Liu C , Xie C , Grant K , Su Z , Gao W , Liu Q , Zhou L .

R01 GM098592 NIGMS NIH HHS , R01 GM109193 NIGMS NIH HHS

	Figure 1. Use ROONS-EGFP as a model to determine the relationship between macroscopic fluorescence intensity and the total number of channels on the membrane patch. (A, top) Two single-channel traces of ROONS-EGFP channel recorded at the potential of 80 mV. Transitions between opening and closing were recorded in the presence of 3.5 µM cGMP, a subsaturating concentration for ROONS-EGFP. (bottom left) Maximal channel opening recorded with 35 µM cGMP. The current level corresponding to the closed state is indicated by a gray line. (bottom right) The current trace of the same patch recorded in the absence of cGMP. The gray line represents the current level of the open state. (B) Histogram of the top recording trace from A. Averaged amplitude of single-channel currents was 4.29 ± 0.57 pA (n = 7). (C, left) Macroscopic current recorded under 35 µM cGMP and a voltage step from 0 to 40 mV. (right) The corresponding brightfield and fluorescence images. (D, left) Histogram of the pixel intensities of the whole fluorescence image collected using four different exposure durations. The Cascade 1K is a 16-bit camera so that the pixel intensity ranges from 0 to 65,535. (right) Total fluorescence intensity from the ROI surrounding the patch of membrane patch (green) and the background (blue), which was separately recorded after moving the recording pipette out of the view. (E) Cross-plots of fluorescence intensity versus current amplitude. Two x axes show the macroscopic current (bottom) and the corresponding number of channels (top). The amplitude of single-channel current, 4.29 pA, was used in the conversion. Linear fit statistics: Pearson correlation coefficient, 0.823. Green curves show the confidence bands (95%).
	Figure 2. PCF recordings of the mHCN2-EGFP channel and cross-plots of fluorescence intensity versus current amplitude. (A, left) Macroscopic current recorded with 3 µM cAMP in the bath (red trace) and a voltage step from −40 to −140 mV. (right) The corresponding brightfield and fluorescence images. (B, left) Histogram of the pixel intensities of the whole image collected with four different exposure durations. Notice longer exposure durations, 100 and 200 ms, lead to saturated pixels. (right) Total fluorescence intensity from the membrane patch (green) and the background (blue). (C) Cross-plots of fluorescence intensity versus current amplitude. Linear fit statistics: Pearson correlation coefficient, 0.907. Green curves show the confidence bands (95%).
	Figure 3. Cross-plots of fluorescence intensity versus current amplitude for mHCN1-EGFP and spHCN-EGFP channels. (A, left) Macroscopic current of the mHCN1-EGFP channel recorded with 3 µM cAMP in the bath. A voltage step from 0 to −120 mV was used for channel activation. Tail current was recorded at −40 mV. (B, left) Macroscopic current of spHCN-EGFP channel recorded with 30 µM cAMP in the bath. A voltage step from 0 to −120 mV was used for channel activation. Tail current was recorded at 40 mV. (A and B, right) The corresponding brightfield and fluorescence images. (C) Cross-plots of fluorescence intensity versus current amplitude for the mHCN1-EGFP channel. Linear fit statistics: Pearson correlation coefficient, 0.973. (D) Cross-plots of fluorescence intensity versus current amplitude for the spHCN-EGFP channel. Linear fit statistics: Pearson correlation coefficient, 0.859. Green curves show the confidence bands (95%).
	Figure 4. NSNA of macroscopic mHCN2-EGFP currents. (A, top) Voltage protocol used for channel activation and deactivation. (bottom) 6 representative traces from 100 repeatedly collected traces. (B) Current variance over the complete time course of a single episode. (C) Current variance versus mean current amplitude. Red, parabola fit of the macroscopic current part. Green, parabola fit of the tail current part. (D) Normal residual after curve fit for macroscopic current (corresponding to the red trace in C). Results: single-channel current, −0.298 pA (−130 mV); single-channel conductance, 2.29 pS; total number of channels, 8,715; Po, 75.5%. Adjusted R2, 0.934. (E) Normal residual after curve fit for tail current (corresponding to the green trace in C). Results: single-channel current, −0.114 pA (−40 mV); single-channel conductance, 2.86 pS; total number of channels, 4,707; Po, 88.4%. Adjusted R2, 0.963.
	Figure 5. Determining the relative ionic selectivity for the mHCN2-EGFP channel. Symmetrical solutions were used on both sides of the membrane. All recordings were collected in the presence of saturating concentration of cAMP (3 µM). (A) Current trace recorded with solutions containing 110 mM Na+ and 5 mM K+ as the charge carriers. (B) Current trace recorded with solutions containing 110 mM NH4+ and 5 mM K+. (C) Cross-plots of fluorescence intensity versus current amplitude. Symmetric solutions contain 110 mM Na+ only. The slope was 0.676 a.u./pA. As the control, the slope of the condition with 110 mM K+ and 2 mM Na+ was 0.0067 a.u./pA. Thus, PK+/PNa+ based on the ratio of two slopes was 101 (in the absence of K+). Linear fit statistics: Pearson correlation coefficient, 0.766. (D) Results of 110 mM Na+ and 5 mM K+. The slope was 0.023 a.u./pA. PK+/PNa+ was 3.4 (in the presence of 5 mM K+). Linear fit statistics: Pearson correlation coefficient, 0.845. (E) Results of 110 mM NH4+ only. The slope was 0.088 a.u./pA. PK+/PNH4+ was 13.1 (in the absence of K+). Linear fit statistics: Pearson correlation coefficient, 0.811. (F) Results of 110 mM NH4+ and 5 mM K+. The slope was 0.081 a.u./pA. PK+/PNH4+ was 12.1 (in the presence of 5 mM K+). Linear fit statistics: Pearson correlation coefficient, 0.832. Green curves show the confidence bands (95%).

