This review addresses the localized regulation of voltage-gated ion channels by phosphorylation. Comprehensive data on channel regulation by associated protein kinases, phosphatases, and related regulatory proteins are mainly available for voltage-gated Ca2+ channels, which form the main focus of this review. Other voltage-gated ion channels and especially Kv7.1-3 (KCNQ1-3), the large- and small-conductance Ca2+-activated K+ channels BK and SK2, and the inward-rectifying K+ channels Kir3 have also been studied to quite some extent and will be included. Regulation of the L-type Ca2+ channel Cav1.2 by PKA has been studied most thoroughly as it underlies the cardiac fight-or-flight response. A prototypical Cav1.2 signaling complex containing the β2 adrenergic receptor, the heterotrimeric G protein Gs, adenylyl cyclase, and PKA has been identified that supports highly localized via cAMP. The type 2 ryanodine receptor as well as AMPA- and NMDA-type glutamate receptors are in close proximity to Cav1.2 in cardiomyocytes and neurons, respectively, yet independently anchor PKA, CaMKII, and the serine/threonine phosphatases PP1, PP2A, and PP2B, as is discussed in detail. Descriptions of the structural and functional aspects of the interactions of PKA, PKC, CaMKII, Src, and various phosphatases with Cav1.2 will include comparisons with analogous interactions with other channels such as the ryanodine receptor or ionotropic glutamate receptors. Regulation of Na+ and K+ channel phosphorylation complexes will be discussed in separate papers. This review is thus intended for readers interested in ion channel regulation or in localization of kinases, phosphatases, and their upstream regulators.
SUMMARY Regulation of neuronal excitability and cardiac excitation-contraction coupling requires proper localization of L-type Ca2+ channels. We show that the actin-binding protein α-actinin binds to the C-terminal surface targeting motif of α11.2, the central pore-forming CaV1.2 subunit, to foster its surface expression. Disruption of α-actinin function by dominant negative or shRNA constructs reduces CaV1.2 surface localization in HEK293 and neuronal cultures, and dendritic spine localization in neurons. We demonstrate that calmodulin displaces α-actinin from their shared binding site on α11.2 upon Ca2+ influx through L-type channels but not through NMDAR, thereby triggering loss of CaV1.2 from spines. Coexpression of a Ca2+-binding deficient calmodulin mutant does not affect basal CaV1.2 surface expression, but inhibits its internalization upon Ca2+ influx. We conclude that α-actinin stabilizes CaV1.2 at the plasma membrane, and that its displacement by Ca2+-calmodulin induces Ca2+-induced endocytosis of CaV1.2, thus providing an important negative feedback mechanism for Ca2+ influx.
The Q Q subunit was initially identified as an auxiliary subunit of the skeletal muscle calcium channel complex. Evidence for the existence of further Q Q subunits arose following the characterization of a genetic defect that induces epileptic seizures in stargazer mice. We present here the first account of a family of at least five putative Q Q subunits that are predominantly expressed in brain. The Q Q-2 and Q Q-4 subunits shift the steady-state inactivation curve to more hyperpolarized potentials upon coexpression with the P/Q type K K 1A subunit. The coexpression of the Q Q-5 subunit accelerates the time course of current activation and inactivation of the K K 1G T-type calcium channel. z 2000 Federation of European Biochemical Societies.
Propofol is the most commonly used sedative-hypnotic drug for noxious procedures, yet the molecular targets underlying either its beneficial or toxic effects remain uncertain. In order to determine targets and thereby mechanisms of propofol, we have synthesized a photoactivateable analogue by substituting an alkyldiazirinyl moiety for one of the isopropyl arms but in the meta position. m-Azipropofol retains the physical, biochemical, GABAA receptor modulatory, and in vivo activity of propofol and photoadducts to amino acid residues in known propofol binding sites in natural proteins. Using either mass spectrometry or radiolabeling, this reagent may be used to reveal sites and targets that underlie the mechanism of both the desirable and undesirable actions of this important clinical compound.
