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Inside-out Patches (inside-out + patch)
Selected AbstractsA Novel Background Potassium Channel in Rat Atrial CellsEXPERIMENTAL PHYSIOLOGY, Issue 4 2000Z. Shui A K+ channel activated by intracellular ATP has been observed in inside-out patches from rat atrial cells. The channel has a slope conductance of 130 ± 5 pS in symmetrical 140 mM K+ solution, and is almost independent of voltage over the range from -80 to +80 mV. There is no detectable inactivation during application of ATP over a few minutes. In the presence of 3 mM intracellular ATP, channel openings occur as bursts with a mean open time of 1.7 ms, a mean closed time of 0.4 ms, a mean burst duration of 18 ms and a mean burst interval of 41 ms. Kinetic analysis suggests that ATP mainly affects the burst duration and the burst interval of the channel. Based on the properties above, the channel differs from other known K+ channels in cardiac cells and may contribute to background K+ current. [source] Chlorotoxin-sensitive Ca2+ -activated Cl, channel in type R2 reactive astrocytes from adult rat brainGLIA, Issue 4 2003Stanislava Dalton Abstract Astrocytes express four types of Cl, or anion channels, but Ca2+ -activated Cl, (ClCa) channels have not been described. We studied Cl, channels in a morphologically distinct subpopulation (, 5% of cells) of small (10,12 ,m, 11.8 ± 0.6 pF), phase-dark, GFAP-positive native reactive astrocytes (NRAs) freshly isolated from injured adult rat brains. Their resting potential, ,57.1 ± 4.0 mV, polarized to ,72.7 ± 4.5 mV with BAPTA-AM, an intracellular Ca2+ chelator, and depolarized to ,30.7 ± 6.1 mV with thapsigargin, which mobilizes Ca2+ from intracellular stores. With nystatin-perforated patch clamp, thapsigargin activated a current that reversed near the Cl, reversal potential, which was blocked by Cl, channel blockers, 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) and Zn2+, by I, (10 mM), and by chlorotoxin (EC50 = 47 nM). With conventional whole-cell clamp, NPPB- and Zn2+ -sensitive currents became larger with increasing [Ca2+]i (10, 150, 300 nM). Single-channel recordings of inside-out patches confirmed Ca2+ sensitivity of the channel and showed open-state conductances of 40, 80, 130, and 180 pS, and outside-out patches confirmed sensitivity to chlorotoxin. In primary culture, small phase-dark NRAs developed into small GFAP-positive bipolar cells with chlorotoxin-sensitive ClCa channels. Imaging with biotinylated chlorotoxin confirmed the presence of label in GFAP-positive cells from regions of brain injury, but not from uninjured brain. Chlorotoxin-tagged cells isolated by flow cytometry and cultured up to two passages exhibit positive labeling for GFAP and vimentin, but not for prolyl 4-hydroxylase (fibroblast), A2B5 (O2A progenitor), or OX-42 (microglia). Expression of a novel chlorotoxin-sensitive ClCa channel in a morphologically distinct subpopulation of NRAs distinguishes these cells as a new subtype of reactive astrocyte. GLIA 42:325,339, 2003. © 2003 Wiley-Liss, Inc. [source] BK channels in human glioma cells have enhanced calcium sensitivity,GLIA, Issue 4 2002Christopher B. Ransom Abstract We have previously demonstrated the expression of large-conductance, calcium-activated potassium (BK) channels in human glioma cells. In the present study, we characterized the calcium sensitivity of glioma BK channels in excised membrane patches. Channels in inside-out patches were activated at ,60 mV by 2.1 × 10,6 M cytosolic Ca2+, were highly K+ -selective, and had a slope conductance of ,210 pS. We characterized the Ca2+ sensitivity of these channels in detail by isolating BK currents in outside-out patches with different free [Ca2+]i. The half-maximal voltage for channel activation, V0.5, of glioma BK currents in outside-out patches was +138 mV with 0 Ca2+/10 EGTA. V0.5 was shifted to +81 mV and ,14 mV with free [Ca2+]i of 1.5 × 10,7 M and 2.1 × 10,6 M, respectively. These results suggest that glioma BK channels have a higher Ca2+ sensitivity than that described in many other human preparations. Data obtained from a cloned BK channel (hbr5) expressed in HEK cells support the conclusion that glioma BK channels have an unusually high sensitivity to calcium. In addition, the sensitivity of glioma BK channels to the BK inhibitor tetrandrine suggests the expression of BK channel auxiliary ,-subunits by glioma cells. Expression of the auxiliary ,-subunit of BK channels by glioma cells may relate to the high Ca2+ sensitivity of glioma BK channels. GLIA 38:281,291, 2002. © 2002 Wiley-Liss, Inc. [source] Obligatory role for phosphatidylinositol 4,5-bisphosphate in activation of native TRPC1 store-operated channels in vascular myocytesTHE JOURNAL OF PHYSIOLOGY, Issue 3 2009Sohag N. Saleh In the present study the effect of phosphatidylinositol 4,5-bisphosphate (PIP2) was studied on a native TRPC1 