1. cGMP-Mediated Facilitation in Nerve Terminals by Enhancement of the Spike Afterhyperpolarization 
Vitaly A. Klyachko, Gerard P. Ahern, Meyer B. Jackson 
Neuron 
Volume 31, Issue 6, Pages (September 2001)
DOI: /S (01)

2. Figure 1
cGMP-Dependent and Independent Components of K+ Current Enhancement by NO
Whole-terminal K+ current was measured at 20 s intervals at the end of 400 ms depolarizing pulses from −80 to 50 mV (the final current shows a far greater change than the peak) (see Figure 2). Patch pipettes contain sodium nitroprusside (0.5 mM), a form of caged NO. After waiting ∼3 min to allow caged NO to perfuse the nerve terminal interior, photolysis released NO (arrow; earlier time points not shown). Control (squares; n = 4) and 50 μM ODQ (circles; n = 5; ODQ is a guanylate cyclase inhibitor added to the patch pipette) data are plotted as percentage change from the initial value. ODQ slows the initial rise in K+ current induced by NO (ANOVA yielded p < 0.01). The difference between these two curves is plotted in the inset to give the guanylate cyclase-dependent component of K+ current enhancement elicited by NO
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3. Figure 2 K+ Current Enhancement by cGMP
(A) In recordings with caged cGMP, flashes of UV light enhance final K+ current (reflecting largely BK current) but leave the peak K+ current (reflecting largely A-current) only slightly changed. Each of five consecutive light flashes, applied at 15–20 s intervals, enhanced current.
(B) Enhancement of K+ current by the membrane-permeable cGMP analog pCPT-cGMP (1 mM) applied by pressure ejection from a nearby micropipette.
(C) Average enhancement of peak and final K+ current for caged cGMP and pCPT-cGMP. The numbers of measurements are above each bar. Pulse protocols are as described in Experimental Procedures
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4. Figure 3 Single-Channel Analysis of cGMP Action
(A) In an excised outside-out patch containing a BK channel (with solutions as in Experimental Procedures), channel openings are infrequent and brief due to the presence of EGTA and the absence of added Ca2+ (control; membrane potential = 50 mV in all traces). Application of 10 μM ionomycin to the outside of the patch increases channel activity (+ Ionomycin). Channel activity recovered to the initial low level after removal of ionomycin (Wash out). Uncaging cGMP inside the patch pipette increased channel activity ∼5-fold (+ cGMP). About 5 min later, activity had recovered to the control levels (Recovery).
(B) The amplitude distribution of the open channel for control (open circles), ionomycin (open triangles), and cGMP (filled squares) were normalized to the maximum frequency within the open-channel peak (∼8 pA). The full distributions are shown in the inset, with the selected region delimited by arrows.
(C) Single-channel current was plotted versus voltage. The patch pipette solution contained 130 mM KCl (see Experimental Procedures), but the outside solution was (in mM) 130 KCl, 10 NaCl, 10 HEPES, 1 MgCl2, and 2 CaCl2 (pH 7.2). Both inward and outward currents extrapolated to 0 mV. The slope conductance was 125 pS for inward current and 212 pS for outward current.
(D) Open probability was determined from single-channel records 20 s in length. Each bar shows the average from the same seven patches before and after uncaging cGMP
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5. Figure 4
Role of PKG
(A) Replacing ATP in the patch pipette solution with the nonhydrolyzable ATP analog AMP-PNP (4 mM) blocked the increase in K+ current induced by uncaging cGMP (for control, see Figure 2A).
(B) KT5823, a PKG inhibitor, (1 μM) added to the patch solution also blocked the action of cGMP.
(C) With HA100 (50 μM), an inhibitor of both PKA and PKC, cGMP enhanced K+ current as well as in controls.
