No abstract

To investigate the roles of K ؉ channels in the regulation and fine-tuning of cellular excitability, we generated a mutant mouse carrying a disrupted gene for the fast activating, voltage-gated K ؉ channel Kv3.1. Kv3

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 voltagedependent K ϩ currents. The currents were mostly blocked by 1 mM TEA, activated rapidly at voltages more positive than Ϫ20 mV, and deactivated quickly, properties reminiscent of the currents carried by the Kv3 subfamily of K ϩ 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 Ϫ20 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 highfrequency 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

During the last few years a variety of genetically encodable optical probes that monitor physiological parameters such as local pH, Ca2+, Cl-, or transmembrane voltage have been developed. These sensors are based on variants of green-fluorescent protein (GFP) and can be synthesized by mammalian cells after transfection with cDNA. To use these sensor proteins in intact brain tissue, specific promoters are needed that drive protein expression at a sufficiently high expression level in distinct neuronal subpopulations. Here we investigated whether the promoter sequence of a particular potassium channel may be useful for this purpose. We produced transgenic mouse lines carrying the gene for enhanced yellow-fluorescent protein (EYFP), a yellow-green pH- and Cl- sensitive variant of GFP, under control of the Kv3.1 K+ channel promoter (pKv3.1). Transgenic mouse lines displayed high levels of EYFP expression, identified by confocal microscopy, in adult cerebellar granule cells, interneurons of the cerebral cortex, and in neurons of hippocampus and thalamus. Furthermore, using living cerebellar slices we demonstrate that expression levels of EYFP are sufficient to report intracellular pH and Cl- concentration using imaging techniques and conditions analogous to those used with conventional ion-sensitive dyes. We conclude that transgenic mice expressing GFP-derived sensors under the control of cell-type specific promoters, provide a unique opportunity for functional characterization of defined subsets of neurons.

Kv3.1 is a voltage-gated, fast activating/deactivating potassium (K(+)) channel with a high-threshold of activation and a large unit conductance. Kv3.1 K(+) channels are expressed in fast-spiking, parvalbumin-containing interneurons in cortex, hippocampus, striatum, the thalamic reticular nucleus (TRN), and in several nuclei of the brain stem. A high density of Kv3.1 channels contributes to short-duration action potentials, fast afterhyperpolarizations, and brief refractory periods enhancing the capability in these neurons for high-frequency firing. Kv3.1 K(+) channel expression in the TRN and cortex also suggests a role in thalamocortical and cortical function. Here we show that fast gamma and slow delta oscillations recorded from the somatomotor cortex are altered in the freely behaving Kv3.1 mutant mouse. Electroencephalographic (EEG) recordings from homozygous Kv3.1(-/-) mice show a three- to fourfold increase in both absolute and relative spectral power in the gamma frequency range (20-60 Hz). In contrast, Kv3.1-deficient mice have a 20-50% reduction of power in the slow delta range (2-3 Hz). The increase in gamma power is most prominent during waking in the 40- to 55-Hz range, whereas the decrease in delta power occurs equally across all states of arousal. Our findings suggest that Kv3. 1-expressing neurons are involved in the generation and maintenance of cortical fast gamma and slow delta oscillations. Hence the Kv3. 1-mutant mouse could serve as a model to study the generation and maintenance of fast gamma and slow delta rhythms and their involvement in behavior and cognition.

Syntaxin1A, a neural-specific N-ethylmaleimide-sensitive factor attachment protein receptor protein essential to neurotransmitter release, in isolation forms a closed conformation with an N-terminal ␣-helix bundle folded upon the SNARE motif (H3 domain), thereby limiting interaction of the H3 domain with cognate SNAREs. Munc18-1, a neural-specific member of the Sec1/Munc18 protein family, binds to syntaxin1A, stabilizing this closed conformation. We used fluorescence resonance energy transfer (FRET) to characterize the Munc18-1/syntaxin1A interaction in intact cells. Enhanced cyan fluorescent protein-Munc18-1 and a citrine variant of enhanced yellow fluorescent proteinsyntaxin1A, or mutants of these proteins, were expressed as donor and acceptor pairs in human embryonic kidney HEK293-S3 and adrenal chromaffin cells. Apparent FRET efficiency was measured using two independent approaches with complementary results that unambiguously verified FRET and provided a spatial map of FRET efficiency. In addition, enhanced cyan fluorescent protein-Munc18-1 and a citrine variant of enhanced yellow fluorescent protein-syntaxin1A colocalized with a Golgi marker and exhibited FRET at early expression times, whereas a strong plasma membrane colocalization, with similar FRET values, was apparent at later times. Trafficking of syntaxin1A to the plasma membrane was dependent on the presence of Munc18-1. Both syntaxin1A(L165A/E166A), a constitutively open conformation mutant, and syntaxin1A(I233A), an H3 domain point mutant, demonstrated apparent FRET efficiency that was reduced ϳ70% from control. In contrast, the H3 domain mutant syntaxin1A(I209A) had no effect. By using phosphomimetic mutants of Munc18-1, we also established that Ser-313, a Munc18-1 protein kinase C phosphorylation site, and Thr-574, a cyclin-dependent kinase 5 phosphorylation site, regulate Munc18-1/ syntaxin1A interaction in HEK293-S3 and chromaffin cells. We conclude that FRET imaging in living cells may allow correlated regulation of Munc18-1/syntaxin1A interactions to Ca 2؉ -regulated secretory events.

