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FIGURE 1. Summary of the structure of a SpIH C-terminal fragment. A, side view showing the SpIH C-terminal structure (amino acids 470–665). Three subunits are shown in a surface representation, and one subunit is in ribbon representation. The tetrameric structure is positioned beneath a schematic diagram representing the transmembrane domain idealized from the space-filled model of Kv1.2 (32). B, ribbon representation of a single SpIH C-terminal structure (Protein Data Bank code 2PTM). Helices are colored red (A′–F′, A–C, and P), β-sheets are colored cyan, and connecting loops are colored black.
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FIGURE 2. The C terminus acts as an autoinhibitory domain. A, schematic of the membrane topology of a single subunit of the SpIH channel. Representative currents were measured from inside-out excised patches of oocyte membrane. Voltage pulses were applied to patches in the range of −30 to −120 mV, followed by a voltage step to −40 mV. The holding voltage was 0 mV. Currents were measured in the absence of cyclic nucleotides (black traces) and in the presence of 1 mm cAMP (red traces). B, schematic representing the topology of the C-terminal deletion mutant (deleted amino acids 471–767). Representative currents were measured over a voltage range of −40 to −120 mV from a 0-mV holding potential in the absence of cyclic nucleotides.
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FIGURE 3. Effects of PIP2 on cyclic nucleotide-modulated SpIH channels. A, representative currents measured in response to a hyperpolarizing voltage step to −120 mV, followed by a voltage step to −40 mV from a holding voltage of 0 mV. Currents were measured in the absence of cyclic nucleotide (dashed black trace) and in the presence of 1 mm cGMP (dashed green trace), 1 mm cAMP (dashed red trace), cAMP + 10 μm diC8-PIP2 (solid red trace), and cGMP + 10 μm diC8-PIP2 (solid green trace). B, normalized G-V relationships for each condition in A. Tail currents at −40 mV were measured and normalized to the tail currents measured in the presence of 1 mm cAMP (dashed red trace in A). Solid and dotted curves represent fits to the data of the Boltzmann equation (see “Experimental Proceduresâ€). Leak currents were subtracted as described under “Experimental Procedures.†C, bar graph showing the mean ± S.E. of the PIP2-induced shift in the midpoint of the voltage dependence (ΔV1/2 = V1/2,PIP2 − V1/2) for both cAMP- and cGMP-modulated channels (n = 5). D, bar graph showing the mean ± S.E. of the relative normalized conductance (G/Gmax,cAMP) for cyclic nucleotide-modulated channels in the absence and presence of PIP2 (n = 5). cA, cAMP; cG, cGMP.
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FIGURE 4. The C-terminal domain mediates PIP2 inhibition and the transmembrane domain mediates PIP2-induced positive shift in voltage. A and B, representative currents recorded from SpIHΔC channels lacking the C-terminal region (deleted amino acids 471–767) in response to voltage steps between −40 and −130 mV in the absence and presence of 30 μm diC8-PIP2. The red trace indicates currents measured at the same voltage, −70 mV. C, G-V relationships for SpIHΔC measured from tail currents at −40 mV in the absence of PIP2 (black circles), in the presence of 30 μm diC8-PIP2 (gray circles), and after PIP2 was washed out (white circles). The curves represent fits of the Boltzmann equation to the data. D and E, representative currents recorded from C-terminal deleted SpIHΔNC channels (deleted amino acids 1–160 and 471–767) in response to voltage steps between −40 and −130 mV in the absence and presence of 30 μm diC8-PIP2. The red trace indicates currents measured at the same voltage, −90 mV. F, G-V relationships for SpIHΔNC measured from tail currents at −90 mV in the absence of PIP2 (black circles), in the presence of 30 μm diC8-PIP2 (gray circles), and after PIP2 was washed out (white circles). G, change in voltage dependence as determined for the difference in V1/2 before and after modulation by PIP2 (ΔV1/2). H, normalized conductance in diC8-PIP2 for SpIHΔC and SpIHΔNC.
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FIGURE 5. Positively charged residues in the A′ helix of SpIH near the membrane. A, sequence alignment of amino acids in the A′ helix of SpIH and related CNG channels. Red arrows indicate the positions of positively charged residues in SpIH. B, model of the atomic structure of SpIH showing positive residues in the A′ helix as sticks on the ribbon backbone (Lys-475, Arg-478, and Lys-482; red). Lys-480 (blue sticks) points away from the membrane and forms an intersubunit salt bridge (dashed red line) with Glu-522 in the neighboring subunit (yellow stick in the yellow subunit). The position of the membrane is shown by the labeled bar.
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FIGURE 6. Effects of 10 μm PIP2 on cyclic nucleotide modulation of mutant SpIH channels (R475C, R478C, and K482C). A, C, and E, representative currents measured in response to a hyperpolarizing voltage step to −120 mV, followed by a voltage step to −40 mV from a holding voltage of 0 mV. Currents were measured in the absence of cyclic nucleotide (dashed black trace) and in the presence of 1 mm cGMP (dashed green trace), 1 mm cAMP (dashed red trace), cAMP + 10 μm diC8-PIP2 (solid red trace), and cGMP + 10 μm diC8-PIP2 (solid green trace). B, D, and F, normalized G-V relationships for each mutant. All currents were measured from tail currents at −40 mV and normalized (see “Experimental Proceduresâ€). Data in the absence of PIP2 are shown as open circles, and those in the presence of PIP2 are shown as closed circles (see legend in B). Solid and dotted curves represent fits to the data of the Boltzmann equation (see “Experimental Proceduresâ€). nn, no nucleotide; cA, cAMP; cG, cGMP.
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FIGURE 7. Change in free energy of opening caused by PIP2 modulation of cGMP-modulated SpIH and mutant channels. The bar graph represents mean ΔΔG values ± S.E. (see “Experimental Proceduresâ€). Data sample sizes are shown inside boxes.
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FIGURE 8. Dose-response relationship of PIP2 and PIP in SpIH and K478C mutant channels. A, representative SpIH currents measured during a −120-mV test pulse in the presence of 1 mm cGMP (black trace) and increasing concentrations of diC8-PIP2 (100 μm = red trace). B, representative K478C currents measured during a −120-mV test pulse in the presence of 1 mm cGMP (black trace) and increasing concentrations of diC8-PIP2 (100 μm = red trace). C and D, data from three patches were averaged and plotted as a function of PIP2 or PIP concentration, respectively. Data were fit with the Hill equation (see “Experimental Proceduresâ€). For SpIH, IPIP2/IcGMP,No PIP2 = 55%, K1/2,PIP2 = 3.6 μm, IPIP/IcGMP,No PIP = 49%, and K1/2,PIP = 13.6 μm.
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