inactivation (
channels and in combination with mutation T449V. These mutants were done as described previously (
2225°C). Electroporation parameters (350 V and 125 µF) yielded typical time constant values of 2426 ms. cDNA of green fluorescent protein included in plasmid pRK5 was cotransfected with the K+ channel
subunits to detect by fluorescence those cells expressing K+ currents. After electroporation, the cells were resuspended in culture medium (McCoy's 5A with supplements), plated on slivers of glass coverslips, and maintained in an incubator at 37°C until use.
Electrophysiology and Data Analysis
Electrical recordings were performed on cells 2472 h after plating. For the experiments, a coverslip was transferred to a recording chamber of 0.2 ml with continuous flow of solution that could be completely replaced in <40 s. Potassium currents were recorded using the whole-cell configuration of the patch-clamp technique as adapted to our laboratory (
), capacity compensation and subtraction of linear leakage, and capacitive currents. Series resistance compensation was between 40 and 50%. Macroscopic current recordings were low-pass filtered with the cutoff frequency between 3 and 10 kHz. Single-channel recordings were done in excised outside-out membrane patches using pipettes of 57 M
. In these experiments, subtraction of leakage and capacity currents was done off-line using the average current generated in the same patch by 20 consecutive hyperpolarizing pulses of 10 mV and scaled to the appropriate value. Single-channel records were low-pass filtered at 500 or 700 Hz. For the calculation of the mean cluster duration, cluster criterion were set at 20 and 5 ms for the E418E and E418Q channels, respectively. The deactivation time course of macroscopic K+ tail currents and the C-type inactivation rate were estimated by fitting the appropriate current traces with a single exponential function. The composition of the standard solutions was (mM): external: 140 NaCl, 2.7 KCl, 2.5 CaCl2, 4 MgCl2, 10 HEPES, pH 7.4; solution in the pipette and inside the cell: 30 KCl, 80 K-glutamate, 20 K-fluoride, 2 Mg-Cl2, 4 ATP-Mg, 10 HEPES, 10 EGTA, pH 7.2. For experiments in which we used external solutions with a different [K+], NaCl was replaced for the same amount of KCl. To study C-type inactivation of inward K+ currents, all the K+ of the internal solution was replaced for NMDG. Unless otherwise noted, the holding potential in the electrophysiological experiments was -80 mV. Although the junction potential of the solutions was not compensated, we estimated the changes of junction potential produced by the use of solution of variable [K+]. Switching from the control (2.7 mM K+) external solution to solutions with 30, 70, and 140 mM K+ produced changes of junction potential of approximately -0.6, -2, and -4.5 mV, respectively. Experiments were performed at room temperature (
2225°C).
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RESULTS |
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Differential Influences of Extracellular Dicarboxylic Residues on K+ Channel Gating
The dicarboxylic amino acids mutated in this study are indicated in Fig 1 A on a scheme of the proposed transmembrane topology of the subunit of voltage-gated K+ channels. Glutamate and aspartate residues located near the transmembrane segments in loops S1-S2 (E251 and D277), S3-S4 (E333, E334, D335, and E336), and S5-S6 (E418 and E422) of the Shaker channel were replaced by glutamine or asparagine. Mutations in loop S3-S4 were all done simultaneously so the channel studied (EEDE333-336QQQQ) had four negative charges neutralized in each
subunit (channel 4Q). On this channel construct, we replaced residues in positions 422 and 418 to obtain the channels EEDE333-336QQQQ, E422Q (5Q), and EEDE333-336QQQQ, E422Q, E418Q (6Q). Representative current traces obtained during depolarizing pulses to +20 mV in cells transfected with either wild-type Shaker B
channels or any of the five mutants are shown in Fig 1 B. Because, in most of the mutants (except channel 6Q), inactivation rates were similar to, or even slightly slower than, the inactivation rate of Shaker B
channels, current decay was not appreciable during the short-lasting depolarizing pulses of the figure. Mean values of inactivation time constant and of other voltage-dependent parameters of the various types of K+ currents recorded are summarized in Table 1. Inactivation kinetics of the 5Q channels was seen to change from one experiment to another, being in some cases two or three times faster than in the wild-type channel. This variability, which could not be attributed to any special modification of the experimental protocol, is illustrated in Fig 1 B with examples of fast- and slow-inactivating current traces (** and*, respectively). Macroscopic currents resulting from the expression of the 6Q channels had normal activation kinetics, but showed an almost 150-fold acceleration of inactivation. Another notable characteristic of the recordings obtained from cells expressing 6Q channels was a >10-fold reduction of peak current amplitude (Table 1). In cells where the efficiency of the 6Q cDNA transfection was not too high, the amplitude of the current in the normal (2.7 mM K+) extracellular solution was almost negligible (see below).
