Biomedical subjects

D R Laver

Publications and source records attributed to D R Laver.

29 records · Page 2Linked to original sources

PubMed1996

A calcium-activated chloride channel in sarcoplasmic reticulum vesicles from rabbit skeletal muscle.

A Ca(2+)-activated Cl- channel is described in sarcoplasmic reticulum (SR) enriched vesicles of skeletal muscle incorporated into lipid bilayers. Small chloride (SCl) channels (n = 20) were rapidly and reversibly activated when cis- (cytoplasmic) [Ca2+] was increased above 10(-7) M, with trans-(luminal) [Ca2+] at either 10(-3) or 10(-7) M. The open probability of single channels increased from zero when cis-[Ca2+] was 10(-7) M to 0.61 +/- 0.12 when [Ca2+] was 10(-4) M. High- and low-conductance levels in single-channel activity were activated at different cis-[Ca2+]. Channel openings to the maximum conductance, 65-75 pS (250/50 mM Cl-, cis/ trans), were active when cis-[Ca2+] was increased above 5 x 10(-6) M. In contrast to the maximum conductance, channel openings to submaximal levels between 5 and 40 pS were activated at a lower cis-[Ca2+] and dominated channel activity between 5 x 10(-7) and 5 x 10(-6) M. Activation of SCl channels was Ca2+ specific and not reproduced by cytoplasmic Mg2+ concentrations of 10(-3) M. We suggest that the SCl channel arises in the SR membrane. The Ca2+ dependence of this channel implies that it is active at [Ca2+] achieved during muscle contraction.

Animals↗

PubMed1995

Cytoplasmic Ca2+ inhibits the ryanodine receptor from cardiac muscle.

Ca(2+)-dependent inhibition of native and isolated ryanodine receptor (RyR) calcium release channels from sheep heart and rabbit skeletal muscle was investigated using the lipid bilayer technique. We found that cytoplasmic Ca2+ inhibited cardiac RyRs with an average Km = 15 mM, skeletal RyRs with Km = 0.7 mM and with Hill coefficients of 2 in both isoforms. This is consistent with measurements of Ca2+ release from the sarcoplasmic reticulum (SR) in skinned fibers and with [3H]-ryanodine binding to SR vesicles, but is contrary to previous bilayer studies which were unable to demonstrate Ca(2+)-inhibition in cardiac RyRs (Chu, Fill, Stefani & Entman (1993) J. Membrane Biol. 135, 49-59). Ryanodine prevented Ca2+ from inhibiting either cardiac or skeletal RyRs. Ca(2+)-inhibition in cardiac RyRs appeared to be the most fragile characteristic of channel function, being irreversibly disrupted by 500 mM Cs+, but not by 500 mM K+, in the cis bath or by solublization with the detergent CHAPS. These treatments had no effect on channel regulation by AMP-PNP, caffeine, ryanodine, ruthenium red, or Ca(2+)-activation. Ca(2+)-inhibition in skeletal RyRs was retained in the presence of 500 mM Cs+. Our results provide an explanation for previous findings in which cardiac RyRs in bilayers with 250 mM Cs+ in the solutions fail to demonstrate Ca(2+)-inhibition, while Ca(2+)-inhibition of Ca2+ release is observed in vesicle studies where K+ is the major cation. A comparison of open and closed probability distributions from individual RyRs suggested that the same gating mechanism mediates Ca(2+)-inhibition in skeletal RyRs and cardiac RyRs, with different Ca2+ affinities for inhibition. We conclude that differences in the Ca(2+)-inhibition in cardiac and skeletal channels depends on their Ca2+ binding properties.

Animals↗

PubMed1994

Surface potentials near the mouth of the large-conductance K+ channel from Chara australis: a new method of testing for diffusion-limited ion flow.

