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1.
Freshly dissociated myocytes from nonpregnant, pregnant, and postpartum rat uteri have been studied with the tight-seal patch-clamp method. The inward current contains both INa and ICa that are vastly different from those in tissue-cultured material. INa is abolished by Na+-free medium and by 1 μM tetrodotoxin. It first appears at ∼−40 mV, reaches maximum at 0 mV, and reverses at 84 mV. It activates with a voltage-dependent τ of 0.2 ms at 20 mV, and inactivates as a single exponential with a τ of 0.4 ms. Na+ conductance is half activated at −21.5 mV, and half inactivated at −59 mV. INa reactivates with a τ of 20 ms. ICa is abolished by Ca2+-free medium, Co2+ (5 mM), or nisoldipine (2 μM), and enhanced in 30 mM Ca2+, Ba2+, or BAY-K 8644. It first appears at ∼−30 mV and reaches maximum at +10 mV. It activates with a voltage-dependent τ of 1.5 ms at 20 mV, and inactivates in two exponential phases, with τ''s of 33 and 133 ms. Ca2+ conductance is half activated at −7.4 mV, and half inactivated at −34 mV. ICa reactivates with τ''s of 27 and 374 ms. INa and ICa are seen in myocytes from nonpregnant estrus uteri and throughout pregnancy, exhibiting complex changes. The ratio of densities of peak INa/ICa changes from 0.5 in the nonpregnant state to 1.6 at term. The enhanced role of INa, with faster kinetics, allows more frequent repetitive spike discharges to facilitate simultaneous excitation of the parturient uterus. In postpartum, both currents decrease markedly, with INa vanishing from most myocytes. Estrogen-enhanced genomic influences may account for the emergence of INa, and increased densities of INa and ICa as pregnancy progresses. Other influences may regulate varied channel expression at different stages of pregnancy.  相似文献   

2.
The epithelial Na+ channel (ENaC), composed of three subunits (α, β, and γ), is expressed in several epithelia and plays a critical role in salt and water balance and in the regulation of blood pressure. Little is known, however, about the electrophysiological properties of this cloned channel when expressed in epithelial cells. Using whole-cell and single channel current recording techniques, we have now characterized the rat αβγENaC (rENaC) stably transfected and expressed in Madin-Darby canine kidney (MDCK) cells. Under whole-cell patch-clamp configuration, the αβγrENaC-expressing MDCK cells exhibited greater whole cell Na+ current at −143 mV (−1,466.2 ± 297.5 pA) than did untransfected cells (−47.6 ± 10.7 pA). This conductance was completely and reversibly inhibited by 10 μM amiloride, with a Ki of 20 nM at a membrane potential of −103 mV; the amiloride inhibition was slightly voltage dependent. Amiloride-sensitive whole-cell current of MDCK cells expressing αβ or αγ subunits alone was −115.2 ± 41.4 pA and −52.1 ± 24.5 pA at −143 mV, respectively, similar to the whole-cell Na+ current of untransfected cells. Relaxation analysis of the amiloride-sensitive current after voltage steps suggested that the channels were activated by membrane hyperpolarization. Ion selectivity sequence of the Na+ conductance was Li+ > Na+ >> K+ = N-methyl-d-glucamine+ (NMDG+). Using excised outside-out patches, amiloride-sensitive single channel conductance, likely responsible for the macroscopic Na+ channel current, was found to be ∼5 and 8 pS when Na+ and Li+ were used as a charge carrier, respectively. K+ conductance through the channel was undetectable. The channel activity, defined as a product of the number of active channel (n) and open probability (P o), was increased by membrane hyperpolarization. Both whole-cell Na+ current and conductance were saturated with increased extracellular Na+ concentrations, which likely resulted from saturation of the single channel conductance. The channel activity (nP o) was significantly decreased when cytosolic Na+ concentration was increased from 0 to 50 mM in inside-out patches. Whole-cell Na+ conductance (with Li+ as a charge carrier) was inhibited by the addition of ionomycin (1 μM) and Ca2+ (1 mM) to the bath. Dialysis of the cells with a pipette solution containing 1 μM Ca2+ caused a biphasic inhibition, with time constants of 1.7 ± 0.3 min (n = 3) and 128.4 ± 33.4 min (n = 3). An increase in cytosolic Ca2+ concentration from <1 nM to 1 μM was accompanied by a decrease in channel activity. Increasing cytosolic Ca2+ to 10 μM exhibited a pronounced inhibitory effect. Single channel conductance, however, was unchanged by increasing free Ca2+ concentrations from <1 nM to 10 μM. Collectively, these results provide the first characterization of rENaC heterologously expressed in a mammalian epithelial cell line, and provide evidence for channel regulation by cytosolic Na+ and Ca2+.  相似文献   

