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S W Jones

Publications and source records attributed to S W Jones.

At least 19 recordsLinked to original sources
PubMed2001

U-type inactivation of Kv3.1 and Shaker potassium channels.

We previously concluded that the Kv2.1 K(+) channel inactivates preferentially from partially activated closed states. We report here that the Kv3.1 channel also exhibits two key features of this inactivation mechanism: a U-shaped voltage dependence measured at 10 s and stronger inactivation with repetitive pulses than with a single long depolarization. More surprisingly, slow inactivation of the Kv1 Shaker K(+) channel (Shaker B Delta 6--46) also has a U-shaped voltage dependence for 10-s depolarizations. The time and voltage dependence of recovery from inactivation reveals two distinct components for Shaker. Strong depolarizations favor inactivation that is reduced by K(o)(+) or by partial block by TEA(o), as previously reported for slow inactivation of Shaker. However, depolarizations near 0 mV favor inactivation that recovers rapidly, with strong voltage dependence (as for Kv2.1 and 3.1). The fraction of channels that recover rapidly is increased in TEA(o) or high K(o)(+). We introduce the term U-type inactivation for the mechanism that is dominant in Kv2.1 and Kv3.1. U-type inactivation also makes a major but previously unrecognized contribution to slow inactivation of Shaker.

Allosteric Regulation↗
PubMed2001

Gating kinetics of the alpha1I T-type calcium channel.

The alpha1I T-type calcium channel inactivates almost 10-fold more slowly than the other family members (alpha1G and alpha1H) or most native T-channels. We have examined the underlying mechanisms using whole-cell recordings from rat alpha1I stably expressed in HEK293 cells. We found several kinetic differences between alpha1G and alpha1I, including some properties that at first appear qualitatively different. Notably, alpha1I tail currents require two or even three exponentials, whereas alpha1G tails were well described by a single exponential over a wide voltage range. Also, closed-state inactivation is more significant for alpha1I, even for relatively strong depolarizations. Despite these differences, gating of alpha1I can be described by the same kinetic scheme used for alpha1G, where voltage sensor movement is allosterically coupled to inactivation. Nearly all of the rate constants in the model are 5-12-fold slower for alpha1I, but the microscopic rate for channel closing is fourfold faster. This suggests that T-channels share a common gating mechanism, but with considerable quantitative variability.

Action Potentials↗
PubMed2000

Mg(2+) block unmasks Ca(2+)/Ba(2+) selectivity of alpha1G T-type calcium channels.

We have examined permeation by Ca(2+) and Ba(2+), and block by Mg(2+), using whole-cell recordings from alpha1G T-type calcium channels stably expressed in HEK 293 cells. Without Mg(o)(2+), inward currents were comparable with Ca(2+) and Ba(2+). Surprisingly, three other results indicate that alpha1G is actually selective for Ca(2+) over Ba(2+). 1) Mg(2+) block is approximately 7-fold more potent with Ba(2+) than with Ca(2+). With near-physiological (1 mM) Mg(o)(2+), inward currents were approximately 3-fold larger with 2 mM Ca(2+) than with 2 mM Ba(2+). The stronger competition between Ca(2+) and Mg(2+) implies that Ca(2+) binds more tightly than Ba(2+). 2) Outward currents (carried by Na(+)) are blocked more strongly by Ca(2+) than by Ba(2+). 3) The reversal potential is more positive with Ca(2+) than with Ba(2+), thus P(Ca) > P(Ba). We conclude that alpha1G can distinguish Ca(2+) from Ba(2+), despite the similar inward currents in the absence of Mg(o)(2+). Our results can be explained by a 2-site, 3-barrier model if Ca(2+) enters the pore 2-fold more easily than Ba(2+) but exits the pore at a 2-fold lower rate.

Animals↗
PubMed2000

Apparent change in ion selectivity caused by changes in intracellular K(+) during whole-cell recording.

In whole-cell recordings from HEK293 cells stably transfected with the delayed rectifier K(+) channel Kv2.1, long depolarizations produce current-dependent changes in [K(+)](i) that mimic inactivation and changes in ion selectivity. With 10 mM K(o)(+) or K(i)(+), and 140-160 mM Na(i,o)(+), long depolarizations shifted the reversal potential (V(R)) toward E(Na). However, similar shifts in V(R) were observed when Na(i,o)(+) was replaced with N-methyl-D-glucamine (NMG(+))(i, o). In that condition, [K(+)](o) did not change significantly, but the results could be quantitatively explained by changes in [K(+)](i). For example, a mean outward K(+) current of 1 nA for 2 s could decrease [K(+)](i) from 10 mM to 3 mM in a 10 pF cell. Dialysis by the recording pipette reduced but did not fully prevent changes in [K(+)](i). With 10 mM K(i,o)(+), 150 mM Na(i)(+), and 140 mM NMG(o)(+), steps to +20 mV produced a positive shift in V(R), as expected from depletion of K(i)(+), but opposite to the shift expected from a decreased K(+)/Na(+) selectivity. Long steps to V(R) caused inactivation, but no change in V(R). We conclude that current-dependent changes in [K(+)](i) need to be carefully evaluated when studying large K(+) currents in small cells.