The kinetics of single K+ channels were derived for patch-clamp recordings of membrane patches excised from cytoplasmic drops from the plant, Chara australis R. Br. Specifically, the "tilt effect" model of MacKinnon, Latorre and Miller (1989. Biochemistry 28:8092-8099) has been used to measure the electrostatic potential (surface PD) and fixed charge at the entrances of the channel. The surface PD is derived from the difference between the trans-pore potential difference (PD) and that between the two bulk phases. The trans-pore PD is probed using three voltage-dependent properties of the channel. These are (1) the association and dissociation rates of Ca2+ binding to the channel, from both the cytoplasmic and vacuolar solutions. These were determined from the mean blocked and unblocked durations of the channel in the presence of either 20 mmol liter-1 vacuolar or 1 mmol liter-1 cytoplasmic Ca2+; (2) the closing rate of the channel's intrinsic gating process. This was determined from the mean channel open time in the absence of vacuolar Ca2+ at membrane PDs more negative than -100 mV; and (3) the effect of Mg2+ on channel conductance when added to solutions initially containing 3 mmol liter-1 KCl. The voltage dependence of properties 1 and 2 shifts along the voltage axis according to the ionic strength of the bathing media, consistent with the presence of negative charge in the channel vestibules. Furthermore, the magnitude of this shift depends on the current in a manner consistent with diffusion-limited ion flow in the channel (i.e., the rate of ion diffusion in the external electrolyte limits the channel conductance). Mg2+ on either side of the membrane alters channel conductance in a voltage-dependent way. A novel feature of the Mg2+ effect is that it reverses, from a block to an enhancement, when the membrane PD is more negative than -70 mV. This reversal only appears in solutions of low ionic strength. The attenuating effect is due to voltage-dependent binding of Mg2+ within the pore, which presumably plugs the channel. The enhancing effect is due to screening by Mg2+ of surface potentials arising from diffusion-limited flow of K+. All experimental approaches give a consistent picture of K+ permeation in which the surface charge and convergence permeability of the cytoplasmic vestibule are the major factors in determining channel conductance. The cytoplasmic vestibule has a charge density of -0.035 C/m2 which is similar to that found for maxi K channels in rat muscle.(ABSTRACT TRUNCATED AT 400 WORDS)

Biological Transport↗

PubMed1994

The barrel-stave model as applied to alamethicin and its analogs reevaluated.

Alamethicin and its analogs from cation selective, multi-conductance channels in lipid bilayers. The conductance levels have been thought to be due to a barrel-stave structure where conducting pores (barrels) are formed by the self-assembly of a variable number of alpha-helical rods (staves). The conductance transitions were then interpreted as the addition or deletion of peptide monomers from the pore-forming complex (Sansom, M.S. 1991. Prog. Biophys. Mol. Biol. 55:139-235). Initially, pore conductances were calculated from that expected of right circular cylinders of "bulk" electrolyte. More recent theories also included the access resistance of the electrolyte outside the pore. However, they all consistently overestimated the observed conductances. The reason for the discrepancy is presented here. Previous theories ignored the effects of ion concentration gradients near the pore. Hence, they only held in the limit of small bilayer potentials (< 25 mV) and so would overestimate measurements that typically used much larger potentials (> 100 mV). This theoretical flaw is corrected by using Läuger's theory of diffusion-limited ion flow (Läuger, P. 1976. Biochim. Biophys. Acta. 455:493-509). Thus, including the effects of ion concentration gradients results in a considerable improvement in predicting pore conductances. It is found that: 1) the effects of ion concentration gradients must be included in the barrel-stave model for it to apply to the available data; 2) previously published explanations for the discrepancy between the model and the data, namely the "distorted bundle" and the "head-to-tail aggregate" hypotheses are not necessary (reviewed by Sansom, 1991).

Alamethicin↗

PubMed1992

Divalent cation block and competition between divalent and monovalent cations in the large-conductance K+ channel from Chara australis.