3.
Plasma membrane large-conductance Ca2+-activated K+ (BKCa) channels and sarcoplasmic reticulum inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs) are expressed in a wide variety of cell types, including arterial smooth muscle cells. Here, we studied BKCa channel regulation by IP3 and IP3Rs in rat and mouse cerebral artery smooth muscle cells. IP3 activated BKCa channels both in intact cells and in excised inside-out membrane patches. IP3 caused concentration-dependent BKCa channel activation with an apparent dissociation constant (Kd) of ∼4 µM at physiological voltage (−40 mV) and intracellular Ca2+ concentration ([Ca2+]i; 10 µM). IP3 also caused a leftward-shift in BKCa channel apparent Ca2+ sensitivity and reduced the Kd for free [Ca2+]i from ∼20 to 12 µM, but did not alter the slope or maximal Po. BAPTA, a fast Ca2+ buffer, or an elevation in extracellular Ca2+ concentration did not alter IP3-induced BKCa channel activation. Heparin, an IP3R inhibitor, and a monoclonal type 1 IP3R (IP3R1) antibody blocked IP3-induced BKCa channel activation. Adenophostin A, an IP3R agonist, also activated BKCa channels. IP3 activated BKCa channels in inside-out patches from wild-type (IP3R1+/+) mouse arterial smooth muscle cells, but had no effect on BKCa channels of IP3R1-deficient (IP3R1−/−) mice. Immunofluorescence resonance energy transfer microscopy indicated that IP3R1 is located in close spatial proximity to BKCa α subunits. The IP3R1 monoclonal antibody coimmunoprecipitated IP3R1 and BKCa channel α and β1 subunits from cerebral arteries. In summary, data indicate that IP3R1 activation elevates BKCa channel apparent Ca2+ sensitivity through local molecular coupling in arterial smooth muscle cells.  相似文献   

4.
The relationship between Ca2+ release (“Ca2+ sparks”) through ryanodine-sensitive Ca2+ release channels in the sarcoplasmic reticulum and KCa channels was examined in smooth muscle cells from rat cerebral arteries. Whole cell potassium currents at physiological membrane potentials (−40 mV) and intracellular Ca2+ were measured simultaneously, using the perforated patch clamp technique and a laser two-dimensional (x–y) scanning confocal microscope and the fluorescent Ca2+ indicator, fluo-3. Virtually all (96%) detectable Ca2+ sparks were associated with the activation of a spontaneous transient outward current (STOC) through KCa channels. A small number of sparks (5 of 128) were associated with currents smaller than 6 pA (mean amplitude, 4.7 pA, at −40 mV). Approximately 41% of STOCs occurred without a detectable Ca2+ spark. The amplitudes of the Ca2+ sparks correlated with the amplitudes of the STOCs (regression coefficient 0.8; P < 0.05). The half time of decay of Ca2+ sparks (56 ms) was longer than the associated STOCs (9 ms). The mean amplitude of the STOCs, which were associated with Ca2+ sparks, was 33 pA at −40 mV. The mean amplitude of the “sparkless” STOCs was smaller, 16 pA. The very significant increase in KCa channel open probability (>104-fold) during a Ca2+ spark is consistent with local Ca2+ during a spark being in the order of 1–100 μM. Therefore, the increase in fractional fluorescence (F/Fo) measured during a Ca2+ spark (mean 2.04 F/Fo or ∼310 nM Ca2+) appears to significantly underestimate the local Ca2+ that activates KCa channels. These results indicate that the majority of ryanodine receptors that cause Ca2+ sparks are functionally coupled to KCa channels in the surface membrane, providing direct support for the idea that Ca2+ sparks cause STOCs.  相似文献   

