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Ionotropic glutamate receptors are the most important excitatory receptors in the central nervous system, and their impairment can lead to multiple neuronal diseases. Here, we show that glutamate-induced currents in oocytes expressing GluA1 are increased by coexpression of the schizophrenia-associated phosphoinositide kinase PIP5K2A. This effect was due to enhanced membrane abundance and was blunted by a point mutation (N251S) in PIP5K2A. An increase in GluA1 currents was also observed upon acute injection of PI(4,5)P, the main product of PIP5K2A. By expression of wild-type and mutant PIP5K2A in human embryonic kidney cells, we were able to provide evidence of impaired kinase activity of the mutant PIP5K2A. We defined the region K813-K823 of GluA1 as critical for the PI(4,5)P effect by performing an alanine scan that suggested PI(4,5)P binding to this area. A PIP strip assay revealed PI(4,5)P binding to the C-terminal GluA1 peptide. The present observations disclose a novel mechanism in the regulation of GluA1. [ABSTRACT FROM AUTHOR]

Tamoxifen (Tmx) is a nonsteroidal selective estrogen receptor antagonist and is frequently used in the treatment and prevention of breast cancer. The compound and its metabolites have been reported to inhibit functions of different classes of membrane proteins, including various ion channels. For members of the inward-rectifying K (Kir) channel family, interference of Tmx with binding of phosphatidylinositol 4,5-bisphosphate (PIP) has been suggested as the mechanism underlying such inhibition. We have studied the inhibition of G protein-activated K (GIRK) current by Tmx in isolated myocytes from hearts of adult rats using whole-cell voltage clamp and experimental conditions for measuring K currents as inward currents ( E −50 mV; holding potential −90 mV). Extracellular Tmx reversibly inhibited GIRK current activated by acetylcholine ( I) with an EC of 7.4 × 10 M. This inhibition was composed of two components, a basal reduction in peak current and a block that required opening of channels by ACh. The open-channel block was partially relieved by depolarizing voltage steps in a voltage- and time-dependent fashion. A voltage-dependent open-channel block was not observed when I was measured as outward current ( E −90 mV; holding potential −40 mV). Intracellular application of Tmx via the patch clamp pipette at a concentration (7 × 10 M) that caused a rapid inhibition of I upon extracellular application did not affect the current. Intracellular application of the HO-soluble PIP analog diC-PIP reduced the voltage-independent component of inhibition but had no effect on voltage-dependent open-channel block. The effects of 4-hydroxy-Tmx, a major active metabolite, tested at 2 × 10 M, had effects on I analogous to those of Tmx. Inhibition of constitutive inward-rectifying K current ( I) in ventricular myocytes, carried by Kir2 complexes, by Tmx was devoid of a voltage-dependent component. This study suggests the voltage-dependent open-channel block of GIRK inward current as a novel mechanism of Tmx action. [ABSTRACT FROM AUTHOR]

G protein-activated K channels composed of Kir3 (GIRK) subunits contribute to regulation of heart rate and excitability. Opening of these channels in myocytes is increased by binding of G upon activation of muscarinic M receptors (M-R) or A adenosine receptors (A-R). It has been shown that saturating activation of A-R resulted in a smaller GIRK current than activation of M-R. Adenovirus-driven overexpression of the A-R caused an increase in current induced by adenosine (I), whereas the M-R-activated current (I) was reduced. Here, we sought to get deeper insight into the mechanism causing this negative crosstalk. GIRK current in cultured rat atrial myocytes was recorded in whole cell mode. Adenovirus-driven RNA interference targeting the M-R resulted in a reduction in I without affecting I, arguing against a competition of the two receptors for common signaling complexes. The negative effect of A-R overexpression on I was reduced by the A-R antagonist DPCPX and augmented by the agonist chloro-N6-cyclopentyladenosin (CCPA). In native myocytes incubation with either CCPA or the muscarinic agonist carbachol resulted in reduction in I and I, suggesting common pathways of A-R and M-R downregulation. In the absence of agonist, inhibition of adenosine deaminase by EHNA or exposure to AMP, less to ADP, but not ATP resulted in reduction of I and I. Our data indicate that atrial myocytes generate adenosine from extracellular AMP, which activates A-R in an autocrine fashion. Chronic activation of A-R causes parallel downregulation of both A-R and M-R. [ABSTRACT FROM AUTHOR]

The R145G amino acid exchange in the inhibitory subunit (cTnI) of cardiac troponin, which regulates muscle contraction, is related to familial hypertrophic cardiomyopathy. Information on its impact on contractility of adult cardiomyocytes is scarce. We studied shortening of adult rat cardiomyocytes before and during ß-adrenergic stimulation using adenovirus-driven expression of human cTnI-wild type (wt) and cTnI-R145G. Baseline sarcomere shortening was significantly decreased by cTnI-R145G expression. Upon ß-adrenergic stimulation using isoproterenol (ISO), nearly identical amplitudes of shortening were obtained with cells expressing cTnI-R145G and control cardiomyocytes (native and cTnI-wt). However, rates of shortening and relengthening were depressed in cTnI-R145G-expressing cells but were comparable to those of control cells upon addition of forskolin or ISO and ICI118,551. This indicates that cTnI-R145G expression influences the response to ß-adrenergic stimulation dependent on the receptor subtype. [ABSTRACT FROM AUTHOR]

Atrial myocytes obtained by enzymatic perfusion of hearts from adult guinea-pigs and cultured for 0-14 days were studied using different configurations of the patch-clamp technique. Activation of muscarinic K current [ I] in whole-cell voltage-clamp mode by strongly diluted sera from various sources could be mimicked by corresponding concentrations of albumin, but not by delipidated ('fatty-acid-free') samples of albumin. In cell-attached membrane patches activity of I channels was significantly higher than basal I channel activity, if the pipette contained serum, whereas application of serum-containing solution to the cell outside the patch did not affect channel activity. In isolated inside-out membrane patches, strong I activation by internal guanosine triphosphate (GTP, 5 μM) was observed if the pipette contained serum. If no activator was presented to the outer face of the membrane, only weak opening activity was observed during bath application of GTP. These results demonstrate that the serum factor which causes activation of I is associated with albumin. Furthermore activation of I by that factor proceeds analogous to ACh or adenosine, i. e. via a membrane-delimited receptor, G-protein, channel interaction. [ABSTRACT FROM AUTHOR]

L-type Ca current ( I) was measured in cultured atrial myocytes from hearts of adult guinea-pigs using whole-cell voltage clamp. Potentiation of I induced by β-adrenergic stimulation (isoprenaline 2· 10 M) could be completely antagonized by diluted sera (1∶100 v/v). Half-maximal inhibition of β-receptorstimulated I occurred at about 1∶1000. Basal I was not affected by serum. Atropine in a concentration (10M) that completely antagonized the anti-adrenergic effect of acetylcholine (ACh, 2·10 M) did not interfere with the effect of serum. In cells dialysed with cyclic adenosine monophosphate (cAMP)-containing (10 M) pipette solution, potentiated I was insensitive to both ACh and serum. Preincubation of the myocytes with pertussis toxin almost completely abolished the anti-adrenergic effects of both ACh and serum. The potency of serum was not reduced by dialysis. It is concluded that serum contains a factor which, like ACh, inhibits β-receptor-stimulated adenylyl cyclase via Gprotein. [ABSTRACT FROM AUTHOR]

The effect of calcium on spontaneous transmitter release and on the release induced by tetanic stimulation and by raising the external potassium concentration ([K]) was studied in sympathetic ganglion cells of Rana esculenta. [ABSTRACT FROM AUTHOR]