The patch-clamp technique is used to investigate divalent ion block of the large-conductance K+ channel from Chara australis. Block by Ba2+, Ca2+, Mg2+, and Pt(NH3)4(2+) from the vacuolar and cytoplasmic sides is used to probe the structure of, and ion interactions within, the pore. Five divalent ion binding sites are detected. Vacuolar Ca2+ reduces channel conductance by binding to a site located 7% along the membrane potential difference (site 1, delta = 0.07; from the vacuolar side); it also causes channel closures with mean a duration of approximately 0.1-1 ms by binding at a deeper site (site 2, delta = 0.3). Ca2+ can exit from site 2 into both the vacuolar and cytoplasmic solutions. Cytoplasmic Ca2+ reduces conductance by binding at two sites (site 3, delta = -0.21; site 4, delta = -0.6; from the cytoplasmic side) and causes closures with a mean duration of 10-100 ms by binding to site 5 (delta = -0.7). The deep sites exhibit stronger ion specificity than the superficial sites. Cytoplasmic Ca2+ binds sequentially to sites 3-5 and Ca2+ at site 5 can be locked into the pore by a second Ca2+ at site 3 or 4. Ca2+ block is alleviated by increasing [K+] on the same side of the channel. Further, Ca2+ occupancy of the deep sites (2, 4, and 5) is reduced by K+, Rb+, NH4+, and Na+ on the opposite side of the pore. Their relative efficacy correlates with their relative permeability in the channel. While some Ca2+ and K+ sites compete for ions, Ca2+ and K+ can simultaneously occupy the channel. Ca2+ binding at site 1 only partially blocks channel conduction. The results suggest the presence of four K+ binding sites on the channel protein. One cytoplasmic facing site has an equilibrium affinity of 10 mM (site 6, delta = -0.3) and one vacuolar site (site 7, delta less than 0.2) has low affinity (greater than 500 mM). Divalent ion block of the Chara channel shows many similarities to that of the maxi-K channel from rat skeletal muscle.

Barium↗

PubMed1991

Activation by Ca2+ and block by divalent ions of the K+ channel in the membrane of cytoplasmic drops from Chara australis.

Patch-clamp studies of cytoplasmic drops from the charophyte Chara australis have previously revealed K+ channels combining high conductance (170 pS) with high selectivity for K+, which are voltage activated. The cation-selectivity sequence of the channel is shown here to be: K+ greater than Rb+ greater than NH4+ much greater than Na+ and Cl-. Divalent cytosolic ions reduce the K+ conductance of this channel and alter its K+ gating in a voltage-dependent manner. The order of blocking potency is Ba2+ greater than Sr2+ greater than Ca2+ greater than Mg2+. The channel is activated by micromolar cytosolic Ca2+, an activation that is found to be only weakly voltage dependent. However, the concentration dependence of calcium activation is quite pronounced, having a Hill coefficient of three, equivalent to three bound Ca2+ needed to open the channel. The possible role of the Ca(2+)-activated K+ channel in the tonoplast of Chara is discussed.

Calcium↗

PubMed1990

Coupling of K(+)-gating and permeation with Ca2+ block in the Ca2(+)-activated K+ channel in Chara australis.

The patch-clamp technique is used here to investigate the kinetics of Ca2+ block in single high-conductance Ca2(+)-activated K+ channels. These channels are detected in the membrane surrounding cytoplasmic drops from Chara australis, a membrane which originates from the tonoplast of the parent cell. The amplitudes and durations of single channel events are measured over a wide range of membrane potential (-300 to 200 mV). Ca2+ on either side of the channel reduces its K+ conductance and alters its ion-gating characteristics in a voltage-dependent manner. This Ca2(+)-induced attenuation of conductance is analyzed using the theory of diffusion-limited ion flow through pores. Interaction of external Ca2+ with the channel's ion-gating mechanism is examined in terms of a kinetic model for ion-gating that includes two voltage-dependent gating mechanisms. The kinetics of channel block by external Ca2+ indicates that (i) external Ca2+ binds at two sites, a superficial site and a deep site, located at 8 and 40% along the trans-pore potential difference, (ii) the external vestibule cannot be occupied by more than one Ca2+ or K+, and (iii) the kinetics of Ca2+ binding at the deep site is coupled with that of a voltage-dependent gate on the external side of the channel. Kinetics of channel block by internal Ca2+ indicates that more than one Ca2+ is involved.

Calcium↗

PubMed1986

The effect of benzyl alcohol and cholesterol on the acyl chain order and alkane solubility of bimolecular phosphatidylcholine membranes.