5.
The kinetic properties of main and subconductance states of a mutant mouse N-methyl-d-aspartate (NMDA) receptor channel were examined. Recombinant receptors made of ζ-ε2 (NR1-NR2B) subunits having asparagine-to-glutamine mutations in the M2 segment (ζN598Q /ε2N589Q) were expressed in Xenopus oocytes. Single channel currents recorded from outside-out patches were analyzed using hidden Markov model techniques. In Ca2+-free solutions, an open receptor channel occupies a main conductance (93 pS) and a subconductance (62 pS) with about equal probability. There are both brief and long-lived subconductance states, but only a single main level state. At −80 mV, the lifetime of the main and the longer-lived sub level are both ∼3.3 ms. The gating of the pore and the transition between conductance levels are essentially independent processes. Surprisingly, hyperpolarization speeds both the sub-to-main and main-to-sub transition rate constants (∼120 mV/e-fold change), but does not alter the equilibrium occupancies. Extracellular Ca2+ does not influence the transition rate constants. We conclude that the subconductance levels arise from fluctuations in the energetics of ion permeation through a single pore, and that the voltage dependence of these fluctuations reflects the modulation by the membrane potential of the barrier between the main and subconductance conformations of the pore.  相似文献   

6.
Single-channel and [3H]ryanodine binding experiments were carried out to examine the effects of imperatoxin activator (IpTxa), a 33 amino acid peptide isolated from the venom of the African scorpion Pandinus imperator, on rabbit skeletal and canine cardiac muscle Ca2+ release channels (CRCs). Single channel currents from purified CRCs incorporated into planar lipid bilayers were recorded in 250 mM KCl media. Addition of IpTxa in nanomolar concentration to the cytosolic (cis) side, but not to the lumenal (trans) side, induced substates in both ryanodine receptor isoforms. The substates displayed a slightly rectifying current–voltage relationship. The chord conductance at −40 mV was ∼43% of the full conductance, whereas it was ∼28% at a holding potential of +40 mV. The substate formation by IpTxa was voltage and concentration dependent. Analysis of voltage and concentration dependence and kinetics of substate formation suggested that IpTxa reversibly binds to the CRC at a single site in the voltage drop across the channel. The rate constant for IpTxa binding to the skeletal muscle CRC increased e-fold per +53 mV and the rate constant of dissociation decreased e-fold per +25 mV applied holding potential. The effective valence of the reaction leading to the substate was ∼1.5. The IpTxa binding site was calculated to be located at ∼23% of the voltage drop from the cytosolic side. IpTxa induced substates in the ryanodine-modified skeletal CRC and increased or reduced [3H]ryanodine binding to sarcoplasmic reticulum vesicles depending on the level of channel activation. These results suggest that IpTxa induces subconductance states in skeletal and cardiac muscle Ca2+ release channels by binding to a single, cytosolically accessible site different from the ryanodine binding site.  相似文献   

7.
In this study, we numerically analyzed the nonlinear Ca2+-dependent gating dynamics of a single, nonconducting inositol 1,4,5-trisphosphate receptor (IP3R) channel, using an exact and fully stochastic simulation algorithm that includes channel gating, Ca2+ buffering, and Ca2+ diffusion. The IP3R is a ubiquitous intracellular Ca2+ release channel that plays an important role in the formation of complex spatiotemporal Ca2+ signals such as waves and oscillations. Dynamic subfemtoliter Ca2+ microdomains reveal low copy numbers of Ca2+ ions, buffer molecules, and IP3Rs, and stochastic fluctuations arising from molecular interactions and diffusion do not average out. In contrast to models treating calcium dynamics deterministically, the stochastic approach accounts for this molecular noise. We varied Ca2+ diffusion coefficients and buffer reaction rates to tune the autocorrelation properties of Ca2+ noise and found a distinct relation between the autocorrelation time τac, the mean channel open and close times, and the resulting IP3R open probability PO. We observed an increased PO for shorter noise autocorrelation times, caused by increasing channel open times and decreasing close times. In a pure diffusion model the effects become apparent at elevated calcium concentrations, e.g., at [Ca2+] = 25 μM, τac = 0.082 ms, the IP3R open probability increased by ≈20% and mean open times increased by ≈4 ms, compared to a zero noise model. We identified the inactivating Ca2+ binding site of IP3R subunits as the primarily noise-susceptible element of the De Young and Keizer model. Short Ca2+ noise autocorrelation times decrease the probability of Ca2+ association and consequently increase IP3R activity. These results suggest a functional role of local calcium noise properties on calcium-regulated target molecules such as the ubiquitous IP3R. This finding may stimulate novel experimental approaches analyzing the role of calcium noise properties on microdomain behavior.  相似文献   