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Because it has been shown in previous studies that dependence of C-type inactivation on external K+ is higher after abolishment of outward current ( outward and inward K+ currents were slower in transfected Chinese hamster ovary cells (this study) than in patches excised from Xenopus oocytes (
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A Conserved Extracellular Glutamate Determines K+ Current Amplitude and Kinetics
Given the pronounced differences in inactivation kinetics between outward currents of channels 5Q and 6Q (Fig 1), presumably ascribable to the mutation of the residue in position 418, we studied the single mutant E418Q. Macroscopic currents recorded from cells expressing this channel type were of small amplitude and exhibited the fast inactivation time course characteristic of channel 6Q (Fig 3 A; Table 1). Inactivation rate of mutant E418Q was also very fast in cells dialyzed with K+-free internal solutions and, in this condition, measurable currents were recorded only when external [K+] was higher than 5 mM (Fig 2 and Table II; see below). The action of glutamate 418 appeared to reside mainly on the negatively charged carboxyl group since its replacement for aspartate, with shorter side chain but maintaining the negative charge (mutant E418D), had almost no effect on channel kinetics and current amplitude (Fig 3 A, Table 1). Inactivation and closing kinetics of channel E418D were even slightly slower than in the wild-type Shaker B channel (Table 1), but the differences were small and were not studied in further detail. Thus, whereas neutralization of the charge of several dicarboxylic amino acids in the extracellular loops of Shaker K+ channels has little affect on channel activity and kinetics, the maintenance of a negatively charged residue in position 418 is absolutely necessary for the normal function of the channels. Fig 3 B illustrates that, although the length and number of negatively charged residues of the turret varies among the different types of K+ channels, the glutamate at position 418 of Shaker is conserved in all the families of delayed rectifier voltage-dependent channels of both Drosophila and mammals. This residue is also maintained in silent, regulatory K+ channel
subunits, such as Kv2.3 (see
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The relatively small size of the K+ current observed in cells transfected with mutants 6Q or E418Q (Table 1) was not due to a decrease in the level of channel expression, but to a reduction in the number of open channels, which was strongly dependent on extracellular [K+] (Fig 4). Superfusion of E418Q-transfected cells with a K+-free solution elicited an immediate abolishment of the voltage-dependent K+ current, which was reversed by reintroduction of K+ in the chamber (Fig 4 A). In contrast, exposure to high (30 mM) external K+ produced, despite the reduction of the K+ driving force, an increase in current amplitude (Fig 4 B). K+ currents recorded from a representative E418Q-transfected cell exposed to various external [K+] are shown in Fig 4 C, and Fig 4 D summarizes the relationship between peak current amplitude and extracellular [K+] with average data obtained from several cells. The dependence of peak current amplitude on external [K+] had a bell-shaped curve. Rising [K+] evoked a gradual increase in current amplitude, with a maximal value obtained with 70 mM. Current amplitude decreased with higher concentrations due to reduction of the K+ driving force. Since the increase of external [K+] had little effect on E418Q single-channel current amplitude (data not shown), these data indicate that the augmentation of the macroscopic E418Q currents induced by extracellular K+ was due to the increase in the number of channels that were simultaneously open during the depolarizing pulses.