Biophysical Phenomena↗
PubMed1999

Fibre type-specific expression of p94, a skeletal muscle-specific calpain.

Members of the calpain proteinase family are present in all mammalian cells, although a novel calpain 94 kDa isoform is found almost exclusively in skeletal muscle. p94 is difficult to purify from muscle and recombinant p94 autolyses rapidly when expressed in COS cells. However, in vivo the enzyme may be stabilised by interaction with titin, which has two well-characterised binding sites for p94 at the N2- and M-lines. Both these titin subdomains are subject to muscle-specific alternative splicing, which could be related to p94 expression level or stability in muscles of different fibre type. In this study, porcine longissimus dorsi (LD), trapezius (TZ) and adductor longus (AL) were characterised as fast, intermediate and slow using commercially available specific anti-human fast- and slow-myosin heavy chain mAbs and also by conventional histochemistry. p94 was quantified both in whole muscle preparations and single fibres by western blotting using an anti-p94 antiserum generated by expressing a recombinant p94 sequence as a GST fusion protein antigen. SDS PAGE and immunoblotting revealed a single band of approximately 94 kDa with identical mobility in all muscle and fibre preparations. The intensity of the 94 kDa band was greater in LD (22 +/- 1.7 densitometric units mean +/- SEM, n = 3) than TZ and AL (10 +/- 2.3 and 6 +/- 0.9 units, respectively). Expressed as a ratio relative to actin immunoreactivity, p94 is present in all types of single fibres isolated from TZ, but at a significantly lower level (P < 0.01) in slow type I (0.08 +/- 0.01, n = 9), compared to fast IIA/IIB fibres (0.22 +/- 0.02, n = 26). No evidence was seen for rapid or variable rate of p94 degradation in either type of fibre. These data suggest a positive correlation between p94 expression level and fast glycolytic characteristics in porcine muscle.

Animals↗
PubMed1999

State-dependent inactivation of the alpha1G T-type calcium channel.

We have examined the kinetics of whole-cell T-current in HEK 293 cells stably expressing the alpha1G channel, with symmetrical Na(+)(i) and Na(+)(o) and 2 mM Ca(2+)(o). After brief strong depolarization to activate the channels (2 ms at +60 mV; holding potential -100 mV), currents relaxed exponentially at all voltages. The time constant of the relaxation was exponentially voltage dependent from -120 to -70 mV (e-fold for 31 mV; tau = 2.5 ms at -100 mV), but tau = 12-17 ms from-40 to +60 mV. This suggests a mixture of voltage-dependent deactivation (dominating at very negative voltages) and nearly voltage-independent inactivation. Inactivation measured by test pulses following that protocol was consistent with open-state inactivation. During depolarizations lasting 100-300 ms, inactivation was strong but incomplete (approximately 98%). Inactivation was also produced by long, weak depolarizations (tau = 220 ms at -80 mV; V(1/2) = -82 mV), which could not be explained by voltage-independent inactivation exclusively from the open state. Recovery from inactivation was exponential and fast (tau = 85 ms at -100 mV), but weakly voltage dependent. Recovery was similar after 60-ms steps to -20 mV or 600-ms steps to -70 mV, suggesting rapid equilibration of open- and closed-state inactivation. There was little current at -100 mV during recovery from inactivation, consistent with </=8% of the channels recovering through the open state. The results are well described by a kinetic model where inactivation is allosterically coupled to the movement of the first three voltage sensors to activate. One consequence of state-dependent inactivation is that alpha1G channels continue to inactivate after repolarization, primarily from the open state, which leads to cumulative inactivation during repetitive pulses.

Algorithms↗
PubMed1998

Multiple channels mediate calcium leakage in the A7r5 smooth muscle-derived cell line.

Ca2+ entry under resting conditions may be important for contraction of vascular smooth muscle, but little is known about the mechanisms involved. Ca2+ leakage was studied in the A7r5 smooth muscle-derived cell line by patch-clamp techniques. Two channels that could mediate calcium influx at resting membrane potentials were characterized. In 110 mM Ba2+, one channel had a slope conductance of 6.0 +/- 0.6 pS and an extrapolated reversal potential of +41 +/- 13 mV (mean +/- SD, n = 8). The current rectified strongly, with no detectable outward current, even at +90 mV. Channel gating was voltage independent. A second type of channel had a linear current-voltage relationship, a slope conductance of 17.0 +/- 3.2 pS, and a reversal potential of +7 +/- 4 mV (n = 9). The open probability increased e-fold per 44 +/- 10 mV depolarization (n = 5). Both channels were also observed in 110 mM Ca2+. Noise analysis of whole-cell currents indicates that approximately 100 6-pS channels and 30 17-pS channels are open per cell. These 6-pS and 17-pS channels may contribute to resting calcium entry in vascular smooth muscle cells.