8.
The stimulation of IP3 production by muscarinic agonists causes both intracellular Ca2+ release and activation of a voltage-independent cation current in differentiated N1E-115 cells, a neuroblastoma cell line derived from mouse sympathetic ganglia. Earlier work showed that the membrane current requires an increase in 3′,5′-cyclic guanosine monophosphate (cGMP) produced through the NO-synthase/guanylyl cyclase cascade and suggested that the cells may express cyclic nucleotide–gated ion channels. This was tested using patch clamp methods. The membrane permeable cGMP analogue, 8-br-cGMP, activates Na+ permeable channels in cell attached patches. Single channel currents were recorded in excised patches bathed in symmetrical Na+ solutions. cGMP-dependent single channel activity consists of prolonged bursts of rapid openings and closings that continue without desensitization. The rate of occurrence of bursts as well as the burst length increase with cGMP concentration. The unitary conductance in symmetrical 160 mM Na+ is 47 pS and is independent of voltage in the range −50 to +50 mV. There is no apparent effect of voltage on opening probability. The dose response curve relating cGMP concentration to channel opening probability is fit by the Hill equation assuming an apparent K D of 10 μm and a Hill coefficient of 2. In contrast, cAMP failed to activate the channel at concentrations as high as 100 μm. Cyclic nucleotide gated (CNG) channels in N1E-115 cells share a number of properties with CNG channels in sensory receptors. Their presence in neuronal cells provides a mechanism by which activation of the NO/cGMP pathway by G-protein–coupled neurotransmitter receptors can directly modify Ca2+ influx and electrical excitability. In N1E-115 cells, Ca2+ entry by this pathway is necessary to refill the IP3-sensitive intracellular Ca2+ pool during repeated stimulation and CNG channels may play a similar role in other neurons.  相似文献   

9.
The Ca2+-dependent gating mechanism of large-conductance calcium-activated K+ (BK) channels from cultured rat skeletal muscle was examined from low (4 μM) to high (1,024 μM) intracellular concentrations of calcium (Ca2+ i) using single-channel recording. Open probability (P o) increased with increasing Ca2+ i (K 0.5 11.2 ± 0.3 μM at +30 mV, Hill coefficient of 3.5 ± 0.3), reaching a maximum of ∼0.97 for Ca2+ i ∼ 100 μM. Increasing Ca2+ i further to 1,024 μM had little additional effect on either P o or the single-channel kinetics. The channels gated among at least three to four open and four to five closed states at high levels of Ca2+ i (>100 μM), compared with three to four open and five to seven closed states at lower Ca2+ i. The ability of kinetic schemes to account for the single-channel kinetics was examined with simultaneous maximum likelihood fitting of two-dimensional (2-D) dwell-time distributions obtained from low to high Ca2+ i. Kinetic schemes drawn from the 10-state Monod-Wyman-Changeux model could not describe the dwell-time distributions from low to high Ca2+ i. Kinetic schemes drawn from Eigen''s general model for a ligand-activated tetrameric protein could approximate the dwell-time distributions but not the dependency (correlations) between adjacent intervals at high Ca2+ i. However, models drawn from a general 50 state two-tiered scheme, in which there were 25 closed states on the upper tier and 25 open states on the lower tier, could approximate both the dwell-time distributions and the dependency from low to high Ca2+ i. In the two-tiered model, the BK channel can open directly from each closed state, and a minimum of five open and five closed states are available for gating at any given Ca2+ i. A model that assumed that the apparent Ca2+-binding steps can reach a maximum rate at high Ca2+ i could also approximate the gating from low to high Ca2+ i. The considered models can serve as working hypotheses for the gating of BK channels.  相似文献   

10.
Cut muscle fibers from Rana temporaria (sarcomere length, 3.5–3.9 μm; 14–16°C) were mounted in a double Vaseline-gap chamber and equilibrated with an external solution that contained tetraethyl ammonium– gluconate and an internal solution that contained Cs as the principal cation, 20 mM EGTA, and 0 Ca. Fibers were stimulated with a voltage-clamp pulse protocol that consisted of pulses to −70, −65, −60, −45, and −20 mV, each separated by 400-ms periods at −90 mV. The change in total Ca that entered into the myoplasm (Δ[CaT]) and the Ca content of the SR ([CaSR]) were estimated with the EGTA/phenol red method (Pape, P.C., D.-S. Jong, and W.K. Chandler. 1995. J. Gen. Physiol. 106:259–336). Fibers were stimulated with the pulse protocol, usually every 5 min, so that the resting value of [CaSR] decreased from its initial value of 1,700–2,300 μM to values near or below 100 μM after 18–30 stimulations. Three main findings for the voltage pulses to −70, −65, and −60 mV are: (a) the depletion-corrected rate of Ca release (release permeability) showed little change when [CaSR] decreased from its highest level (>1,700 μM) to ∼1,000 μM; (b) as [CaSR] decreased below 1,000 μM, the release permeability increased to a maximum level when [CaSR] was near 300 μM that was on average about sevenfold larger than the values observed for [CaSR] > 1,000 μM; and (c) as [CaSR] decreased from ∼300 μM to <100 μM, the release permeability decreased, reaching half its maximum value when [CaSR] was ∼110 μM on average. It was concluded that finding b was likely due to a decrease in Ca inactivation, while finding c was likely due to a decrease in Ca-induced Ca release.  相似文献   

