A small viral potassium ion channel with an inherent inward rectification

Abstract

Some algal viruses have coding sequences for proteins with structural and functional characteristics of pore modules of complex K⁺ channels. Here we exploit the structural diversity among these channel orthologs to discover new basic principles of structure/function correlates in K⁺ channels. The analysis of three similar K⁺ channels with ≤ 86 amino acids (AA) shows that one channel (Kmpv₁) generates an ohmic conductance in HEK293 cells while the other two (Kmpv_SP1, Kmpv_PL1) exhibit typical features of canonical Kir channels. Like Kir channels, the rectification of the viral channels is a function of the K⁺ driving force. Reconstitution of Kmpv_SP1 and Kmpv_PL1 in planar lipid bilayers showed rapid channel fluctuations only at voltages negative of the K⁺ reversal voltage. This rectification was maintained in KCl buffer with 1 mM EDTA, which excludes blocking cations as the source of rectification. This means that rectification of the viral channels must be an inherent property of the channel. The structural basis for rectification was investigated by a chimera between rectifying and non-rectifying channels as well as point mutations making the rectifier similar to the ohmic conducting channel. The results of these experiments exclude the pore with pore helix and selectivity filter as playing a role in rectification. The insensitivity of the rectifier to point mutations suggests that tertiary or quaternary structural interactions between the transmembrane domains are responsible for this type of gating.

Keywords: Ba2+ block; Kir channels; Viral K+ channel; inward rectification.

Figure 1.

Similar channels with partially different functional properties. (a) Alignment of three channel proteins Kmpv₁, Kmpv_PL1 and Kmpv_SP1. The identical filter domain is highlighted in grey. The amino acids in Kmpv₁, which are different from the two other proteins, are indicated in yellow. The amino acids in which Kmpv_PL1 differs from Kmpv_SP1 are marked in green. The estimated transmembrane domains are underlined. A crucial AA 52 in Kmpv_SP1 is marked by an arrow. Representative current responses to voltage steps between +65 mV and −135 mV of mock transfected HEK293 cells in buffer with 50 mM K⁺ (b) and HEK293 cells expressing Kmpv₁ in 50 mM K⁺ (c) or 50 mM Na⁺ (d). The corresponding steady state I/V relations on the right are shown in (e). Same experiments with cell expressing Kmpv_SP1 in 50 mM K⁺ (f) and 50 mM Na⁺ (g) plus corresponding I/V relations in (h). The currents for I/V relations were sampled at end of voltage pulses (marked by arrows). Symbols at current traces correspond to symbols in respective I/V curves. In both I/V relations open symbols report measurements in K⁺ and closed symbols report measurements in Na⁺. Note that Kmpv₁ generates an ohmic conductance in K⁺ and Kmpv_SP1 exhibits a strong inward rectification. Asterisks (*) indicate conserved residues, colons (:) indicate residues with very similar properties, and periods (.) indicate residues with weakly similar properties.

Figure 2.

Kmpv₁ and Kmpv_SP1 exhibit a different sensitivity to extracellular Ba²⁺. Exemplar currents and corresponding I/V relations as in Figure 1 from HEK293 cells expressing Kmpv₁ (a-c) or Kmpv_SP1 (d-f). Currents were recorded in bath solution with 50 mM K⁺ in absence (a,d) and presence of 1 mM BaCl₂ (b,e). The corresponding I/V relations for Kmpv₁ (c) and Kmpv_SP1 (f) show steady state currents in the absence (open symbols) and presence (closed symbols) of 1 mM Ba²⁺. The currents for I/V relations were sampled at end of voltage pulses (marked by arrows). Symbols at current traces correspond to symbols in respective I/V curves. (g) Relative block of Kmpv_SP1 (black) or Kmpv₁ (red) at −115 mV as a function of extracellular Ba²⁺ concentration. Data (mean ± sd; n ≥ 4) were fitted with the Hill equation (Equation 1). The K_D values for Ba²⁺ block of both channels from fits as in G are plotted as a function of voltage (h). The K_D value increases ten fold per 114 mV (Kmpv_SP1, black) and 59 mV (Kmpv₁, red) of negative voltage.

Figure 3.

Inward rectification of Kmpv_SP1 shifts with driving force for K⁺. (a) Exemplar current responses of one HEK293 cell expressing Kmpv_SP1 to voltage ramp between 50 and −160 mV with 5 mM (red) or 50 mM (black) K⁺ in the bath solution. Large currents occur negative of K⁺ reversal voltages, which are indicated by arrows for 5 and 50 mM K⁺. (b) Slope conductance of Kmpv_SP1 generated inward current between −95 and −135 mV as a function of the extracellular K⁺ concentration. Data from n = 4 measurements were normalized to conductance in 5 mM K⁺ and jointly fitted with eqn. 1 yielding a value of 0.46 for n.