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The Effects of Mutation E418Q Are Similar to Those Produced by Channel Deprivation of K+ Ions
The modulatory effect of extracellular K+ on E418Q current amplitude resembles the effect of the cation on the currents mediated by Kv1.4 channels (
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Comparison of single-channel currents recorded with the standard asymmetrical [K+] indicated that, as expected, E418Q channels have smaller single-channel current amplitude and conductance than wild-type, E418E, channels (Fig 6). We estimated the average duration of well-defined clusters of openings recorded from excised patches that appeared to contain a single E418E or E418Q channel. Mean burst duration was voltage independent in the range between +20 and +50 mV, averaging 270 ± 188 ms (mean ± SD, n = 135 in five cells) in Shaker B channels (E418E), but only 10.5 ± 5.5 ms (n = 29 in four cells) in E418Q channels. We also observed clear differences between the two channel types in the number of clusters during each depolarizing pulse, a parameter that at positive membrane potentials indicates the reversibility of the C-type inactivated state. In response to a large depolarization, E418Q channels produced normally only a single cluster of openings (1.18 ± 0.39, mean ± SD, n = 38 clusters in seven cells), whereas two or three clusters were observed in Shaker B
channels with pulses of 0.5 or 1 s, even though they lasted less than the time required for inactivation to be complete. The reduction of cluster duration and the number of clusters per trace produced by mutation E418Q is in accord with the acceleration of the rate of macroscopic C-type inactivation described above, and further suggests that neutralization of charge at position 418 destabilizes the open conformation of the channels and favors their transition to the C-inactivated state.
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Neutralization of residue E418 had effects opposed to those produced by pore mutations, such as T449V, which abolish modulation by extracellular K+ and result in channels with ultraslow C-type inactivation ( channels, the single mutant T449V, and the double mutant T449V, E418Q. The marked slowing of inactivation time course produced by mutation T449V (~18-fold reduction of inactivation rate) was partially compensated when the mutation E418Q was done on the T449V channel (channel E418Q, T449V). These data indicate that mutation E418Q alters channel gating in a way similar to deprivation of the channels of K+ and that the effects of this mutation can be compensated by external K+ or mutations in the pore (such as T449V), with slow C-type inactivation.
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DISCUSSION |
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The major finding in this study is the identification of a molecular determinant for the stabilization of conductiving versus closed and of conductiving vs. C-inactivated conformations of voltage-dependent K+ channels. Among the various dicarboxylic residues in the extracellular loops of the K+ channels, E418 of Shaker, conserved in most families of voltage-dependent K+ channels, is absolutely necessary for the channels to be functional at the normal extracellular [K+]. The role of glutamate 418 appears to depend on its negatively charged side chain since a mutation conserving the charge (E418D) has no major functional consequences, whereas neutralization of the charge (mutation E418Q) results in almost complete loss of conductance. In a previous work on toxin binding to Shaker K+ channels, the mutant E418K was reported to be unable to express currents in Xenopus oocytes (
Mutation E418Q causes a decrease of single-channel conductance and reduction of the time that the channels stay open during a maintained depolarization. In parallel with these changes, macroscopic currents mediated by the mutated channels exhibit a marked acceleration of C-type inactivation and closing kinetics. Due to these biophysical alterations, macroscopic K+ currents in E418Q-transfected cells were barely appreciable, although the number of channels expressed was normal. In these same experiments, vigorous K+ currents were recorded when extracellular [K+] was elevated to 30 or 70 mM. The effects of mutation E418Q are qualitatively similar to the changes produced in Shaker K+ channels by removal of extracellular K+ or by mutations reducing the occupation of the pore by permeating ions (
Despite the similarity between the gating modifications produced by mutation E418Q and by depletion of the pore of permeating ions, the location of residue 418 makes it unlikely that its primary role is to increase the local [K+] in the external mouth of the channels. Glutamate 418 is in the transition between the membrane-spanning segment S5 and the base of the turret, and therefore far from the entryway of the channel pore (
This last role of glutamate 418 is described diagrammatically in Fig 8 with a scheme of a sectioned K+ channel inspired by the model of
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Good candidates for having a part in the electrostatic interaction between S4 and E418 are the positively charged arginines (R362 and R365) located in the external side of the voltage sensor. Naturally, the effect of neutralization of these arginines must be tested in future experimental work; however, it is quite interesting that some of the properties of channel mutants R362Q and R365Q, studied by
The loss of conductance observed at low external K+ in E418Q channels might be related to the defunct state caused by removal of K+ from the two sides of the membrane (
Given the fundamental role of glutamate 418 in gating, its conservation or replacement might have contributed to the evolution of K+ channels with kinetic properties adapted to specific functional roles. As indicated before, a glutamate residue in the position equivalent to 418 of Shaker is conserved in all the voltage-dependent K+ channels where stabilization of the S4 sensor in the "depolarized" state is probably necessary to ensure channel opening. Interestingly, glutamate 418 is replaced by neutral amino acids in most K+ channels with two membrane-spanning segments lacking the S4 voltage sensor (
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Acknowledgements |
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This study was supported by grants from the Spanish Ministry of Education, Andalusian Government, and Fundaciones La Caixa and R. Areces. P. Ortega-Sáenz and R. Pardal were recipients of fellowships of the Programa de Formación del Personal Investigador of the Spanish Ministry of Education and Fondo de Investigación Sanitaria.