11.
Modulation of L-type Ca2+ channels by tonic elevation of cytoplasmic Ca2+ was investigated in intact cells and inside-out patches from human umbilical vein smooth muscle. Ba2+ was used as charge carrier, and run down of Ca2+ channel activity in inside-out patches was prevented with calpastatin plus ATP. Increasing cytoplasmic Ca2+ in intact cells by elevation of extracellular Ca2+ in the presence of the ionophore A23187 inhibited the activity of L-type Ca2+ channels in cell-attached patches. Measurement of the actual level of intracellular free Ca2+ with fura-2 revealed a 50% inhibitory concentration (IC50) of 260 nM and a Hill coefficient close to 4 for Ca2+- dependent inhibition. Ca2+-induced inhibition of Ca2+ channel activity in intact cells was due to a reduction of channel open probability and availability. Ca2+-induced inhibition was not affected by the protein kinase inhibitor H-7 (10 μM) or the cytoskeleton disruptive agent cytochalasin B (20 μM), but prevented by cyclosporin A (1 μg/ ml), an inhibitor of protein phosphatase 2B (calcineurin). Elevation of Ca2+ at the cytoplasmic side of inside-out patches inhibited Ca2+ channels with an IC50 of 2 μM and a Hill coefficient close to unity. Direct Ca2+-dependent inhibition in cell-free patches was due to a reduction of open probability, whereas availability was barely affected. Application of purified protein phosphatase 2B (12 U/ml) to the cytoplasmic side of inside-out patches at a free Ca2+ concentration of 1 μM inhibited Ca2+ channel open probability and availability. Elevation of cytoplasmic Ca2+ in the presence of PP2B, suppressed channel activity in inside-out patches with an IC50 of ∼380 nM and a Hill coefficient of ∼3; i.e., characteristics reminiscent of the Ca2+ sensitivity of Ca2+ channels in intact cells. Our results suggest that L-type Ca2+ channels of smooth muscle are controlled by two Ca2+-dependent negative feedback mechanisms. These mechanisms are based on (a) a protein phosphatase 2B-mediated dephosphorylation process, and (b) the interaction of intracellular Ca2+ with a single membrane-associated site that may reside on the channel protein itself.  相似文献   

12.
Ba2+ block of large conductance Ca2+-activated K+ channels was studied in patches of membrane excised from cultures of rat skeletal muscle using the patch clamp technique. Under conditions in which a blocking Ba2+ ion would dissociate to the external solution (150 mM N-methyl-d-glucamine+ o, 500 mM K+ i, 10 μM Ba2+ i, +30 mV, and 100 μM Ca2+ i to fully activate the channel), Ba2+ blocks with a mean duration of ∼2 s occurred, on average, once every ∼100 ms of channel open time. Of these Ba2+ blocks, 78% terminated with a single step in the current to the fully open level and 22% terminated with a transition to a subconductance level at ∼0.26 of the fully open level (preopening) before stepping to the fully open level. Only one apparent preclosing was observed in ∼10,000 Ba2+ blocks. Thus, the preopenings represent Ba2+-induced time-irreversible subconductance gating. The fraction of Ba2+ blocks terminating with a preopening and the duration of preopenings (exponentially distributed, mean = 0.75 ms) appeared independent of changes in [Ba2+]i or membrane potential. The fractional conductance of the preopenings increased from 0.24 at +10 mV to 0.39 at +90 mV. In contrast, the average subconductance level during normal gating in the absence of Ba2+ was independent of membrane potential, suggesting different mechanisms for preopenings and normal subconductance levels. Preopenings were also observed with 10 mM Ba2+ o and no added Ba2+ i. Adding K+, Rb+, or Na+ to the external solution decreased the fraction of Ba2+ blocks with preopenings, with K+ and Rb+ being more effective than Na+. These results are consistent with models in which the blocking Ba2+ ion either induces a preopening gate, and then dissociates to the external solution, or moves to a site located on the external side of the Ba2+ blocking site and acts directly as the preopening gate.  相似文献   