An investigation was made of the effects of cholesterol and benzyl alcohol on the partitioning of n-alkanes between lipid bilayer membranes and bulk lipid/alkane solutions (in the torus). Bilayers were formed from solutions containing alkanes of different chain lengths, together with phosphatidylcholine and cholesterol in varying proportions. The partitioning of the alkanes was determined from measurements of the very low frequency (1 Hz) capacitance of the membranes. Perturbation of the internal membrane structure by the inclusion of cholesterol and benzyl alcohol produced very significant changes in the n-alkane partition coefficient, cholesterol causing a decrease and benzyl alcohol an increase in the alkane partitioning into the bilayer. A correlation exists between the effects of these compounds on the alkane partitioning and their effect on the segmental chain order of the acyl chains in the bilayer and this correlation is consistent with a statistical-mechanical model of the lipid/alkane bilayers in the liquid crystalline state. The perturbation by cholesterol and benzyl alcohol of the internal structure of membranes bears on the conflicting reports of the effects of these substances on artificial lipid bilayers and could also be relevant to their known physiological effects.

Alkanes↗

PubMed1986

The effect of temperature on lipid-n-alkane interactions in lipid bilayers.

The partitioning of n-alkanes between an egg-phosphatidylcholine bilayer and its torus was studied for alkanes with ten to sixteen carbon atoms using measurements of membrane capacitance. The partition coefficient was found to decrease with increasing alkane chainlength and to increase with increasing temperature. This is consistent with a well-known statistical model of lipid alkane bilayers in the liquid crystalline state. It was found that n-decane was unsuitable as a solvent in these experiments because significant partitioning of n-decane into the aqueous phase and atmosphere occurred and this could not be adequately controlled. Egg-phosphatidylcholine bilayers containing negligible amounts of solvent could be produced using a method similar to the 'freeze-out' method of White (Biochim. Biophys. Acta 356 (1974) 8-16). Bilayers formed using n-hexadecane were found to be virtually solvent-free at temperatures below 30 degrees C. The partition coefficient of n-alkanes in the bilayer was also found to depend on the alkane mole fraction. Thus it was concluded that the assumption of ideal mixing between acyl and alkane chains in the bilayer was not valid when the alkane mole fraction exceeded 40% (with respect to the acyl chains of the lipid). The variation of the standard chemical potential with temperature was measured for alkanes of different chainlengths and it was concluded that the enthalpy and entropy of the alkanes in the bilayer are in themselves temperature-dependent. This indicates that the state of the hydrophobic interior of lipid bilayers varies with temperature.

Alkanes↗

PubMed1982

Effect of 2H2O/H2O replacement on the dielectric structure of lipid bilayer membranes.

Dielectric dispersion measurements were made on planar egg phosphatidylcholine and egg phosphatidylcholine/cholesterol bilayer membranes in H2O and 2H2O electrolyte solutions. The properties of the dielectrically distinct substructural layers of these membranes, which can be characterised by this technique, were found to be insensitive to replacement of H2O by 2H2O.

Chemical Phenomena↗

PubMedDate not supplied

Interactions between dihydropyridine receptors and ryanodine receptors in striated muscle.

Excitation-contraction coupling in both skeletal and cardiac muscle depends on structural and functional interactions between the voltage-sensing dihydropyridine receptor L-type Ca(2+) channels in the surface/transverse tubular membrane and ryanodine receptor Ca(2+) release channels in the sarcoplasmic reticulum membrane. The channels are targeted to either side of a narrow junctional gap that separates the external and internal membrane systems and are arranged so that bi-directional structural and functional coupling can occur between the proteins. There is strong evidence for a physical interaction between the two types of channel protein in skeletal muscle. This evidence is derived from studies of excitation-contraction coupling in intact myocytes and from experiments in isolated systems where fragments of the dihydropyridine receptor can bind to the ryanodine receptors in sarcoplasmic reticulum vesicles or in lipid bilayers and alter channel activity. Although micro-regions that participate in the functional interactions have been identified in each protein, the role of these regions and the molecular nature of the protein-protein interaction remain unknown. The trigger for Ca(2+) release through ryanodine receptors in cardiac muscle is a Ca(2+) influx through the L-type Ca(2+) channel. The Ca(2+) entering through the surface membrane Ca(2+) channels flows directly onto underlying ryanodine receptors and activates the channels. This was thought to be a relatively simple system compared with that in skeletal muscle. However, complexities are emerging and evidence has now been obtained for a bi-directional physical coupling between the proteins in cardiac as well as skeletal muscle. The molecular nature of this coupling remains to be elucidated.

Amino Acid Sequence↗