(D) A summary of the actions of protein kinase inhibitors on enhancement of K+ current at the end of a depolarizing pulse. Numbers of experiments are above each bar. The fifth bar shows the result with the PKC inhibitor bisindolylmaleimide (100 nM). The increases in K+ current with AMP-PNP and KT5823 were not statistically significant (p > 0.7 and p > 0.3, respectively), indicating effective block. cGMP-induced current increases with HA100 and bisindolylmaleimide were indistinguishable from controls (p > 0.5 and p > 0.7, respectively), indicating no block
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6. Figure 5 Reversibility of K+ Current Enhancement by cGMP
K+ current at the end of depolarizing pulses was measured at 20 s intervals. Photorelease of cGMP at the arrow shows that BK channels are enhanced within ∼20 s. In control experiments, K+ current decayed more rapidly (circles; τ = 72 ± 7 s, n = 7) than in experiments with 250 μM IBMX in the pipette solution (squares; τ = 264 ± 21 s, n = 7). Because IBMX is a broad-spectrum phosphodiesterase inhibitor, this experiment indicates that cGMP hydrolysis contributes to the recovery of K+ current
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7. Figure 6 Voltage-Dependence of Final K+ Current Enhancement by cGMP
(A) Conductance was plotted versus voltage for a series of voltage steps (holding potential −80 mV). K+ current was converted to conductance by dividing by the K+ driving force, based on a computed K+ Nernst potential of −88.7 mV. For each terminal, a control curve was recorded prior to uncaging cGMP. Circles represent the control experiment, and squares represent cGMP. Because of the decay of the cGMP effect on K+ current (see Figure 5), two separate uncagings were performed in each terminal, and two sets of measurements were made, one with an ascending sequence of voltage steps and the other with a descending sequence. For each recording, the results for the two sequences were averaged, and the plot shows the average from five such experiments.
(B) An inactivation curve was measured in a separate set of terminals (n = 5) by a series of pulses from various prepulse potentials followed by a voltage jump to +50 mV. As in (A), two uncaging experiments were performed in each nerve terminal in ascending and descending pulse sequences
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8. Figure 7 cGMP and Action Potentials
(A). After uncaging cGMP, action potentials repolarized more rapidly and had more prominent afterhyperpolarizations. Inset: Na+ channel inactivation function, h∞ (a Boltzmann function with V1/2 = −67.6 mV and slope factor = 6.9 mV, parameters from Ahern et al., 2000). The dashed lines show an increase in Na+ channel availability of 0.15 resulting from a 4.0 mV hyperpolarization.
(B) Response to a 10 s, 15 Hz pulse train. Most pulses fail to evoke spikes toward the end of the control train. Uncaging cGMP in the same nerve terminal reduced the failure rate.
(C) Average increases in action potential duration during 10 s, 15–30 Hz trains. cGMP narrows the first action potential by a small but statistically significant amount. At the end of the train, action potentials are considerably broader in both controls and cGMP. The last cGMP and control action potential durations were not significantly different.
(D) Percentage of stimuli that evoke an action potential in the last 2 s of a 10 s train such as shown in (B) (number of experiments above each bar). Action potential success rate is more than doubled by cGMP
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9. Figure 8 cGMP Increases Ca2+ Entry during Spike Trains
(A) During a 30 Hz train, action potential failure becomes very frequent within ∼2 s. After photolysis of caged cGMP, in the same nerve terminal a greater percentage of current pulses evoked action potentials (see Figure 7D).
(B) Fluorescence of the Ca2+ indicator Fluo-4 during trains of action potentials in (A) shows a rise in cytosolic Ca2+. Uncaging cGMP enhanced Ca2+ entry, increasing the fluorescence change of Fluo-4 by an average of 102%
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10. Figure 9
Repetitive Firing Activated the NO/cGMP Cascade and Enhanced Action Potentials
Recordings were made under current clamp using the NOS solution (see Experimental Procedures). Trains of current pulses at 25 Hz (duration 10 s) were applied at 1 min intervals, starting 2–3 min after break-in.
(A) The first train (0 min) and the sixth train (6 min) illustrates the increase in the action potential success rate.
(B) When ODQ was added to the patch-pipette solution, the action potential success rate decreased.
(C) The fraction of current pulses eliciting action potentials was determined in the final 2 s of a train and normalized to the success rate determined in the first train. The final success rate was plotted versus time, starting with the first train. The control success rate increased but declined when ODQ or 7-NI were added to the patch pipette to block guanylate cyclase or NOS, respectively (control, n = 6; ODQ, n = 5; 7-NI, n = 5). ANOVA gave p < 10−5 for ODQ versus control and p < 0.04 for ODQ versus 7-NI
Neuron  , DOI: ( /S (01) )