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. 1997 Mar;109(3):301-11.
doi: 10.1085/jgp.109.3.301.

Transfer of voltage independence from a rat olfactory channel to the Drosophila ether-à-go-go K+ channel

C Y Tang  1 , D M Papazian
Affiliations

Transfer of voltage independence from a rat olfactory channel to the Drosophila ether-à-go-go K+ channel

C Y Tang et al. J Gen Physiol. 1997 Mar.

Abstract

The S4 segment is an important part of the voltage sensor in voltage-gated ion channels. Cyclic nucleotide-gated channels, which are members of the superfamily of voltage-gated channels, have little inherent sensitivity to voltage despite the presence of an S4 segment. We made chimeras between a voltage-independent rat olfactory channel (rolf) and the voltage-dependent ether-à-go-go K+ channel (eag) to determine the basis of their divergent gating properties. We found that the rolf S4 segment can support a voltage-dependent mechanism of activation in eag, suggesting that rolf has a potentially functional voltage sensor that is silent during gating. In addition, we found that the S3-S4 loop of rolf increases the relative stability of the open conformation of eag, effectively converting eag into a voltage-independent channel. A single charged residue in the loop makes a significant contribution to the relative stabilization of the open stage in eag. Our data suggest that cyclic nucleotide-gated channels such as rolf contain a voltage sensor which, in the physiological voltage range, is stabilized in an activated conformation that is permissive for pore opening.

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Figures

Figure 1
Figure 1
Comparison of the structural organization, sequence, and gating properties of eag and rolf. (A) A model for the topology of the eag and rolf subunits in the membrane. Both eag and rolf contain putative transmembrane segments S1-S6, the pore-forming P loop, and a cyclic nucleotide binding domain (solid box). Also shown are the approximate positions of charged residues in transmembrane segments S2, S3, and S4 that are conserved among members of the superfamily of voltage-gated channels (Jan and Jan, 1990; Chandy and Gutman, 1994). Single letters indicate the residues present in both eag and rolf. Pairs of letters indicate the residues found in eag (first) or rolf (second). (B) An alignment of the eag, rolf, and Shaker sequences including the S3 segment, S3-S4 loop, and S4 segment. The numbers at the top indicate the position in the eag sequence. The positions of mutations that are discussed in the text are underlined. Boxes indicate the positions of residues conserved among members of the superfamily of voltage-gated K+ channels. Gaps have been introduced to maintain the alignment. The S3-S4 loop in Shaker is substantially longer than in eag and rolf and is shown in loops to conserve space. Overall, the sequences of eag and rolf are 23% identical. (C) Representative eag (left) and rolf (right) currents after expression in Xenopus oocytes. Eag currents were recorded in an 89 mM KCl bath solution using a two electrode voltage clamp. From a holding potential of −90 mV, currents were evoked by 150-ms pulses to test potentials from −80 to +70 mV, in 10-mV increments. Rolf currents were recorded in an excised, inside-out macropatch with 100 mM KCl in the pipette and bath solutions. The patch was held at +40 mV. Currents were evoked by perfusion of 1 mM cGMP on the cytoplasmic face of the patch.
Figure 2
Figure 2
The low net charge of the S4 segment does not account for the lack of sensitivity to voltage in rolf. (A) The S4 sequences of eag, rolf, and the S4-1 and S4-2 chimeras. Numbers at top correspond to the position in the eag sequence. Residues that have been exchanged in the chimeras are underlined. The net charge of the S4 segment of each sequence, shown at the right, was calculated assuming R or K = +1, E or D = −1, and H = 0. (B) Representative current traces from the S4-1 (left) and S4-2 (right) chimeras expressed in Xenopus oocytes. Currents were recorded in 89 mM KCl using a two-electrode voltage clamp. From a holding potential of −90 mV, 150-ms pulses to test potentials from −30 to +120 mV were applied in 10-mV increments. (C) Steady-state activation curves of eag (○) and the S4-1 (•) and the S4-2 (▵) chimeras. After a series of depolarizing pulses to the indicated test potentials, the amplitudes of isochronal tail currents at −90 mV were normalized to the maximum amplitude to obtain the fraction of open channels (P o) at the test potential. Data points, shown as mean ± SEM (n = 6–8), were fit with a Boltzmann equation (solid curves). If error bars are not present, the SEM was smaller than the symbol shown. The activation curves of the S4-1 and S4-2 chimeras did not saturate in the tested voltage range.
Figure 3
Figure 3
S3+loop and loop chimeras shift the activation of eag to hyperpolarized potentials. (A) Representative ramp-evoked currents from the S3+loop chimera, wild-type eag, and an uninjected oocyte. Currents were recorded in 89 mM KCl using a two electrode voltage clamp. From a holding potential of −10 mV, a 500-ms voltage ramp from −220 to +120 mV was applied. Large capacitive transients evoked at the beginning and end of the ramp are not shown. (B) Representative ramp-evoked currents from the loop chimera and an uninjected oocyte from the same batch have been superimposed. Quinidine (800 μM), which blocks eag channels, was added where indicated. Current traces were obtained as in A.
Figure 4
Figure 4
The mutation A345E in the S3-S4 loop of eag shifts activation in the hyperpolarized direction. (A) Representative current was evoked by a 500-ms voltage ramp from −220 to +60 mV. The arrow indicates that inward current is first detected at about −90 mV. (B) Representative, leak-subtracted current traces from A345E expressed in oocytes. Currents were recorded in 89 mM KCl using a two-electrode voltage clamp. From a holding potential of −130 mV, 200-ms pulses to potentials ranging from −120 to +30 mV were applied in 10-mV increments. Leakage currents were subtracted using the P/−4 protocol (Bezanilla and Armstrong, 1977). (C) Steady-state activation curves of eag (○) and A345E (•). After a series of depolarizing pulses to the indicated test potentials, the amplitudes of isochronal tail currents at −90 mV (eag) or −130 mV (A345E) were normalized to the maximum amplitude to obtain the P o at the test potential. Data points, shown as mean ± SEM (n = 7–8), were fit with a Boltzmann equation (solid curves). If error bars are not present, the SEM was smaller than the symbol shown.
Figure 5
Figure 5
Mutations I317K, D333-337, and L342H do not shift the activation of eag in the hyperpolarized direction. The P o-V curves of eag (○), I317K (▪), Δ333–337 (♦), and L342H (▾) were obtained from the analysis of isochronal tail currents recorded at −90 mV, as in Figs. 2 and 4.
Figure 6
Figure 6
The mutation A345R in the S3-S4 loop of eag shifts activation in the hyperpolarized direction. (A) Representative current was evoked by a voltage ramp as in Fig. 4. (B) Representative current traces from A345R expressed in oocytes. Currents were recorded in 89 mM KCl using a two electrode voltage clamp. From a holding potential of −150 mV, 250-ms pulses to potentials ranging from −130 to +70 mV were applied in 20-mV increments. The current reversed at about −5 mV. Leakage currents were not subtracted.
Figure 7
Figure 7
Rolf, and the S3+loop and loop chimeras display similar quasi-linear current-voltage relationships in the physiological range of membrane potentials. Representative currents were evoked by a 500-ms voltage ramp from −120 to +120 mV. The holding potential was 0 mV for rolf and −10 mV for the two chimeras. Rolf currents were recorded in an excised, inside-out macropatch in the presence or absence of 1 mM cGMP (left). Ramp-evoked currents from the S3+loop (middle) and loop (right) chimeras were recorded using a two-electrode voltage clamp; also shown are ramp-evoked currents from uninjected oocytes.
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