Animals↗
PubMed1998

Inactivation of Kv2.1 potassium channels.

We report here several unusual features of inactivation of the rat Kv2.1 delayed rectifier potassium channel, expressed in Xenopus oocytes. The voltage dependence of inactivation was U-shaped, with maximum inactivation near 0 mV. During a maintained depolarization, development of inactivation was slow and only weakly voltage dependent (tau = 4 s at 0 mV; tau = 7 s at +80 mV). However, recovery from inactivation was strongly voltage dependent (e-fold for 20 mV) and could be rapid (tau = 0.27 s at -140 mV). Kv2.1 showed cumulative inactivation, where inactivation built up during a train of brief depolarizations. A single maintained depolarization produced more steady-state inactivation than a train of pulses, but there could actually be more inactivation with the repeated pulses during the first few seconds. We term this phenomenon "excessive cumulative inactivation." These results can be explained by an allosteric model, in which inactivation is favored by activation of voltage sensors, but the open state of the channel is resistant to inactivation.

Allosteric Regulation↗
PubMed1998

Surface charge and lanthanum block of calcium current in bullfrog sympathetic neurons.

The density of surface charge associated with the calcium channel pore was estimated from the effect of extracellular ionic strength on block by La3+. Currents carried by 2 mM Ba2+ were recorded from isolated frog sympathetic neurons by the whole-cell patch-clamp technique. In normal ionic strength (120 mM N-methyl-D-glucamine, NMG), La3+ blocked the current with high affinity (IC50 = 22 nM at 0 mV). La3+ block was relieved by strong depolarization in a time- and voltage-dependent manner. After unblocking, open channels reblocked rapidly at 0 mV, allowing estimation of association and dissociation rates for La3+: k(on) = (7.2 +/- 0.7) x 10(8) M(-1) s(-1), k(off) = 10.0 +/- 0.5 s(-1). To assess surface charge effects, La3+ block was also measured in low ionic strength (12.5 mM NMG) and high ionic strength (250 mM NMG). La3+ block was higher affinity and faster by two- to threefold in 12.5 mM NMG, with little effect of 250 mM NMG. The data could be described by Gouy-Chapman theory with a surface charge density of approximately 1 e-/3000-4000 A2. These results indicate that there is a small but detectable surface charge associated with the pore of voltage-dependent calcium channels.

Animals↗
PubMed1998

Overview of voltage-dependent calcium channels.

Voltage-dependent calcium channels couple electrical signals to cellular responses in excitable cells. Calcium channels are crucial for excitation-secretion coupling in neurons and endocrine cells, and excitation-contraction coupling in muscle. Several pharmacologically and kinetically distinct calcium channel types have been identified at the electrophysiological and molecular levels. This review summarizes the basic properties of voltage-dependent calcium channels, including mechanisms of ion permeation and block, gating kinetics, and modulation by G proteins and second messengers.

Animals↗
PubMed1998

Facilitation of L-type calcium current in thalamic neurons.

We have studied facilitation of the L-type calcium current in neurons acutely isolated from the ventrobasal nucleus of the rat thalamus. Currents were recorded after pretreatment with 1 microM omega-conotoxin GVIA and 5 microM omega-conotoxin MVIIC, to better isolate L-current. Long, strong depolarizations induced slow tail currents at negative voltages, but did not affect currents at voltages where channels were strongly activated. The initial peak tail current was not measurably increased. The time course of recovery from facilitation paralleled the time course of the tail current, indicating that facilitation does not outlast channel closing. The kinase inhibitors staurosporine and H-7 and the phosphatase inhibitor okadaic acid had no significant effect on L-current facilitation compared with control, but facilitation was greater with H-7 than with okadaic acid. The guanosine 5'-triphosphate (GTP) analogs GTP-gamma-S and GDP-beta-S did not affect facilitation. We conclude that L-current facilitation in thalamic neurons does not result from Ser/Thr phosphorylation, although phosphorylation may modulate facilitation. This form of facilitation differs kinetically and pharmacologically from facilitation induced by activation of G protein-coupled receptors.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1998

Activation kinetics of the delayed rectifier potassium current of bullfrog sympathetic neurons.