X11 proteins have been shown to modulate metabolism of the amyloid precursor protein (APP) and to reduce the secretion of ␤-amyloid peptides (A␤) that are associated with Alzheimer's disease. Whereas X11␣ interacts with APP via its phosphotyrosine-binding domain, recent reports indicate that additional regulatory interactions involve the N terminus of X11. Here we report that the syntaxin-1a-binding protein Munc18a, which interacts with the Munc18a-interacting domain (MID) at the N terminus of X11, strongly regulates the actions of X11 on APP metabolism. When co-expressed with X11␣, Munc18a potentiated the retention of APP and suppression of A␤ secretion by X11␣. As a result, the constitutive release of A␤40 was nearly abolished. Experiments using N terminus deletion mutants of X11␣/␤ and the MID-deficient X11␥ revealed that the majority of the regulatory effect by Munc18a occurred independent of a direct interaction of Munc18a with X11, although the presence of X11 was required. Munc18a expression induced a small increase in ␤-secretase activity, whereas it also intensified the reduction in A␤40 secretion by X11␣. These data indicate that Munc18a in concert with X11 acts to suppress ␥-secretase processing. We conclude that Munc18a acts through direct and indirect interactions with X11 proteins and powerfully regulates APP metabolism and A␤ secretion.The two major pathological features in the brains of patients with Alzheimer's disease are the presence of ␤-amyloid peptide (␤-AP or A␤) 1 containing senile plaques and neurofibrillary tangles (1). A␤ peptides were derived from proteolytic processing of a precursor protein, namely amyloid precursor protein (APP). The cleavage sites and extent of processing depend on its trafficking pathways as different APP derivatives have been mapped to distinct compartments of the cell, presumably where specific APP processing enzymes, or secretases, reside (2-6). A major pathway for A␤ production involves internalization of APP, as directed by an ENPTY sequence at the C terminus of APP, following its delivery to the plasma membrane (7). This internalization motif has been found to interact with several phosphotyrosine-binding domain (PTB) containing proteins such as Fe65 and X11, although phosphorylation within this motif is not required for binding (8 -12). The interaction between the PTB domains of Fe65 or X11 proteins and the C terminus of APP has been shown to effect the distribution and turnover of APP, and the secretion of A␤ (13-16). Mammalian X11 proteins (X11␣, -␤, and -␥) are homologues of lin-10 in Caenorhabditis elegans. In conjunction with lin-2 and lin-7, lin-10 has been shown to be required for the precise targeting and localization of certain membrane proteins, such as the GLR-1 glutamate receptor (17, 18). Similar to lin-10, X11 proteins possess multiple protein interacting domains (see Fig. 1A) and have been ascribed to function as adaptor proteins that were critical for protein trafficking (19 -23). It has therefore been postulated that X11 may modulate APP ...

Rho family GTPases are primary mediators of cytoskeletal reorganization, although they have also been reported to regulate cell secretion. Yet, the extent to which Rho family GTPases are activated by secretory stimuli in neural and neuroendocrine cells remains unknown. In bovine adrenal chromaffin cells, we found Rac1, but not Cdc42, to be rapidly and selectively activated by secretory stimuli using an assay selective for the activated GTPases. To examine effects of activated Rac1 on secretion, constitutively active mutants of Rac1 (Rac1‐V12, Rac1‐L61) were transiently expressed in adrenal chromaffin cells. These mutants facilitated secretory responses elicited from populations of intact and digitonin‐permeabilized cells as well as from cells under whole cell patch clamp. A dominant negative Rac1 mutant (Rac1‐N17) produced no effect on secretion. Expression of RhoGDI, a negative regulator of Rac1, inhibited secretory responses while overexpression of effectors of Rac1, notably, p21‐activated kinase (Pak1) and actin depolymerization factor (ADF) promoted evoked secretion. In addition, expression of effector domain mutants of Rac1‐V12 that exhibit reduced activation of the cytoskeletal regulators Pak1 and Partner of Rac1 (POR1) resulted in a loss of Rac1‐V12‐mediated enhancement of evoked secretion. These findings suggest that Rac1, in part, functions to modulate secretion through actions on the cytoskeleton. Consistent with this hypothesis, the actin modifying drugs phalloidin and jasplakinolide enhanced secretion, while latrunculin‐A inhibited secretion and eliminated the secretory effects of Rac1‐V12. In summary, Rac1 was activated by secretory stimuli and modulated the secretory pathway downstream of Ca2+ influx, partly through regulation of cytoskeletal organization.