Figure 4.

Kmpv₁ generates a voltage independent channel with small unitary conductance in planar lipid bilayers. (a) Exemplar current traces of Kmpv₁ activity over a range of clamp voltages in symmetrical KCl (100 mM + 10 mM HEPES, pH 7). For sake of presentation the data were filtered with 100 Hz. The channel protein was translated in vitro into nanodiscs and recorded in a vertical planar DPhPC bilayer [21]. The channel exhibits a high open probability with only some longer-lasting closures. Individual closing events, which are marked on the traces, are enlarged on the right. (b) Representative current traces of experiments as in A but with nanodiscs, which contained no channel protein. (c) Mean unitary channel voltage relation (top), open probability voltage relation (middle) and time averaged I/V relation (bottom) from n = 2 to 5 recordings as in A. The latter were obtained by multiplying values from unitary I/V and P_o/V relations.

Figure 5.

Kmpv_SP1 has an inherent inward rectification. (a) Exemplar current traces of Kmpv_SP1 activity in bilayer recordings over a range of clamp voltages in symmetrical KCl (100 mM KCl + 10 mM HEPES, pH 7) in absence or presence of BaCl₂ (+1 mM Ba²⁺). Clamp voltages are given (in mV) on the left of current traces. The channel protein was translated in vitro as in Figure 4(a). The channel exhibits a high open probability with flicker type fluctuations. Individual clear-cut closing events, which are marked on the traces, are enlarged on the right. (b) I/V relation of mean currents measured over clamp steps of 60 sec in absence (circles, mean ± sd; n = 9) and presence of Ba²⁺ (squares). (c) Normalized mean I/V relations of Kmpv_SP1 currents (mean ± sd; n ≥ 4) measured in bilayers (open circles) and in HEK293 cells as in Figure 1 but with 100 mM K⁺ in bath solution (closed circles). (d) Chord conductance (Gc)/voltage relation of mean current from b fitted with Boltzmann function (orange line) using z value of = 0.9. (e) I/V relation of Kmpv_SP1 channel from bilayer recordings as in B with 10 mM EDTA in cis and trans chamber (triangles) or after removing HEPES from the bath solution (open circles). For comparison the data from B are re-plotted (closed circles).

Figure 6.

The filter domain of Kmpv₁ is responsible for low Ba²⁺ sensitivity. (a) Chimera (ChA) comprising the pore domain of Kmpv₁ (red) and the TM domains of Kmpv_SP1 (black). The selectivity filter sequence is highlighted in grey; the estimated transmembrane domains are underlined. Exemplar currents as in Figure 1 from HEK293 cells expressing ChA in bath solution with 50 mM K⁺ in the absence (b) and presence of 1 mM BaCl₂ (c). The corresponding I/V relations (d) show steady state currents in the absence (open circles) and presence (closed circles) of 1 mM Ba²⁺. (e) Relative block of Kmpv_SP1 (black), Kmpv₁ (red), chimera ChA (grey) and Kmpv_SP1 mutant S53F (blue) at −115 mV as a function of extracellular Ba²⁺ concentration. Data (mean ± sd; n ≥ 4) were fitted with the Hill equation. The K_D values for the Ba²⁺ block of both channels obtained from fits as in E are plotted as a function of voltage (f). The K_D value increases ten fold per 48 mV (Kmpv_SP1-S53F, blue) and 85 mV (ChA, grey) of negative voltage. For comparison the corresponding data of the wt channels are re-plotted.

Figure 7.

Rectification properties of Kmpv1, KmpvSP1, chimera and point mutants. Rectification index (R.I.) of Kmpv1 (V, red) KmpvSP1 (SP, black), chimera with Kmpv1 pore and KmpvSP1 TM domains (ChA, blue) as well as mutants of KmpvSP1 (grey). The latter comprise AAs in which Kmpv1 differs from the inward rectifiers KmpvPL1 and KmpvSP1 (see Figure 1). The relevant AAs were mutated individually in KmpvSP1 to match the sequence of the ohmic Kmpv1 channel. In the mutant dNSP1 three AAs (T2-I4) in the N-terminus of KmpvSP1 were deleted. All constructs were expressed in HEK293 cells as in Figures 1 and 2. For mutant channels, which conducted significantly more inward current than un-transfected cells, the rectification index R.I. was calculated. R.I. is the ratio of the chord conductance at +25 mV (G₂₅) and −75 mV (G_-75) (R.I. = G₂₅/G-75). The mean R.I. values (± sd; number of recordings in brackets) are plotted. The R.I. value for Kmpv1 (V) is significantly (P < 0.001; ***) larger than that of all other constructs. a one way anova test showed that the r.i. values of kmpvsp1 (sp) and all mutants/chimera are not significantly different (p > 0.1); none of the mutations converted a rectifier into an ohmic conductor.