The auxiliary calcium channel alpha2delta subunit comprises a family of three genes, alpha2delta-1 to 3, which are expressed in a tissue-specific manner. alpha2delta-2 mRNA is found in the heart, skeletal muscle, brain, kidney, liver and pancreas. We report here for the first time the identification and functional characterization of alpha2delta-2 splice variants and their mRNA distribution in the mouse brain. The splice variants differ in the alpha2 and delta protein by eight and three amino acid residues, respectively, and are differentially expressed in cardiac tissue and human medullary thyroid carcinoma (hMTC) cells. In situ hybridization of mouse brain sections revealed the highest expression of alpha2delta-2 mRNA in the Purkinje cell layer of the cerebellum, habenulae and septal nuclei, and a lower expression in the cerebral cortex, olfactory bulb, thalamic and hypothalamic nuclei, as well as the inferior and superior colliculus. As the in situ data did not suggest a specific colocalization with any alpha1 subunit, coexpression studies of alpha2delta-2 were carried out either with the high-voltage-gated calcium channels, alpha1C, alpha1E or alpha1A, or with the low-voltage-gated calcium channel, alpha1G. Coexpression of alpha2delta-2 increased the current density, shifted the voltage dependence of channel activation and inactivation of alpha1C, alpha1E and alpha1A subunits in a hyperpolarizing direction, and accelerated the decay and shifted the steady-state inactivation of the alpha1G current.
Volatile anesthetics and alcohols enhance transmission mediated by ␥-aminobutyric acid type A receptors (GABA A Rs) in the central nervous system, an effect that may underlie some of the behavioral actions of these agents. Substituting a critical serine residue within the GABA A R ␣ 1 subunit at position 270 with the larger residue histidine eliminated receptor modulation by isoflurane, but it also affected receptor gating (increased GABA sensitivity). To correct the shift in GABA sensitivity of this mutant, we mutated a second residue, leucine at position 277 to alanine. The double mutant ␣ 1 (S270H,L277A) 2 ␥ 2S GABA A R was expressed in Xenopus laevis oocytes and human embryonic kidney (HEK)293 cells, and it had near-normal GABA sensitivity. However, rapid application of a brief GABA pulse to receptors expressed in HEK293 cells revealed that the deactivation was faster in double mutant than in wild-type receptors. In all heterologous systems, the enhancing effect of isoflurane and ethanol was greatly decreased in the double mutant receptor. Homozygous knockin mice harboring the double mutation were viable and presented no overt abnormality, except hyperactivity. This knockin mouse line should be useful in determining which behavioral actions of volatile anesthetics and ethanol are mediated by the GABA A Rs containing the ␣ 1 subunit.
Lung adenocarcinomas (LUAD) arise from precancerous lesions such as atypical adenomatous hyperplasia, which progress into adenocarcinoma in situ and minimally invasive adenocarcinoma, then finally into invasive adenocarcinoma. The cellular heterogeneity and molecular events underlying this stepwise progression remain unclear. In this study, we perform single-cell RNA sequencing of 268,471 cells collected from 25 patients in four histologic stages of LUAD and compare them to normal cell types. We detect a group of cells closely resembling alveolar type 2 cells (AT2) that emerged during atypical adenomatous hyperplasia and whose transcriptional profile began to diverge from that of AT2 cells as LUAD progressed, taking on feature characteristic of stem-like cells. We identify genes related to energy metabolism and ribosome synthesis that are upregulated in early stages of LUAD and may promote progression. MDK and TIMP1 could be potential biomarkers for understanding LUAD pathogenesis. Our work shed light on the underlying transcriptional signatures of distinct histologic stages of LUAD progression and our findings may facilitate early diagnosis.
A positive selection technique was used to identify novel auxiliary calcium channel subunits that are similar to the skeletal muscle gamma subunit. A new rat gamma subunit cDNA was found, which was highly expressed in skeletal muscle tissue and was detected by RT-PCR in cardiac tissue. The 223-amino-acid-protein shares 84% and 79% identity, respectively, with the human and rabbit skeletal muscle subunits. Northern blot analysis revealed a single transcript of 1.5 kb in rat skeletal muscle, but not in cardiac tissue. Transient coexpression with the cardiac calcium channel complex demonstrated that the gamma subunit shifted the inactivation curve to negative potentials and accelerated current inactivation without changing other voltage-dependent properties of the channel.
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