store-operated channel (SOC) in freshly dispersed rabbit portal vein myocytes. Application of diC8-PIP2, a water soluble form of PIP2, to quiescent inside-out patches evoked single channel currents with a unitary conductance of 1.9 pS. DiC8-PIP2 -evoked channel currents were inhibited by anti-TRPC1 antibodies and these characteristics are identical to SOCs evoked by cyclopiazonic acid (CPA) and BAPTA-AM. SOCs stimulated by CPA, BAPTA-AM and the phorbol ester phorbol 12,13-dibutyrate (PDBu) were reduced by anti-PIP2 antibodies and by depletion of tissue PIP2 levels by pre-treatment of preparations with wortmannin and LY294002. However, these reagents did not alter the ability of PIP2 to activate SOCs in inside-out patches. Co-immunoprecipitation techniques demonstrated association between TRPC1 and PIP2 at rest, which was greatly decreased by wortmannin and LY294002. Pre-treatment of cells with PDBu, which activates protein kinase C (PKC), augmented SOC activation by PIP2 whereas the PKC inhibitor chelerythrine decreased SOC stimulation by PIP2. Co-immunoprecipitation experiments provide evidence that PKC-dependent phosphorylation of TRPC1 occurs constitutively and was increased by CPA and PDBu but decreased by chelerythrine. These novel results show that PIP2 can activate TRPC1 SOCs in native vascular myocytes and plays an important role in SOC activation by CPA, BAPTA-AM and PDBu. Moreover, the permissive role of PIP2 in SOC activation requires PKC-dependent phosphorylation of TRPC1. [source] Electrophysiological properties of BK channels in Xenopus motor nerve terminalsTHE JOURNAL OF PHYSIOLOGY, Issue 1 2004Xiao-Ping Sun Single channel properties of Ca2+ -activated K+ (BK or Maxi-K) channels have been investigated in presynaptic membranes in Xenopus motoneurone,muscle cell cultures. The occurrence and density of BK channels increased with maturation/synaptogenesis and was not uniform: highest at the release face of bouton-like synaptic varicosities in contact with muscle cells, and lowest in varicosities that did not contact muscle cells. The Ca2+ affinity of the channel (Kd= 7.7 ,m at a membrane potential of +20 mV) was lower than those of BK channels that have been characterized in other terminals. Hill coefficients varied between 1.5 and 2.8 at different potentials and open probability increased e-fold per 16 mV change in membrane potential over a range of [Ca2+]i from 1 ,m to 1 mm. The maximal activation rate of ensembled single BK channel currents was in the submillisecond range at ,+20 mV. The activation rate increased ,10-fold in response to a [Ca2+]i increase from 1 to 100 ,m, but increased only ,2-fold with a voltage change from +20 to +130 mV. The fastest activation kinetics of BK channels in cell-attached patches resembled that in inside-out patches with [Ca2+]i of 100 ,m or more, suggesting that many BK channels are located very close to calcium channels. Given the low Ca2+ affinity and rapid Ca2+ binding/unbinding properties, we conclude that BK channels in this preparation are adapted to play an important role in regulation of neurotransmitter release, and they are ideal reporters of local [Ca2+] at the inner membrane surface. [source] Direct interaction of Na-azide with the KATP channelBRITISH JOURNAL OF PHARMACOLOGY, Issue 6 2000Stefan Trapp The effects of the metabolic inhibitor sodium azide were tested on excised macropatches from Xenopus oocytes expressing cloned ATP-sensitive potassium (KATP) channels of the Kir6.2/SUR1 type. In inside-out patches from oocytes expressing Kir6.2,C36 (a truncated form of Kir6.2 that expresses in the absence of SUR), intracellular Na-azide inhibited macroscopic currents with an IC50 of 11 mM. The inhibitory effect of Na-azide was mimicked by the same concentration of NaCl, but not by sucrose. Na-azide and NaCl blocked Kir6.2/SUR1 currents with IC50 of 36 mM and 19 mM, respectively. Inhibition was abolished in the absence of intracellular Mg2+. In contrast, Kir6.2,C36 currents were inhibited by Na-azide both in the presence or absence of intracellular Mg2+. Kir6.2/SUR1 currents were less sensitive to 3 mM Na-azide in the presence of MgATP. This apparent reduction in sensitivity is caused by a small activatory effect of Na-azide conferred by SUR. We conclude that, in addition to its well-established inhibitory effect on cellular metabolism, which leads to activation of KATP channels in intact cells, intracellular Na-azide has direct effects on the KATP channel. Inhibition is intrinsic to Kir6.2, is mediated by Na+, and is modulated by SUR. There is also a small, ATP-dependent, stimulatory effect of Na-azide mediated by the SUR subunit. The direct effects of 3 mM Na-azide on KATP channels are negligible in comparison to the metabolic activation produced by the same Na-azide concentration. British Journal of Pharmacology (2000) 131, 1105,1112; doi:10.1038/sj.bjp.0703680 [source] | ||||||||||