Submitted: 22 February 2000
Revised: 15 May 2000
Accepted: 26 June 2000
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References |
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Baukrowitz, Y., Yellen, G. 1995. Modulation of K+ currents by frequency and external [K+]: a tale of two inactivating mechanisms. Neuron 15:951-960[Medline].
Baukrowitz, Y., Yellen, G. 1996. Use-dependent blockers and exit rate of the last ion from the multi-ion pore of a K+ channel. Science 271:653-656[Abstract].
Castellano, A., López-Barneo, J. 1991. Sodium and calcium currents in dispersed septal neurons. J. Gen. Physiol. 97:303-319[Abstract].
Castellano, A., Chiara, M.D., Mellstrom, B., Molina, A., Monje, F., Naranjo, J.R., López-Barneo, J. 1997. Identification and functional characterization of a K+ channel -subunit with regulatory properties specific of brain. J. Neurosci. 17:4652-4661
Cha, A., Bezanilla, F. 1998. Structural implications of fluorescence quenching in the Shaker K+ channel. J. Gen. Physiol. 112:391-408
Cha, A., Snyder, G.E., Selvin, P.R., Bezanilla, F. 1999. Atomic scale movement of the voltage-sensing region in a potassium channel measured via spectroscopy. Nature 402:809-813[Medline].
Chen, T.Y., Peng, Y.W., Dhallan, R.S., Ahamed, B., Reed, R.R., Yau, K.W. 1993. A new subunit of the cyclic nucleotide-gated cation channel in retinal rods. Nature 362:764-767[Medline].
Demo, S., Yellen, G. 1992. Ion effects on gating of the Ca2+-activated K+ channels correlate with occupancy of the pore. Biophys. J. 61:639-649[Abstract].
Doyle, D.A., Morais Cabral, J.H., Pfuetzner, R.A., Kuo, A., Gulbis, J.M., Cohen, S.L., Chait, B.T., MacKinnon, R. 1998. The structure of the potassium channel: molecular basis of K+ conduction and selectivity. Science 280:69-77
Glauner, K.S., Mannuzzu, L.M., Gandhi, C.S., Isacoff, E.Y. 1999. Spectroscopic mapping of voltage sensor movement in the Shaker potassium channel. Nature 402:813-817[Medline].
Goldstein, S.A.N., Pheasant, D.J., Miller, C. 1994. The charybdotoxin receptor of a Shaker K+ channel: peptide and channel residues mediating molecular recognition. Neuron 12:1377-1388[Medline].
Gómez-Lagunas, F. 1997. Shaker B K+ conductance in Na+ solutions: a remarkably stable non-conducting state produced by membrane depolarizations. J. Physiol 499:3-15[Abstract].
Hamill, O.P., Marty, A., Neher, E., Sakmann, B., Sigworth, F. 1981. Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflügers Arch 391:85-100[Medline].