We examined the activation kinetics of the delayed rectifier K+ current of bullfrog sympathetic neurons, primarily using whole cell recording. On depolarization, currents activated with a sigmoid delay but did not show a Cole-Moore shift. The time course of activation differed systematically from an exponential raised to a power. At most voltages, a power of 2 gave the best overall fit but a power of 3 better described the initial delay. After the delay, the time course could be fitted by a single exponential. Time constants were 15-20 ms at 0 mV and decreased to a limiting tau = 7 ms at +50 to +100 mV. Tail currents were well fitted by single exponential functions and accelerated with hyperpolarization, from tau = 15-20 ms at 0 mV to tau = 2 ms at -110 mV (e-fold for 40 mV). Eleven kinetic models were evaluated for their ability to describe the activation kinetics of the delayed rectifier. Hodgkin-Huxley-like models did not fit the data well. A linear model where voltage sensor movement is followed by a distinct channel opening step, allosteric models based on the Monod-Wyman--Changeux model, and an unconstrained C-C-C-O model could describe whole cell data from -100 to +40 mV. After including whole cell data at +60 and +80 mV, and a maximal p(open) of 0.8 from noise analysis of cell-attached patches, an allosteric model fit the data best, as the other models had difficulty describing qualitative features of the data. However, some more complex schemes (with additional free parameters) cannot be excluded. We propose the allosteric model as an empirical description of macroscopic ionic currents, and as a model worth considering in future studies on the molecular mechanism of potassium channel gating.

Animals↗
PubMed1997

Bradykinin antagonists in human systems: correlation between receptor binding, calcium signalling in isolated cells, and functional activity in isolated ileum.

The determination of the relationship between ligand affinity and bioactivity is important for the understanding of receptor function in biological systems and for drug development. Several physiological and pathophysiological functions of bradykinin (BK) are mediated via the B2 receptor. In this study, we have examined the relationship between B2 receptor (soluble and membrane-bound) binding of BK peptidic antagonists, inhibition of calcium signalling at a cellular level, and in vitro inhibition of ileum contraction. Only human systems were employed in the experiments. Good correlations between the studied activities of BK antagonists were observed for a variety of different peptidic structures. The correlation coefficients (r) were in the range of 0.905 to 0.955. In addition, we analyzed the effect of the C-terminal Arg9 removal from BK and its analogs on B2 receptor binding. The ratios of binding constants (Ki(+Arg)/Ki(-Arg)) for the Arg9 containing compounds and the corresponding des-Arg9 analogs varied from about 10 to 250,000. These ratios strongly depend on the chemical structures of the compounds. The highest ratios were observed for two natural agonist pairs, BK/des-Arg9-BK and Lys0-BK/des-Arg9-Lys0-BK.

Bradykinin↗
PubMed1997

Delayed rectifier current of bullfrog sympathetic neurons: ion-ion competition, asymmetrical block and effects of ions on gating.

1. The delayed rectifier (DR) K+ channel pore was probed using different permeant and blocking ions applied intra- and extracellularly. Currents were recorded from bullfrog sympathetic neurons using whole-cell patch-clamp techniques. 2. With intra- and extracellular Cs+ (0 K+), there were large, tetraethylammonium (TEA)-sensitive currents. Adding K+ back to the extracellular solution revealed that the current with Cs+i was K+ selective (permeability ratio PCs/PK = 0.17 +/- 0.02, n = 4) and showed a strong anomalous mole fraction effect. 3. There were also large non-inactivating currents with Na+i and Na+o (0 K+). The current with Na+i was K+ selective (Na+o vs. K+o: PNa/PK = 0.022 +/- 0.005, n = 5), and was TEA sensitive with K+o but not with Na+o. 4. Permeant ions affected gating kinetics. DR currents activated faster in K+ than in Cs+, and activated faster with increasing concentrations of either K+ or Cs+. Deactivation was slowed by increased K+ or Cs+ concentration, with no difference between K+ and Cs+. 5. The pore was also characterized using intracellular blocking ions. A wide variety of monovalent cations (TEA, N-methyl-D-glucamine, arginine, choline, CH3NH3+, Li+, Cs+ and Na+) blocked DR channels from the inside in a voltage-dependent manner: KD at 0 mV was 2.9 mM for TEA and 134-487 mM for the others, at apparent electrical distances (delta) of 0.33-0.79. There was no detectable block by 10 mM Mgi2+. Apart from TEA, the organic cations did not block from the outside. 6. The permeability to Na+ in the absence of K+, and the strong anomalous mole fraction effects observed for Cs+o + K+o mixtures, suggest that DR channels select for K+ using ion-ion competition. The block by large intracellular cations shows that the pore is asymmetrical. The loss of high affinity TEAo block with Na+i and Na+o, and the effects of permeant ions on gating, suggest that channel conformation may be affected by ions in the pore.

Animals↗