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Figure 1. Counting the number of channels in excised giant membrane patches. (A) Currents recorded at ±60 mV in the presence of 1 mmol/L cGMP in symmetrical 110 mmol/L NaCl using patch pipette with a diameter of 1–2 μm. (B) Currents recorded at ±10 mV in the presence of 1 mmol/L cGMP in symmetrical 110 mmol/L NaCl using patch pipette with a diameter of 12 μm. (C) blockage by extracellular TEACl in outside-out patches. Black traces represent currents at ±60 mV in symmetrical 110 mmol/L NaCl and in green the same but with TEACl in the bath solution and NaCl + 1 mmol/L cGMP inside the patch pipette. (D) Currents recorded at ±60 mV in the presence of 1 mmol/L cGMP with 110 mmol/L NaCl in bath solution and 110 mmol/L TEACl inside a large patch pipette. A, B, and D are excised patches in inside-out configuration, whereas C is in an outside-out patch. Red broken lines indicate 0 current level.
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Figure 2. Comparison of the dependence of G/G−150 and Q/Q−120 on voltage in the spHCN channels. (A) Current recordings from giant patches excised from oocytes injected with the mRNA coding for the spHCN channels (lower panel). Voltage steps are shown in the upper panel. Current recordings obtained in the presence of 1 mmol/L cAMP and in symmetrical 110 mmol/L KCl conditions. The inset in the box represents the tail current (tail potential at +50 mV) and the arrow corresponds to the isochronal currents used to calculate the conductance (G). (B) Gating current for spHCN channel in response to voltage steps from −10 to −120 mV, from a holding potential of −10 mV, tail potential +50 mV. (C) Ionic conductance (G/G−150 filled circles) and normalized charge movement (Q/Q−120 open circles) for the electrical recordings in A and B; the conductance (G) was calculated from tail current (arrow in panel A) normalized as G(V) = [I(V)−Imin]/(Imax−Imin); the gating current (Fig. 2A) was integrated in time so to obtain the charge (Q). Gray broken lines in panels A and B indicate 0 current level.
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Figure 3. Macroscopic and tail currents in symmetrical Li+, Na+, K+, Rb+, and Cs+ conditions. (A–E) Macroscopic currents recorded from excised patches in symmetrical solutions of Li+ (A), Na+ (B), K+ (C), Rb+ (D), and Cs+ (E) with 1 mmol/L cGMP in the intracellular medium. Leak and capacitative components were removed subtracting from the cGMP-activated current those records obtained in response to the same voltage protocol but without cGMP. The voltage commands were stepped from a holding potential of 0 mV to prepulses varying between −100 and +200 mV in 20 mV steps. At the end, the voltage command was moved to −200 mV for 5 msec in order to elicit tail currents It(V). Gray broken line indicates 0 current level. (F–J), Enlargement of tail currents (boxed areas in A–E) in Li+ (F), Na+ (G), K+ (H), Rb+ (I), and Cs+ (J). Current recordings were filtered at 10 kHz and sampled at 50 kHz to resolve rapid transients.
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Figure 4. Macroscopic and tail currents in symmetrical Na+ and Rb+ conditions at low pHo. (A) Macroscopic currents recorded from excised patches in symmetrical solutions of Na+ with 1 mmol/L cGMP on the cytoplasmic side and pHo 5 at the extracellular side of the membrane (pHi = 7.4). Tail currents at −200 mV evoked by prepulses at voltages from −200 to +200 mV (ΔV=20 mV); current recordings were filtered at 10 kHz and sampled at 50 kHz to resolve rapid transients. (B) Dependence of G/G+200 on V for Na+ at pHo 7.4 (black dots) and 5 (red squares). (C) Estimation of Po/Po_max from tail currents. Po/Po_max was estimated as It/It_max. (D) as in A but in symmetrical solutions of Rb+. E, as in B but for Rb+. F, as in C but for Rb+.
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Figure 5. G/G+200, Po/Po+200, γ/γ+200 against V relationships and noise analysis in symmetrical Li+, Na+, K+, Rb+, and Cs+ conditions. (A–E) Relationship between σI2/I and V for Li+, (A), Na+ (B), K+ (C), Rb+ (D), and Cs+ (E). The background noise observed in the absence of cGMP was subtracted; (n ≥ 6). F, Dependence of G/G+200 on V for Li+, Na+, K+, Rb+, and Cs+. G/G+200 is the fractional steady conductance obtained from macroscopic currents elicited by voltage commands from −200 to +200 mV (ΔV = 20 mV). G, Dependence of Po/Po+200 on V for Li+, Na+, K+, Rb+, and Cs+ obtained from tail currents as It/It+200. (H) Dependence of γsc/γsc+200 on V for Li+, Na+, K+, Rb+, and Cs+ ions. The γsc/γsc+200 relationship for various ions was computed from the G/G+200 and Po/Po+200 plots as described in the text. Li+ (filled diamonds), Na+ (open squares), K+ (open circles), Rb+ (filled triangles), and Cs+ (open triangles); (n ≥ 4).
