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Ca2+ Dependent (ca2+ + dependent)
Selected AbstractsCalcium-dependent K current in plasma membranes of dermal cells of developing bean cotyledonsPLANT CELL & ENVIRONMENT, Issue 2 2004W.-H. ZHANG ABSTRACT In developing seeds of bean (Phaseolus vulgaris L.), phloem-imported assimilates (largely sucrose and potassium) are released from coats to seed apoplasm and subsequently retrieved by the dermal cell complexes of cotyledons. To investigate the mechanisms of K+ uptake by the cotyledons, protoplasts of dermal cell complexes were isolated and whole-cell currents across their plasma membranes were measured with the patch-clamp technique. A weakly rectified cation current displaying a voltage-dependent blockade by external Ca2+ and acidic pH, dominated the conductance of the protoplasts. The P haseolus v ulgaris Cotyledon Dermal-cell pH and Calcium-dependent Cation Conductance (Pv-CD-pHCaCC) was highly selective for K+ over Ca2+ and Cl,. For K+ current through Pv-CD-pHCaCC a sigmoid shaped current,voltage (I,V) curve was observed with negative conductance at voltages between ,200 and ,140 mV. This negative K+ conductance was Ca2+ dependent. With other univalent cations (Na+, Rb+, NH4+) the currents were smaller and were not Ca2+ dependent. Reversal potentials remained constant when external K+ was substituted with these cations, suggesting that Pv-CD-pHCaCC channels were non-selective. The Pv-CD-pHCaCC would provide a pathway for K+ and other univalent cation influx into developing cotyledons. These cation influxes could be co-ordinated with sucrose influx via pH and Ca2+dependence. [source] Calmodulin binding to M-type K+ channels assayed by TIRF/FRET in living cellsTHE JOURNAL OF PHYSIOLOGY, Issue 9 2008Manjot Bal Calmodulin (CaM) binds to KCNQ2,4 channels within their carboxy termini, where it regulates channel function. The existing data have not resolved the Ca2+ dependence of the interaction between the channels and CaM. We performed glutathione S-transferase (GST)-pull-down assays between purified KCNQ2,4 carboxy termini and CaM proteins to determine the Ca2+ dependence of the interaction in vitro. The assays showed substantial Ca2+ dependence of the interaction of the channels with wild-type (WT) CaM, but not with dominant-negative (DN) CaM. To demonstrate CaM,channel interactions in individual living cells, we performed fluorescence resonance energy transfer (FRET) between ECFP-tagged KCNQ2,4 channels and EYFP-tagged CaM expressed in CHO cells, performed under total internal reflection fluorescence (TIRF) microscopy, in which excitation light only penetrates several hundred nanometres into the cell, thus isolating membrane events. FRET was assayed between the channels and either WT or DN CaM, performed under conditions of normal [Ca2+]i, low [Ca2+]i or high [Ca2+]i induced by empirically optimized bathing solutions. The FRET data suggest a strong Ca2+ dependence for the interaction between WT CaM and KCNQ2, but less so for KCNQ3 and KCNQ4. FRET between all KCNQ2,4 channels and DN CaM was robust, and not significantly Ca2+ dependent. These data show interactions between CaM and KCNQ channels in living cells, and suggest that the interactions between KCNQ2,4 channels and CaM are likely to have Ca2+ -dependent and Ca2+ -independent components. [source] Apical SK potassium channels and Ca2+ -dependent anion secretion in endometrial epithelial cellsTHE JOURNAL OF PHYSIOLOGY, Issue 3 2008Melissa L. Palmer Apical uridine triphosphate (UTP) stimulation was shown to increase short circuit current (Isc) in immortalized porcine endometrial gland epithelial monolayers. Pretreatment with the bee venom toxin apamin enhanced this response. Voltage-clamp experiments using amphotericin B-permeablized monolayers revealed that the apamin-sensitive current increased immediately after UTP stimulation and was K+ dependent. The current,voltage relationship was slightly inwardly rectifying with a reversal potential of ,52 ± 2 mV, and the PK/PNa ratio was 14, indicating high selectivity for K+. Concentration,response relationships for apamin and dequalinium had IC50 values of 0.5 nm and 1.8 ,m, respectively, consistent with data previously reported for SK3 channels in excitable cells and hepatocytes. Treatment of monolayers with 50 ,m BAPTA-AM completely blocked the effects of UTP on K+ channel activation, indicating that the apamin-sensitive current was also Ca2+ dependent. Moreover, channel activation was blocked by calmidazolium (IC50= 5 ,m), suggesting a role for calmodulin in Ca2+ -dependent regulation of channel activity. RT-PCR experiments demonstrated expression of mRNA for the SK1 and SK3 channels, but not SK2 channels. Treatment of monolayers with 20 nm oestradiol-17, produced a 2-fold increase in SK3 mRNA, a 2-fold decrease in SK1 mRNA, but no change in GAPDH mRNA expression. This result correlated with a 2.5-fold increase in apamin-sensitive K+ channel activity in the apical membrane. We speculate that SK channels provide a mechanism for rapidly sensing changes in intracellular Ca2+ near the apical membrane, evoking immediate hyperpolarization necessary for increasing the driving force for anion efflux following P2Y receptor activation. [source] Regulation of rat mesencephalic GABAergic neurones through muscarinic receptorsTHE JOURNAL OF PHYSIOLOGY, Issue 2 2004François J. Michel Central dopamine neurones are involved in regulating cognitive and motor processes. Most of these neurones are located in the ventral mesencephalon where they receive abundant intrinsic and extrinsic GABAergic input. Cholinergic neurones, originating from mesopontine nuclei, project profusely in the mesencephalon where they preferentially synapse onto local GABAergic neurones. The physiological role of this cholinergic innervation of GABAergic neurones remains to be determined, but these observations raise the hypothesis that ACh may regulate dopamine neurones indirectly through GABAergic interneurones. Using a mesencephalic primary culture model, we studied the impact of cholinergic agonists on mesencephalic GABAergic neurones. ACh increased the frequency of spontaneous IPSCs (151 ± 49%). Selective activation of muscarinic receptors increased the firing rate of isolated GABAergic neurones by 67 ± 13%. The enhancement in firing rate was Ca2+ dependent since inclusion of BAPTA in the pipette blocked it, actually revealing a decrease in firing rate accompanied by membrane hyperpolarization. This inhibitory action was prevented by tertiapin, a blocker of GIRK-type K+ channels. In addition to its excitatory somatodendritic effect, activation of muscarinic receptors also acted presynaptically, inhibiting the amplitude of unitary GABAergic synaptic currents. Both the enhancement in spontaneous IPSC frequency and presynaptic inhibition were abolished by 4-DAMP (100 nm), a preferential M3 muscarinic receptor antagonist. The presence of M3-like receptors on mesencephalic GABAergic neurones was confirmed by immunocytochemistry. Taken together, these results demonstrate that mesencephalic GABAergic neurones can be regulated directly through muscarinic receptors. Our findings provide new data that should be helpful in better understanding the influence of local GABAergic neurones during cholinergic activation of mesencephalic circuits. [source] | ||||||||||