Channels from KCNQ2 and KCNQ3 genes have been suggested to underlie the neuronal M-type K ϩ current. The M current is modulated by muscarinic agonists via G-proteins and an unidentified diffusible cytoplasmic messenger. Using wholecell clamp, we studied tsA-201 cells in which cloned KCNQ2/ KCNQ3 channels were coexpressed with M 1 muscarinic receptors. Heteromeric KCNQ2/KCNQ3 currents were modulated by the muscarinic agonist oxotremorine-M (oxo-M) in a manner having all of the characteristics of modulation of native M current in sympathetic neurons. Oxo-M also produced obvious intracellular Ca 2ϩ transients, observed by using indo-1 fluorescence. However, modulation of the current remained strong even when Ca 2ϩ signals were abolished by the combined use of strong intracellular Ca 2ϩ buffers, an inhibitor of IP 3 receptors, and thapsigargin to deplete Ca 2ϩ stores. Muscarinic modulation was not blocked by staurosporine, a broad-spectrum protein kinase inhibitor, arguing against involvement of protein kinases. The modulation was not associated with a shift in the voltage dependence of channel activation. Homomeric KCNQ2 and KCNQ3 channels also expressed well and were modulated individually by oxo-M, suggesting that the motifs for modulation are present on both channel subtypes. Homomeric KCNQ2 and KCNQ3 currents were blocked, respectively, at very low and at high concentrations of tetraethylammonium ion. Finally, when KCNQ2 subunits were overexpressed by intranuclear DNA injection in sympathetic neurons, total M current was fully modulated by the endogenous neuronal muscarinic signaling mechanism. Our data further rule out Ca 2ϩ as the diffusible messenger. The reconstitution of muscarinic modulation of the M current that uses cloned components should facilitate the elucidation of the muscarinic signaling mechanism. Key words: K ϩ channel; muscarinic receptor; G-protein; calcium; patch clamp; M current A diverse family of neurotransmitters and hormones regulates Ca 2ϩ and K ϩ channels via G-protein-mediated signaling pathways (Wickman and Clapham, 1995;Brown et al., 1997;Dolphin, 1998). Nearly 20 years ago, several investigators gave the name M current to a noninactivating K ϩ current with slow kinetics in sympathetic neurons that is strongly suppressed by muscarinic acetylcholine receptor (mAChR) agonists (Brown and Adams, 1980;Constanti and Brown, 1981). The M current is thought to play an important role in neuronal excitability, and its suppression increases responses to excitatory synaptic inputs (Jones et al., 1995;Wang and McKinnon, 1995). Modulation of the M current by muscarinic receptor agonists and by angiotensin (Constanti and Brown, 1981; Marrion, 1997;Shapiro et al., 1994a) is mediated by a G-protein of the G q /11 class (Delmas et al., 1998; Haley et al., 1998) via a diffusible cytoplasmic second messenger (Selyanko et al., 1992) that is yet to be identified.Although the buffering of intracellular free Ca 2ϩ ([Ca 2ϩ ] i ) to very low levels prevents muscarinic suppression of the M current in rat sym...

The inositol 1,4,5-trisphosphate (InsP3)-gated Ca channel in cerebellum is tightly regulated by Ca (Bezprozvanny, I., J. Watras, and B.E. Ehrlich. 1991. Nature (Lond.). 351:751–754; Finch, E.A., T.J. Turner, and S.M. Goldin. 1991. Science (Wash. DC). 252:443–446; Hannaert-Merah, Z., J.F. Coquil, L. Combettes, M. Claret, J.P. Mauger, and P. Champeil. 1994. J. Biol. Chem. 269:29642–29649; Iino, M. 1990. J. Gen. Physiol. 95:1103–1122; Marshall, I., and C. Taylor. 1994. Biochem. J. 301:591–598). In previous single channel studies, the Ca dependence of channel activity, monitored at 2 μM InsP3, was described by a bell-shaped curve (Bezprozvanny, I., J. Watras, and B.E. Ehrlich. 1991. Nature (Lond.). 351:751–754). We report here that, when we used lower InsP3 concentrations, the peak of the Ca-dependence curve shifted to lower Ca concentrations. Unexpectedly, when we used high InsP3 concentrations, channel activity persisted at Ca concentrations as high as 30 μM. To explore this unexpected response of the channel, we measured InsP3 binding over a broad range of InsP3 concentrations. We found the well-characterized high affinity InsP3 binding sites (with K d < 1 and 50 nM) (Maeda, N., M. Niinobe, and K. Mikoshiba. 1990. EMBO (Eur. Mol. Biol. Organ.) J. 9:61–67; Mignery, G., T.C. Sudhof, K. Takei, and P. De Camilli. 1989. Nature (Lond.). 342:192–195; Ross, C.A., J. Meldolesi, T.A. Milner, T. Satoh, S. Supattapone, and S.H. Snyder. 1989. Nature (Lond.). 339:468–470) and a low affinity InsP3 binding site (K d = 10 μM). Using these InsP3 binding sites, we developed a new model that accounts for the shift in the Ca-dependence curve at low InsP3 levels and the maintained channel activity at high Ca and InsP3 levels. The observed Ca dependence of the InsP3-gated Ca channel allows the cell to abbreviate the rise of intracellular Ca in the presence of low levels of InsP3, but also provides a means of maintaining high intracellular Ca during periods of prolonged stimulation.