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Figure 6. Tail currents, isc /isc+200 against V relationship and predicted macroscopic current noise in symmetrical Rb+ and Cs+ conditions. (A,B) Macroscopic currents in symmetrical solutions of Rb+ (A) and Cs+ (B). Voltage prepulse held at +200 mV was followed by test potentials ranging from −200 mV to +200 mV (ΔV = 20 mV). Gray broken line indicates 0 current level. (C,D) Dependence of inward transient kinetics on V in symmetrical Rb+ (C) and Cs+ (D). These transients decay with a single time constant between 0.3 and 1.2 msec. (E,F) Dependence of isc /isc+200 on V for Rb+ (E) and Cs+ (F) obtained from instantaneous I–V relationship measured immediately after the depolarizing voltage prepulse (see arrows in A,B). Solid gray lines represent the expected isc /isc+200 curves from the γsc /γsc+200 plots in 5H; (n ≥ 4). G,H, Dependence of isc(1-Po) on V for Rb+ (G) and Cs+ (H). In the presence of 1 mmol/L cGMP, WT CNGA1 channels have a dominant single-channel conductance (γsc). Under these conditions, the noise variance (σI2) is related to the amplitude of the mean current (I) by: σI2 = iscI−I2/n. If isc is the single-channel current then the ratio σI2/I of the variance of current fluctuations (σI2) and of the mean current (I) is isc(1−Po). Filled circles show the experimentally observed noise (σI2/I) in macroscopic currents at different voltages (see also Fig. 5A–E). Solid lines represent the expected isc(1−Po) curves computed from the Po/Po+200 and isc /isc+200 plots as described in the text.
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Figure 7. Macroscopic and tail currents in symmetrical MA+, DMA+, and EA+ conditions. (A–C) Macroscopic currents recorded from excised patches in symmetrical solutions of MA+ (A), DMA+ (B), and EA+ (C) with 1 mmol/L cGMP in the intracellular medium. Voltage prepulses from −180 to +200 mV (ΔV = 20 mV) were followed by a tail potential at −200 mV. Gray broken line indicates 0 current level. (D–F) Enlargement of tail currents in MA+ (D), DMA+ (E), and EA+ (F). Current recordings were filtered at 10 kHz and sampled at 50 kHz. (G) Dependence of G/G+200 on V for MA+, DMA+, and EA+. MA+ open circles, DMA+ filled circles, EA+ filled squares. (H) Dependence of the slow activation time constants (τact_s) on V in the presence of EA+. The bell-shaped distribution of τact_s was fitted to the following relation: τact_s = 1/[α(V)+β(V)], where α(V) and β(V) are the voltage-dependent forward and backward rate constants, respectively. Dependence of It−Imin/I+200, obtained from tail currents, on V for MA+, DMA+, and EA+. Symbols as in G; (n ≥ 4).
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Figure 8. Noise analysis and single-channel recordings in symmetrical DMA+ conditions. (A) Macroscopic currents fluctuations in symmetrical solutions of DMA+ at −200, −150, and −100 mV from the same excised patch. Current r.m.s. (σ) were obtained after background noise subtraction as σcGMP − σseal, where σcGMP and σseal indicate the r.m.s recorded in the presence and absence of 1 mmol/L cGMP, respectively. The corresponding current amplitude histogram after mean current subtraction is shown on the right. Crosses and open triangles refer to traces obtained in the presence and absence of cGMP, respectively. Data were fitted with a Gaussian distribution yielding σcGMP and σseal (solid and dashed lines, respectively). Red broken line indicates 0 current level. (B) Relationship between σI2/I and V for DMA+ (n = 3). (C) Relationship between the expected relative single-channel current (isc/isc-200) and V. Filled circles show the experimentally observed normalized noise (rel σI2/I) at different voltages. Solid line represents the isc(V)/isc-200 curve (est) computed as described in the text. (D) Single-channel recordings at −200, −150, and −100 mV from the same patch in the presence of symmetrical DMA+. Current records were acquired at 50 kHz, and filtered at 10 kHz. For presentation purposes, traces were digitally filtered at 5 kHz and 1 kHz (left and right panels, respectively). Dashed lines in single-channel recordings indicate the closed state (C) of the channel. (E) All-point amplitude histograms from recordings as in (D) after offline digital filtering at 1 kHz. Dashed lines refer to all-point amplitude histograms Gaussian fit from records obtained in the same experimental conditions as in (D) but in the absence of cGMP. Single-channel current amplitude that were estimated from the peak current analysis procedure are indicated (Marchesi et al. 2012). The vertical dotted line indicates 0 current level.
