Intracellular Uncaging of cGMP with Blue Light

We have made a new caged cGMP that is photolyzed with blue light. Using our recently developed derivative of 7-diethylaminocourmarin (DEAC) called DEAC450, we synthesized coumarin phosphoester derivatives of cGMP with two negative charges appended to the DEAC450 moiety. DEAC450-cGMP is freely soluble in physiological buffer without the need for any organic cosolvents. With a photolysis quantum yield of 0.18 and an extinction coefficient of 43 000 M^–1 cm^–1 at 453 nm, DEAC450-cGMP is the most photosensitive caged cGMP made to date. In patch-clamped neurons in acutely isolated brain slices, blue light effectively uncaged cGMP from DEAC450 and facilitated activation of hyperpolarization and cyclic nucleotide gated cation (HCN) channels in cholinergic interneurons. Thus, DEAC450-cGMP has a unique set of optical and chemical properties that make it a useful addition to the optical arsenal available to neurobiologists

Structure–Activity Relationship of N,N′-Disubstituted Pyrimidinetriones as Ca_V1.3 Calcium Channel-Selective Antagonists for Parkinson’s Disease

Ca_V1.3 L-type calcium channels (LTCCs) have been a potential target for Parkinson’s disease since calcium ion influx through the channel was implicated in the generation of mitochondrial oxidative stress, causing cell death in the dopaminergic neurons. Selective inhibition of Ca_V1.3 over other LTCC isoforms, especially Ca_V1.2, is critical to minimize potential side effects. We recently identified pyrimidinetriones (PYTs) as a Ca_V1.3-selective scaffold; here we report the structure–activity relationship of PYTs with both Ca_V1.3 and Ca_V1.2 LTCCs. By variation of the substituents on the cyclopentyl and arylalkyl groups of PYT, SAR studies allowed characterization of the Ca_V1.3 and Ca_V1.2 LTCCs binding sites. The SAR also identified four important moieties that either retain selectivity or enhance binding affinity. Our study represents a significant enhancement of the SAR of PYTs at Ca_V1.3 and Ca_V1.2 LTCCs and highlights several advances in the lead optimization and diversification of this family of compounds for drug development

Transient Activation of GABA_B Receptors Suppresses SK Channel Currents in Substantia Nigra Pars Compacta Dopaminergic Neurons

<div><p>Dopaminergic (DA) neurons in the substantia nigra pars compacta (SNc) are richly innervated by GABAergic neurons. The postsynaptic effects of GABA on SNc DA neurons are mediated by a mixture of GABA_A and GABA_B receptors. Although activation of GABA_A receptors inhibits spike generation, the consequences of GABA_B receptor activation are less well characterized. To help fill this gap, perforated patch recordings were made from young adult mouse SNc DA neurons. Sustained stimulation of GABA_B receptors hyperpolarized SNc DA neurons, as previously described. However, transient stimulation of GABA_B receptors by optical uncaging of GABA did not; rather, it reduced the opening of small-conductance, calcium-activated K⁺ (SK) channels and increased the irregularity of spiking. This modulation was attributable to inhibition of adenylyl cyclase and protein kinase A. Thus, because suppression of SK channel activity increases the probability of burst spiking, transient co-activation of GABA_A and GABA_B receptors could promote a pause-burst pattern of spiking.</p></div

VGCC contribution to SK.

<p>(A) Application of 10 μM isradipine (orange) to inhibit Ca_V1 channels did not reduce total SK charge (n = 8, Wilcoxon signed rank test, p = 0.3828), while inhibiting Ca_V3 channels with 10 μM mibefradil (blue) inhibited roughly half the charge (n = 8, Wilcoxon signed rank test, p = 0.0078). (B) 5 μM baclofen (green) application did not inhibit T-type calcium current (n = 12, Wilcoxon signed rank test, p = 0.1099).</p

RuBi-GABA uncaging.

<p>(A) Top, plot of the normalized firing rate before during and after a 60 s uncaging pulse (blue bar) of 5 μM RuBi-GABA in the presence of 25 μM gabazine (n = 4). Bottom, plot of normalized firing rate (black line) and running standard deviation (grey area) before, during and after a 50 ms uncaging pulse in the presence of 5 μM RuBi-GABA and 25 μM gabazine (n = 12); application of 2 μM CGP 55845 blunted the changes in spiking induced by RuBi-GABA uncaging (orange line, n = 4). Example raster plots are shown at the top of the panel. (B) Left, two different time scales showing action potentials just prior to and after GABA uncaging. Right, overlaid action potentials from just prior to and after GABA uncaging showing a clear reduction in the mAHP. (C-D) As in panel A (bottom) and B, but in the absence of gabazine (n = 9).</p

PKA activation prevents GABA_B modulation of SK.

<p>(A) Schematic diagram showing the hypothesized signaling pathway from GABA_B receptor activation to SK channels, and the site of action of 8-bromo-cAMP and H-89 in that pathway. (B) A 50 ms uncaging pulse elicited an immediate and significant change in SOP variance (black, n = 10, Wilcoxon signed rank test, p = 0.002). (C) The same was not seen when cells were incubated in 1 μM 8-bromo-cAMP (orange, n = 4, Wilcoxon signed rank test, p = 0.875). (D) Directly activating PKA with 1 μM 8-bromo-cAMP does not have an effect on SOP variance (n = 4, Wilcoxon signed rank test, p = 0.75). (E) Inhibiting PKA with 10 μM H89 increases SOP variance (n = 6, Wilcoxon signed rank test, p = 0.0938). (F) Summary data for panels A-B. (G) Summary data for panels C-D. (H) Top, inhibiting PKA with 10 μM H89 significantly decreases SK current (n = 6, Wilcoxon signed rank test, p = 0.0313). Bottom, directly activating PKA with 1 μM 8-bromo-cAMP does not have an effect on SK current (n = 5, Wilcoxon signed rank test, p = 1.00). (I) Summary data for PKA modulators from panel H and Rp-8-CPT-cAMPS (n = 3, Wilcoxon signed rank test, p = 0.25).</p

SNc DA neuron physiology.

<p>(A) 2P reconstruction of SNc DA neuron. (B) Left, normal pacemaking of a SNc DA neuron. Middle, TTX (1 μM) application uncovered slow oscillatory potential (SOP). Right, 5 μM baclofen application hyperpolarized the cell. (C) Summary of hyperpolarization due to application of 5 μM baclofen (n = 8, median = -25.24 mV). (D) Sustained uncaging of 5 μM RuBi-GABA in the presence of 25 μM gabazine (to block GABA_A receptors) hyperpolarized cells in a manner similar to that seen following baclofen application. (E) Summary of hyperpolarization due to sustained 5 μM RuBi-GABA uncaging (n = 7, median = -11.71 mV).</p

Schematic diagram depicting hypothesized signaling pathways involved in the GABA_B receptor-mediated inhibition of SK channels.

<p>GABA_B receptor inhibition of AC by G_i signaling is hypothesized to be responsible for reduced cAMP levels and PKA signaling. The reduction in PKA activity is hypothesized to reduce SK channel opening through mechanism that are independent of either plasma membrane Ca²⁺ channels or release from intracellular stores.</p