Medium-Chain Lipid Conjugation Facilitates Cell-Permeability and Bioactivity.

The majority of bioactive molecules act on membrane proteins or intracellular targets and therefore needs to partition into or cross biological membranes. Natural products often exhibit lipid modifications to facilitate critical molecule-membrane interactions, and in many cases their bioactivity is markedly reduced upon removal of a lipid group. However, despite its importance in nature, lipid-conjugation of small molecules is not commonly used in chemical biology and medicinal chemistry, and the effect of such conjugation has not been systematically studied. To understand the composition of lipids found in natural products, we carried out a chemoinformatic characterization of the "natural product lipidome". According to this analysis, lipidated natural products predominantly contain saturated medium-chain lipids (MCLs), which are significantly shorter than the long-chain lipids (LCLs) found in membranes and lipidated proteins. To study the usefulness of such modifications in probe design, we systematically explored the effect of lipid conjugation on five different small molecule chemotypes and find that permeability, cellular retention, subcellular localization, and bioactivity can be significantly modulated depending on the type of lipid tail used. We demonstrate that MCL conjugation can render molecules cell-permeable and modulate their bioactivity. With all explored chemotypes, MCL-conjugates consistently exhibited superior uptake or bioactivity compared to LCL-conjugates and either comparable or superior uptake or bioactivity to short-chain lipid (SCL)-conjugates. Together, our findings suggest that conjugation of small molecules with MCLs could be a powerful strategy for the design of probes and drugs.

A unique role for Kv3 voltage-gated potassium channels in starburst amacrine cell signaling in mouse retina.

Direction-selective retinal ganglion cells show an increased activity evoked by light stimuli moving in the preferred direction. This selectivity is governed by direction-selective inhibition from starburst amacrine cells occurring during stimulus movement in the opposite or null direction. To understand the intrinsic membrane properties of starburst cells responsible for direction-selective GABA release, we performed whole-cell recordings from starburst cells in mouse retina. Voltage-clamp recordings revealed prominent voltage-dependent K(+) currents. The currents were mostly blocked by 1 mm TEA, activated rapidly at voltages more positive than -20 mV, and deactivated quickly, properties reminiscent of the currents carried by the Kv3 subfamily of K+ channels. Immunoblots confirmed the presence of Kv3.1 and Kv3.2 proteins in retina and immunohistochemistry revealed their expression in starburst cell somata and dendrites. The Kv3-like current in starburst cells was absent in Kv3.1-Kv3.2 knock-out mice. Current-clamp recordings showed that the fast activation of the Kv3 channels provides a voltage-dependent shunt that limits depolarization of the soma to potentials more positive than -20 mV. This provides a mechanism likely to contribute to the electrical isolation of individual starburst cell dendrites, a property thought essential for direction selectivity. This function of Kv3 channels differs from that in other neurons where they facilitate high-frequency repetitive firing. Moreover, we found a gradient in the intensity of Kv3.1b immunolabeling favoring proximal regions of starburst cells. We hypothesize that this Kv3 channel gradient contributes to the preference for centrifugal signal flow in dendrites underlying direction-selective GABA release from starburst amacrine cells

Synaptic regulation of the light-dependent oscillatory currents in starburst amacrine cells of the mouse retina.

Responses of on-center starburst amacrine cells to steady light stimuli were recorded in the dark-adapted mouse retina. The response to spots of dim white light appear to show two components, an initial peak that correspond to the onset of the light stimulus and a series of oscillations that ride on top of the initial peak relaxation. The frequency of oscillations during light stimulation was three time higher than the frequency of spontaneous oscillations recorded in the dark. The light-evoked responses in starburst cells were exclusively dependent on the release of glutamate likely from presynaptic bipolar axon terminals and the binding of glutamate to AMPA/kainate receptors because they were blocked by 6-cyano-7-nitroquinoxalene-2,3-dione. The synaptic pathway responsible for the light responses was blocked by AP4, an agonist of metabotropic glutamate receptors that hyperpolarize on-center bipolar cells on activation. Light responses were inhibited by the calcium channel blockers cadmium ions and nifedipine, suggesting that the release of glutamate was calcium dependent. The oscillatory component of the response was specifically inhibited by blocking the glutamate transporter with d-threo-beta-benzyloxyaspartic acid, suggesting that glutamate reuptake is necessary for the oscillatory release. GABAergic antagonists bicuculline, SR 95531, and picrotoxin increased the amplitude of the initial peak while they inhibit the frequency of oscillations. TTX had a similar effect. Strychnine, the blocker of glycine receptors did not affect the initial peak but strongly decreased the oscillations frequency. These inhibitory inputs onto the bipolar axon terminals shape and synchronize the oscillatory component.

Spontaneous oscillatory activity of starburst amacrine cells in the mouse retina.

Using patch-clamp techniques, we investigated the characteristics of the spontaneous oscillatory activity displayed by starburst amacrine cells in the mouse retina. At a holding potential of -70 mV, oscillations appeared as spontaneous, rhythmic inward currents with a frequency of approximately 3.5 Hz and an average maximal amplitude of approximately 120 pA. Application of TEA, a potassium channel blocker, increased the amplitude of oscillatory currents by >70% but reduced their frequency by approximately 17%. The TEA effects did not appear to result from direct actions on starburst cells, but rather a modulation of their synaptic inputs. Oscillatory currents were inhibited by 6-cyano-7-nitroquinoxalene-2,3-dione (CNQX), an antagonist of AMPA/kainate receptors, indicating that they were dependent on a periodic glutamatergic input likely from presynaptic bipolar cells. The oscillations were also inhibited by the calcium channel blockers cadmium and nifedipine, suggesting that the glutamate release was calcium dependent. Application of AP4, an agonist of mGluR6 receptors on on-center bipolar cells, blocked the oscillatory currents in starburst cells. However, application of TEA overcame the AP4 blockade, suggesting that the periodic glutamate release from bipolar cells is intrinsic to the inner plexiform layer in that, under experimental conditions, it can occur independent of photoreceptor input. The GABA receptor antagonists picrotoxin and bicuculline enhanced the amplitude of oscillations in starburst cells prestimulated with TEA. Our results suggest that this enhancement was due to a reduction of a GABAergic feedback inhibition from amacrine cells to bipolar cells and the resultant increased glutamate release. Finally, we found that some ganglion cells and other types of amacrine cell also displayed rhythmic activity, suggesting that oscillatory behavior is expressed by a number of inner retinal neurons.