13.
Two classes of small homologous basic proteins, mamba snake dendrotoxins (DTX) and bovine pancreatic trypsin inhibitor (BPTI), block the large conductance Ca2+-activated K+ channel (BKCa, KCa1.1) by production of discrete subconductance events when added to the intracellular side of the membrane. This toxin-channel interaction is unlikely to be pharmacologically relevant to the action of mamba venom, but as a fortuitous ligand-protein interaction, it has certain biophysical implications for the mechanism of BKCa channel gating. In this work we examined the subconductance behavior of 9 natural dendrotoxin homologs and 6 charge neutralization mutants of δ-dendrotoxin in the context of current structural information on the intracellular gating ring domain of the BKCa channel. Calculation of an electrostatic surface map of the BKCa gating ring based on the Poisson-Boltzmann equation reveals a predominantly electronegative surface due to an abundance of solvent-accessible side chains of negatively charged amino acids. Available structure-activity information suggests that cationic DTX/BPTI molecules bind by electrostatic attraction to site(s) on the gating ring located in or near the cytoplasmic side portals where the inactivation ball peptide of the β2 subunit enters to block the channel. Such an interaction may decrease the apparent unitary conductance by altering the dynamic balance of open versus closed states of BKCa channel activation gating.  相似文献   

14.
We used the patch-clamp technique to study the voltage-dependent properties of the swelling-activated Cl current (I Cl,swell) in BC3H1 myoblasts. This Cl current is outwardly rectifying and exhibits time-dependent inactivation at positive potentials (potential for half-maximal inactivation of +75 mV). Single-channel Cl currents with similar voltage-dependent characteristics could be measured in outside-out patches pulled from swollen cells. The estimated single-channel slope conductance in the region between +60 and +140 mV was 47 pS. The time course of inactivation was well described by a double exponential function, with a voltage-independent fast time constant (∼60 ms) and a voltage-dependent slow time constant (>200 ms). Recovery from inactivation, which occurred over the physiological voltage range, was also well described by a double exponential function, with a voltage-dependent fast time constant (10–80 ms) and a voltage-dependent slow time constant (>100 ms). The inactivation process was significantly accelerated by reducing the pH, increasing the Mg2+ concentration or reducing the Cl concentration of the extracellular solution. Replacing extracellular Cl by other permeant anions shifted the inactivation curve in parallel with their relative permeabilities (SCN > I > NO3 > Cl >> gluconate). A leftward shift of the inactivation curve could also be induced by channel blockers. Additionally, the permeant anion and the channel blockers, but not external pH or Mg2+, modulated the recovery from inactivation. In conclusion, our results show that the voltage-dependent properties of I Cl,swell are strongly influenced by external pH , external divalent cations, and by the nature of the permeant anion.  相似文献   

15.
It has been reported that protamine (>10 µg/ml) blocks single skeletal RyR1 channels and inhibits RyR1-mediated Ca2+ release from sarcoplasmic reticulum microsomes. We extended these studies to cardiac RyR2 reconstituted into planar lipid bilayers. We found that protamine (0.02–20 µg/ml) added to the cytosolic surface of fully activated RyR2 affected channel activity in a voltage-dependent manner. At membrane voltage (Vm; SR lumen - cytosol) = 0 mV, protamine induced conductance transitions to several intermediate states (substates) as well as full block of RyR2. At Vm>10 mV, the substate with the highest level of conductance was predominant. Increasing Vm from 0 to +80 mV, decreased the number of transitions and residence of the channel in this substate. The drop in current amplitude (full opening to substate) had the same magnitude at 0 and +80 mV despite the ∼3-fold increase in amplitude of the full opening. This is more similar to rectification of channel conductance induced by other polycations than to the action of selective conductance modifiers (ryanoids, imperatoxin). A distinctive effect of protamine (which might be shared with polylysines and histones but not with non-peptidic polycations) is the activation of RyR2 in the presence of nanomolar cytosolic Ca2+ and millimolar Mg2+ levels. Our results suggest that RyRs would be subject to dual modulation (activation and block) by polycationic domains of neighboring proteins via electrostatic interactions. Understanding these interactions could be important as such anomalies may be associated with the increased RyR2-mediated Ca2+ leak observed in cardiac diseases.  相似文献   