Parkinson's disease (PD) pathology is characterized by the formation of intra-neuronal inclusions called Lewy bodies, which are comprised of alpha-synuclein (α-syn). Duplication, triplication or genetic mutations in α-syn (A53T, A30P and E46K) are linked to autosomal dominant PD; thus implicating its role in the pathogenesis of PD. In both PD patients and mouse models, there is increasing evidence that neuronal dysfunction occurs before the accumulation of protein aggregates (i.e., α-syn) and neurodegeneration. Characterization of the timing and nature of symptomatic dysfunction is important for understanding the impact of α-syn on disease progression. Furthermore, this knowledge is essential for identifying pathways and molecular targets for therapeutic intervention. To this end, we examined various functional and morphological endpoints in the transgenic mouse model expressing the human A53T α-syn variant directed by the mouse prion promoter at specific ages relating to disease progression (2, 6 and 12 months of age). Our findings indicate A53T mice develop fine, sensorimotor, and synaptic deficits before the onset of age-related gross motor and cognitive dysfunction. Results from open field and rotarod tests show A53T mice develop age-dependent changes in locomotor activity and reduced anxiety-like behavior. Additionally, digigait analysis shows these mice develop an abnormal gait by 12 months of age. A53T mice also exhibit spatial memory deficits at 6 and 12 months, as demonstrated by Y-maze performance. In contrast to gross motor and cognitive changes, A53T mice display significant impairments in fine- and sensorimotor tasks such as grooming, nest building and acoustic startle as early as 1–2 months of age. These mice also show significant abnormalities in basal synaptic transmission, paired-pulse facilitation and long-term depression (LTD). Combined, these data indicate the A53T model exhibits early- and late-onset behavioral and synaptic impairments similar to PD patients and may provide useful endpoints for assessing novel therapeutic interventions for PD.

Our study highlights molecular and immune phenotypes that warrant further analysis for their roles in clinical outcomes and personalized immune-based therapy of LUAD.

Ryanodine receptors (RyRs) are intracellular channels that regulate the release of Ca2+ from the endoplasmic reticulum of many cell types. The RyRs are physically associated with FK506-binding proteins (FKBPs); immunophilins, with cis-trans peptidyl-prolyl isomerase activity. FKBP12 copurifies with RyR1 (skeletal isoform) and modulates its gating. A different form of FKBP with a slightly higher molecular weight copurifies with RyR2 (cardiac isoform). Previous studies have demonstrated that FKBP stablizes gating of the skeletal Ca(2+)-release channel. In the present study, we measured the activity of cardiac RyRs incorporated into planar lipid bilayers to show that rapamycin, a drug that inhibits the prolyl isomerase activity of FKBP and dissociates FKBP from the RyR, increases the open probability and reduces the current amplitude of cardiac muscle Ca(2+)-release channels. These experiments show for the first time that submicromolar concentrations of rapamycin can alter channel function. Our results provide support for the hypotheses that FKBP functionally associates with the RyR and that the immunosuppressant drug, rapamycin, alters the function of both cardiac and skeletal muscle isoforms of the Ca(2+)-release channel. Our findings suggest that FKBP-dependent modulation of channel function may be generally applicable to all members of the intracellular Ca(2+)-release channel family and that FKBPs may play important regulatory roles in many cell processes, ranging from long-term depression in neurons to contractility in cardiomyocytes.

The biological determinants of sensitivity and resistance to immune checkpoint blockers are not completely understood. To elucidate the role of intratumoral T-cells and their association with the tumor genomic landscape, we perform paired whole exome DNA sequencing and multiplexed quantitative immunofluorescence (QIF) in pre-treatment samples from non-small cell lung carcinoma (NSCLC) patients treated with PD-1 axis blockers. QIF is used to simultaneously measure the level of CD3+ tumor infiltrating lymphocytes (TILs), in situ T-cell proliferation (Ki-67 in CD3) and effector capacity (Granzyme-B in CD3). Elevated mutational load, candidate class-I neoantigens or intratumoral CD3 signal are significantly associated with favorable response to therapy. Additionally, a “dormant” TIL signature is associated with survival benefit in patients treated with immune checkpoint blockers characterized by elevated TILs with low activation and proliferation. We further demonstrate that dormant TILs can be reinvigorated upon PD-1 blockade in a patient-derived xenograft model.

Our findings pinpoint mutated genes that may impact clinical outcome as well as personalized strategies for targeted immunotherapies in early-stage LUSC.

EXPERIMENTAL PROCEDURESPituitary Cell Isolation-Anterior pituitary glands were removed from 4 -6-week-old male rats that had been castrated at week 2 by the supplier. Rats were euthanized by placing them in an empty chamber that was then filled with CO 2 . Tissue was enzymatically dispersed (24), and cells were cultured for up to 4 days.[Ca 2ϩ ] Measurements-The loading of membrane-permeant Ca 2ϩ indicators, and subsequent photometry was described previously (8). In experiments where mitochondrial membrane potential was monitored, cells were incubated in TMRE (600 nM) for 5 min following loading with Ca 2ϩ indicators, and TMRE (100 nM) was present in the bath throughout the experiment. Corrections were applied to fluorescence signals to * This work was supported in part by National Institutes of Health Grant AR17803, by Cooperative Agreement U54 HD12629 (as part of the Specialized Cooperative Centers Program in Reproductive Research), and by funds from the the W. M. Keck Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.§ Supported by a fellowship from the Lalor Foundation and by National Institutes of Health Training Grant 2T32HD07453. To whom correspondence should be addressed: G-424 Health Sciences Bldg., Dept. of Physiology and Biophysics, University of Washington, Seattle, 1 The abbreviations used are: [Ca 2ϩ ] cyt , free cytoplasmic calcium concentration; GnRH, gonadotropin-releasing hormone; [Ca 2ϩ ] mit , free mitochondrial calcium concentration; CCCP, carbonyl cyanide m-chlorophenyl-hydrazone; IP 3 , inositol 1,4,5-trisphosphate; TMRE, tetramethylrhodamine ethyl ester.