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Figure 9. Estimating the number of channels underlying current fluctuations in symmetric DMA+ (A) Schematic depicting an excised patch in bi-ionic conditions of intracellular Na+ (Nai+) and extracellular DMA+ (DMA0+) with two CNGA1 channels present in the membrane patch. The arrows indicate the direction of the electrochemical driving force at +60 mV. In bi-ionic conditions, the cGMP-activated current at +60 mV was carried almost by Na+ ions moving from the bath solution toward the patch pipette. (B) Single-channel recording from the experimental conditions shown in A. Well-resolved openings slightly larger than those usually observed in symmetrical Na+ could be detected. Dashed line indicates the closed (C) state of the channel; O1 and O2 refer to the two open states. (C) All-point amplitude histogram from the electrical recording shown in B. Data were fitted with a three component Gaussian function (solid red line) indicating that two CNGA1 channels were present in the membrane patch.
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Figure 10. Macroscopic and tail currents in symmetrical DMA+ conditions for WT and the R2Q_WT construct. (A, B) Macroscopic currents recorded from excised patches in symmetrical solutions of the DMA+ for WT channel (A) and for the R2Q_WT mutant channel (B) with 1 mmol/L cGMP in the intracellular medium. Voltage prepulses from −180 to +200 mV (ΔV = 20 mV) were followed by a tail potential at −200 mV. Gray broken line indicates 0 current level. (C–D) Enlargement of tail currents (boxed areas in A,B) in DMA+ for WT channel (C) and for the R2Q_WT mutant channel (D). Current recordings were filtered at 10 kHz and sampled at 50 kH. (E,F) Dependence of It−Imin/I+200 on V for DMA+ in WT channel (E) and in the R2Q_WT mutant channel (F). Recordings from different patches are shown in different symbols. (G) Dependence of G/G+200 on V in symmetrical DMA+ solutions for the WT channel and for the R2Q_WT mutant channel. WT (open circles), R2Q_WT (filled circles); (n ≥ 5). (H) Dependence of It−Imin/I+200 on V in DMA+ for WT channel and for the R2Q_WT mutant channel. WT (open circles), R2Q (filled circles); (n ≥ 5). Data were fitted with a modified two components Boltzmann function (see Methods) yielding the following activation parameters: z1 = 1.71, 1.04; Vmid1 = −93.64, −53.01; z2 = 1.07, 0.98; Vmid2 = +130.73, +120.37; for the WT and the R2Q_WT channels, respectively.
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Figure 11. Gating currents measurements in WT CNGA1 channels. (A) Gating currents measurements in the presence of symmetrical TEA+ (110 mmol/L) and in the absence (left panels) and presence (right panels) of 1 mmol/L cGMP from a representative membrane patch, containing ≈30,000 ion channels. Only fast (<300 μsec) nonlinear capacitive artifacts were visible after P/−4 procedure, similar to those recorded in uninjected oocytes. Voltage commands P were as shown in the upper panels. Similar results were observed in five additional giant patches. (B) Contour plot depicting the predicted gating currents color-coded signal-to-noise ratio (S/N < 4, blue; 4 ≤ S/N ≤ 6, green, S/N > 6, red) as a function of z and gating currents time constant τ for a membrane patch containing approximately 3 × 104 ion channels. Off-gating currents for a simple two state model representing two possible states of a voltage sensor (resting and activated) were simulated. S/N was measured at the peak of the simulated off-gating currents. Background r.m.s. was assumed to be 1 pA.
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Figure 12. Partial sequence alignment among different ion channels and molecular structures of the KcsA and CNG mimicking NaK chimera featuring the interactions within the selectivity filter and surrounding scaffolding. (A) Dependence of I-200/I+200 on the permeating ions. ANOVA and Bonferroni post hoc tests were performed to compare each ion against Na+. Asterisks point out a statistical significance with a P < 0.01 (n ≥ 4.). (B) Partial sequence alignment in the pore region of the bovine alfa subunits (CNGA1-CNGA3), CNG mimicking NaK chimera (NaK2CNG-E) and K+ channels (KcsA, Shaker, Kv1.2). The conserved Glycine residue in the selectivity filter is highlighted in blue. The Tyrosine of the K+ selective channels signature sequence is replaced by a ring of Glutamate residues in CNG channels, while the aspartate immediately following the filter is substituted by a proline; highlighted in light gray. Residues involved in H-bonding in the KcsA and CNG chimera structures are marked in green. (C, D) Partial molecular structure of the KcsA channel (C) and NaK2CNG-E channel (D) as deposited in the Protein Data Bank (PDB entry code 1K4C and 3K0D, respectively) featuring the selectivity filter and the P-helix in two adjacent subunits. Crucial intersubunit and intrasubunit H bonds stabilizing the selectivity filter architecture are shown in both structures as dashed green lines. Residues side chains involved in H bonds are shown in ball-and-stick format while aromatic residues constraining the pore structure are shown in space-filling format. The K+ ions in the selectivity filter are shown as white spheres. The selectivity filter of the NaK2CNG-E chimera is stabilized by fewer hydrogen's bonds compared to KcsA channel.
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