16.
The neuropeptide Phe-Met-Arg-Phe-amide (FMRFa) dose dependently (ED50 = 23 nM) activated a K+ current in the peptidergic caudodorsal neurones that regulate egg laying in the mollusc Lymnaea stagnalis. Under standard conditions ([K+]o = 1.7 mM), only outward current responses occurred. In high K+ salines ([K+]o = 20 or 57 mM), current reversal occurred close to the theoretical reversal potential for K+. In both salines, no responses were measured below −120 mV. Between −120 mV and the K+ reversal potential, currents were inward with maximal amplitudes at ∼−60 mV. Thus, U-shaped current–voltage relations were obtained, implying that the response is voltage dependent. The conductance depended both on membrane potential and extracellular K+ concentration. The voltage sensitivity was characterized by an e-fold change in conductance per ∼14 mV at all [K+]o. Since this result was also obtained in nearly symmetrical K+ conditions, it is concluded that channel gating is voltage dependent. In addition, outward rectification occurs in asymmetric K+ concentrations. Onset kinetics of the response were slow (rise time ∼650 ms at −40 mV). However, when FMRFa was applied while holding the cell at −120 mV, to prevent activation of the current but allow activation of the signal transduction pathway, a subsequent step to −40 mV revealed a much more rapid current onset. Thus, onset kinetics are largely determined by steps preceding channel activation. With FMRFa applied at −120 mV, the time constant of activation during the subsequent test pulse decreased from ∼36 ms at −60 mV to ∼13 ms at −30 mV, confirming that channel opening is voltage dependent. The current inactivated voltage dependently. The rate and degree of inactivation progressively increased from −120 to −50 mV. The current is blocked by internal tetraethylammonium and by bath- applied 4-aminopyridine, tetraethylammonium, Ba2+, and, partially, Cd2+ and Cs+. The response to FMRFa was affected by intracellular GTPγS. The response was inhibited by blockers of phospholipase A2 and lipoxygenases, but not by a cyclo-oxygenase blocker. Bath-applied arachidonic acid induced a slow outward current and occluded the response to FMRFa. These results suggest that the FMRFa receptor couples via a G-protein to the lipoxygenase pathway of arachidonic acid metabolism. The biophysical and pharmacological properties of this transmitter operated, but voltage-dependent K+ current distinguish it from other receptor-driven K+ currents such as the S-current- and G-protein-dependent inward rectifiers.  相似文献   

17.
The liberation of calcium ions sequestered in the endoplasmic reticulum through inositol 1,4,5-trisphosphate receptors/channels (IP3Rs) results in a spatiotemporal hierarchy of calcium signaling events that range from single-channel openings to local Ca2+ puffs believed to arise from several to tens of clustered IP3Rs to global calcium waves. Using high-resolution confocal linescan imaging and a sensitive Ca2+ indicator dye (fluo-4-dextran), we show that puffs are often preceded by small, transient Ca2+ elevations that we christen “trigger events”. The magnitude of triggers is consistent with their arising from the opening of a single IP3 receptor/channel, and we propose that they initiate puffs by recruiting neighboring IP3Rs within the cluster by a regenerative process of Ca2+-induced Ca2+ release. Puff amplitudes (fluorescence ratio change) are on average ~6 times greater than that of the triggers, suggesting that at least six IP3Rs may simultaneously be open during a puff. Trigger events have average durations of ~12 ms, as compared to 19 ms for the mean rise time of puffs, and their spatial extent is ~3 times smaller than puffs (respective widths at half peak amplitude 0.6 and 1.6 μm). All these parameters were relatively independent of IP3 concentration, although the proportion of puffs showing resolved triggers was greatest (~80%) at low [IP3]. Because Ca2+ puffs constitute the building blocks from which cellular IP3-mediated Ca2+ signals are constructed, the events that initiate them are likely to be of fundamental importance for cell signaling. Moreover, the trigger events provide a useful yardstick by which to derive information regarding the number and spatial arrangement of IP3Rs within clusters.  相似文献   