Alvarez, Counting channels: a tutorial guide on ion channel fluctuation analysis. 2002, Pubmed

Alvarez, Counting channels: a tutorial guide on ion channel fluctuation analysis. 2002, Pubmed
Armstrong, Inactivation of the sodium channel. II. Gating current experiments. 1977, Pubmed
Armstrong, Charge movement associated with the opening and closing of the activation gates of the Na channels. 1974, Pubmed
Barrow, Low-conductance HCN1 ion channels augment the frequency response of rod and cone photoreceptors. 2009, Pubmed
Benndorf, Unraveling subunit cooperativity in homotetrameric HCN2 channels. 2012, Pubmed
Biel, Hyperpolarization-activated cation channels: from genes to function. 2009, Pubmed
Boron, Regulation of intracellular pH. 2004, Pubmed
Carrisoza-Gaytán, The hyperpolarization-activated cyclic nucleotide-gated HCN2 channel transports ammonium in the distal nephron. 2011, Pubmed , Xenbase
Dekker, Cooperative gating between single HCN pacemaker channels. 2006, Pubmed
DiFrancesco, Characterization of single pacemaker channels in cardiac sino-atrial node cells. , Pubmed
Flynn, Structure and rearrangements in the carboxy-terminal region of SpIH channels. 2007, Pubmed , Xenbase
Frace, External K+ increases Na+ conductance of the hyperpolarization-activated current in rabbit cardiac pacemaker cells. 1992, Pubmed
Goldman, POTENTIAL, IMPEDANCE, AND RECTIFICATION IN MEMBRANES. 1943, Pubmed
Goulding, Molecular cloning and single-channel properties of the cyclic nucleotide-gated channel from catfish olfactory neurons. 1992, Pubmed
Goulding, Molecular mechanism of cyclic-nucleotide-gated channel activation. 1994, Pubmed , Xenbase
Goulding, Role of H5 domain in determining pore diameter and ion permeation through cyclic nucleotide-gated channels. 1993, Pubmed , Xenbase
Harnett, Distribution and function of HCN channels in the apical dendritic tuft of neocortical pyramidal neurons. 2015, Pubmed
HODGKIN, The effect of sodium ions on the electrical activity of giant axon of the squid. 1949, Pubmed
Huang, Presynaptic HCN channels regulate vesicular glutamate transport. 2014, Pubmed
Jan, A superfamily of ion channels. 1990, Pubmed
Johnson, The carboxyl-terminal region of cyclic nucleotide-modulated channels is a gating ring, not a permeation path. 2005, Pubmed , Xenbase
Kaupp, Cyclic nucleotide-gated ion channels. 2002, Pubmed
Ko, Serotonin modulates spike probability in the axon initial segment through HCN channels. 2016, Pubmed
Kole, Single Ih channels in pyramidal neuron dendrites: properties, distribution, and impact on action potential output. 2006, Pubmed
Kusch, Interdependence of receptor activation and ligand binding in HCN2 pacemaker channels. 2010, Pubmed , Xenbase
Kusch, Patch-clamp fluorometry: electrophysiology meets fluorescence. 2014, Pubmed
Li, Single-channel kinetics of the rat olfactory cyclic nucleotide-gated channel expressed in Xenopus oocytes. 1999, Pubmed , Xenbase
Ludwig, A family of hyperpolarization-activated mammalian cation channels. 1998, Pubmed
Matthews, Activation of single ion channels from toad retinal rod inner segments by cyclic GMP: concentration dependence. 1988, Pubmed
Moore, An upper limit to the number of sodium channels in nerve membrane? 1967, Pubmed
Musa-Aziz, Concentration-dependent effects on intracellular and surface pH of exposing Xenopus oocytes to solutions containing NH3/NH4(+). 2009, Pubmed , Xenbase
Neher, Single-channel currents recorded from membrane of denervated frog muscle fibres. 1976, Pubmed
Ruiz, Single cyclic nucleotide-gated channels locked in different ligand-bound states. 1997, Pubmed , Xenbase
Salpeter, Nicotinic acetylcholine receptors in vertebrate muscle: properties, distribution and neural control. 1985, Pubmed
Schneider, NIH Image to ImageJ: 25 years of image analysis. 2012, Pubmed
Sigworth, Single Na+ channel currents observed in cultured rat muscle cells. 1980, Pubmed
Thon, Elementary functional properties of single HCN2 channels. 2013, Pubmed , Xenbase
Tibbs, Allosteric activation and tuning of ligand efficacy in cyclic-nucleotide-gated channels. 1997, Pubmed , Xenbase
Ulbrich, Subunit counting in membrane-bound proteins. 2007, Pubmed , Xenbase
Wollmuth, Ionic selectivity of Ih channels of rod photoreceptors in tiger salamanders. 1992, Pubmed
Wu, Inner activation gate in S6 contributes to the state-dependent binding of cAMP in full-length HCN2 channel. 2012, Pubmed , Xenbase
Wu, State-dependent cAMP binding to functioning HCN channels studied by patch-clamp fluorometry. 2011, Pubmed
Zagotta, Structure and function of cyclic nucleotide-gated channels. 1996, Pubmed
Zheng, Gating rearrangements in cyclic nucleotide-gated channels revealed by patch-clamp fluorometry. 2000, Pubmed
Zheng, Patch-clamp fluorometry recording of conformational rearrangements of ion channels. 2003, Pubmed , Xenbase