Hille, B. 1992. Ionic Channels of Excitable Membranes. 2nd ed Sunderland, MA, Sinauer Associates, Inc, pp. 607.
Hoshi, T., Zagotta, W.N., Aldrich, R.W. 1991. Two types of inactivation in Shaker K+ channels: effects of alterations in the carboxyl terminal region. Neuron 7:547-556[Medline].
Jäger, H., Rauer, H., Nguen, A.N., Aiyar, J., Chandy, K.G., Grissmer, S. 1998. Regulation of mammalian Shaker-related K+ channels: evidence of non-conducting closed and non-conducting inactivated states. J. Physiol. 506:291-301
Kiss, L., Korn, S.J. 1998. Modulation of C-type inactivation by K+ at the potassium channel selectivity filter. Biophys. J 74:1840-1849
Kubo, Y., Reuveny, E., Slesinger, P.A., Jan, Y.N., Jan, L. 1993. Primary structure and functional expression of the rat G-protein coupled muscarinic K+ channel. Nature 364:802-806[Medline].
Liu, Y., Jurman, E., Yellen, G. 1996. Dynamic rearrangement of the outer mouth of a K+ channel during gating. Neuron 16:859-867[Medline].
López-Barneo, J., Hoshi, T., S.H.Heinemann,, Aldrich, R.W. 1993. Effects of external cations and mutations in the pore region on C-type inactivation of Shaker potassium channels. Receptors Channels 1:61-71[Medline].
Loots, E., Isacoff, E.Y. 1998. Protein rearrangements underlying slow inactivation of the Shaker K+ channel. J. Gen. Physiol 112:377-389
Marom, S., Levitan, I. 1994. State-dependent inactivation in Kv3 potassium channel. Biophys. J 67:579-589[Abstract].
MacKinnon, R., Miller, C. 1989. Mutant potassium channels with altered binding of charybdotoxin, a pore-blocking peptide inhibitor. Science 245:1382-1385[Medline].
Melishchuk, A., Loboda, A., Armstrong, C.M. 1998. Loss of Shaker K channel conductance in 0 K+ solutions: role of the voltage sensor. Biophys. J 75:1828-1835
Molina, A., Castellano, A.G., López-Barneo, J. 1997. Pore mutations in Shaker K+ channels distinguish between the sites of TEA blockade and C-type inactivation. J. Physiol. 499:361-367[Abstract].
Molina, A., Ortega-Sáenz, P., López-Barneo, J. 1998. Pore mutations alter closing and opening kinetics in Shaker K+ channels. J. Physiol 509:327-337
Pardo, L.A., Heinemann, S.H., Terlau, H., Ludewig, U., Lorra, C., Pongs, O., Stühmer, W. 1992. Extracellular K+ specifically modulates a rat brain K+ channel. Proc. Natl. Acad. Sci. USA 89:2466-2470[Abstract].
Schönherr, R., Heinemann, S.H. 1996. Molecular determinants for activation and inactivation of HERG a human inward rectifier potassium channel. J. Physiol 493:635-642[Abstract].
Seoh, S.-A., Sigg, D., Papazian, D., Bezanilla, F. 1996. Voltage-sensing residues in the S2 and S4 segment of the Shaker K+ channels. Neuron 16:1159-1167[Medline].
Smith, P.L., Baukrowitz, Y., Yellen, G. 1996. The inward rectification mechanism of the HERG cardiac potassium channel. Science 379:833-836.
Stanfield, P.R., Ashcroft, F.M., Plant, T.D. 1981. Gating of a muscle K+ channel and its dependence on the permeating ion species. Nature 289:509-511[Medline].
Swenson, R.P., Armstrong, C.M. 1981. K+ channels close more slowly in the presence of external K+ and Rb+. Nature 291:427-429[Medline].
Warmke, J.W., Ganetzky, B. 1994. A family of K+ channel genes related to eag in Drosophila and mammals. Proc. Natl. Acad. Sci. USA. 91:3438-3442[Abstract].
Yellen, G. 1997. Single channel seeks permeant ion for brief but intimate relationship. J. Gen. Physiol 110:83-85