18.
Sustained elevation of intracellular calcium by Ca2+ release–activated Ca2+ channels is required for lymphocyte activation. Sustained Ca2+ entry requires endoplasmic reticulum (ER) Ca2+ depletion and prolonged activation of inositol 1,4,5-trisphosphate receptor (IP3R)/Ca2+ release channels. However, a major isoform in lymphocyte ER, IP3R1, is inhibited by elevated levels of cytosolic Ca2+, and the mechanism that enables the prolonged activation of IP3R1 required for lymphocyte activation is unclear. We show that IP3R1 binds to the scaffolding protein linker of activated T cells and colocalizes with the T cell receptor during activation, resulting in persistent phosphorylation of IP3R1 at Tyr353. This phosphorylation increases the sensitivity of the channel to activation by IP3 and renders the channel less sensitive to Ca2+-induced inactivation. Expression of a mutant IP3R1-Y353F channel in lymphocytes causes defective Ca2+ signaling and decreased nuclear factor of activated T cells activation. Thus, tyrosine phosphorylation of IP3R1-Y353 may have an important function in maintaining elevated cytosolic Ca2+ levels during lymphocyte activation.  相似文献   

19.
Over the past few years, it has become clear that an important mechanism by which large-conductance Ca2+-activated K+ channel (BKCa) activity is regulated is the tissue-specific expression of auxiliary β subunits. The first of these to be identified, β1, is expressed predominately in smooth muscle and causes dramatic effects, increasing the apparent affinity of the channel for Ca2+ 10-fold at 0 mV, and shifting the range of voltages over which the channel activates −80 mV at 9.1 μM Ca2+. With this study, we address the question: which aspects of BKCa gating are altered by β1 to bring about these effects: Ca2+ binding, voltage sensing, or the intrinsic energetics of channel opening? The approach we have taken is to express the β1 subunit together with the BKCa α subunit in Xenopus oocytes, and then to compare β1''s steady state effects over a wide range of Ca2+ concentrations and membrane voltages to those predicted by allosteric models whose parameters have been altered to mimic changes in the aspects of gating listed above. The results of our analysis suggest that much of β1''s steady state effects can be accounted for by a reduction in the intrinsic energy the channel must overcome to open and a decrease in its voltage sensitivity, with little change in the affinity of the channel for Ca2+ when it is either open or closed. Interestingly, however, the small changes in Ca2+ binding affinity suggested by our analysis (Kc 7.4 μM → 9.6 μM; Ko = 0.80 μM → 0.65 μM) do appear to be functionally important. We also show that β1 affects the mSlo conductance–voltage relation in the essential absence of Ca2+, shifting it +20 mV and reducing its apparent gating charge 38%, and we develop methods for distinguishing between alterations in Ca2+ binding and other aspects of BKCa channel gating that may be of general use.  相似文献   

20.
Serotonin (5-hydroxytryptamine, 5-HT) is a potent pulmonary vasoconstrictor that promotes pulmonary artery smooth muscle cell (PASMC) proliferation. 5-HT-induced K+ channel inhibition increases [Ca2+]i in PASMCs, which is a major trigger for pulmonary vasoconstriction and development of pulmonary arterial hypertension (PAH). This study investigated whether KMUP-1 reduces pulmonary vasoconstriction in isolated pulmonary arteries (PAs) and attenuates 5-HT-inhibited K+ channel activities in PASMCs. In endothelium-denuded PA rings, KMUP-1 (1 μM) dose-dependently reduced 5-HT (100 μM) mediated contractile responses. Responses to KMUP-1 were reversed by K+ channel inhibitors (TEA, 10 mM, 4-aminopyridine, 5 mM, and paxilline, 10 μM). In primary PASMCs, KMUP-1 also dose-dependently restored 5-HT-inhibited voltage-gated K+-channel (Kv1.5 and Kv2.1) and large-conductance Ca2+-activated K+-channel (BKCa) proteins, as confirmed by immunofluorescent staining. Furthermore, 5-HT (10 μM)-inhibited Kv1.5 protein was unaffected by the PKA inhibitor KT5720 (1 μM) and the PKC activator PMA (1 μM), but these effects were reversed by KMUP-1 (1 μM), 8-Br-cAMP (100 μM), chelerythrine (1 μM), and KMUP-1 combined with a PKA/PKC activator or inhibitor. Notably, KMUP-1 reversed 5-HT-inhibited Kv1.5 protein and this response was significantly attenuated by co-incubation with the PKC activator PMA, suggesting that 5-HT-mediated PKC signaling can be modulated by KMUP-1. In conclusion, KMUP-1 ameliorates 5-HT-induced vasoconstriction and K+-channel inhibition through the PKC pathway, which could be valuable to prevent the development of PAH.  相似文献   

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