Saxitoxin blocks batrachotoxin-modified sodium channels in the node of Ranvier in a voltage-dependent manner.

The inhibition by saxitoxin (STX) of single Na channels incorporated into planar lipid bilayers and modified by batrachotoxin (BTX) previously has been shown to be voltage dependent (Krueger, B.K.,J.F. Worley, and R. J. French, 1983, Nature [Lond.], 303:172-175; Moczydlowski, E., S. Hall, S. S. Garber, G. S. Strichartz, and C. Miller, 1984, J. Gen. Physiol., 84:687-704). We tested for such a voltage dependence of STX block of the Na current in voltage-clamped frog nodes of Ranvier. The block by STX of normal Na channels showed no modulation in response to maintained (20 s) changes of the membrane potential or to a train of brief pulses to potentials more positive than the holding potential. However, when the nodal channels were modified by BTX, the train of pulses produced a modulation of the block of the Na current by STX. The modulation of STX block depended on the voltage of the conditioning pulses and this voltage dependence agreed well with that predicted from the single channel studies over the membrane potential range used in those studies. In addition, we found that the voltage dependence of STX block was manifest only at potentials equal to or more positive than required to activate the channels. Most of the apparent differences among data from single channels in bilayers, equilibrium binding studies of STX, and the experiments described here are resolved by the hypotheses that (a) STX binding to open channels is voltage dependent, and (b) the affinities of STX for closed and inactivated channels are independent of voltage, equal, and less than the open channel affinity at potentials less than 0 mV. Whether these hypotheses apply to the STX block of all Na channels or just of BTX-modified channels remains to be determined.     

Single sodium channels from the squid giant axon

Since the work of A. L. Hodgkin and A. F. Huxley (1952. J. Physiol. [Lond.].117:500-544) the squid giant axon has been considered the classical preparation for the study of voltage-dependent sodium and potassium channels. In this preparation much data have been gathered on macroscopic and gating currents but no single sodium channel data have been available. This paper reports patch clamp recording of single sodium channel events from the cut-open squid axon. It is shown that the single channel conductance in the absence of external divalent ions is approximately 14 pS, similar to sodium channels recorded from other preparations, and that their kinetic properties are consistent with previous results on gating and macroscopic currents obtained from the perfused squid axon preparation.     

Sarcoballs: direct access to sarcoplasmic reticulum Ca2+-channels in skinned frog muscle fibers.

In skeletal muscle, twitch contraction is caused by the rapid release of Ca2+ from the sarcoplasmic reticulum (SR) (Endo, M. 1977. Physiol. Rev. 57:71-108) via Ca2+ conducting channels in the SR membrane (Smith, J. S., R. Coronado, and G. Meissner, 1985. Nature (Lond.). 316:446-449; Suarez-Isla, B. A., G. Orozco, P. F. Heller, and J. P. Froehlich. 1986. Proc. Natl. Acad. Sci. USA. 83:7741-7745). To facilitate study of these and other intracellular channels, we have developed a method which allows direct patch-clamp recording of currents through SR channels in native membrane. The Ca2+-release channel studied using this method exhibits two predominant conductance levels (80-100 pS and 120-160 pS), conducts Ca2+ preferentially over K+ (PCa/Pk = 6.5), is highly voltage sensitive, blocked on one side by ruthenium red (1 microM), and displays enhanced activity in the presence of caffeine (5 mM). Studied in skinned fibers, this channel appears fundamentally similar to homologous channels from isolated rabbit SR incorporated into bilayers, with some distinct differences.     

Trapping of an open-channel blocker at the frog neuromuscular acetylcholine channel.

At the ganglionic nicotinic acetylcholine channel (Gurney, A. M., and H. P. Rang, 1984, Br. J. Pharmacol., 82:623-642) and on some cholinergic neuromuscular synapses of Crustacea (Lingle, C., 1983a, J. Physiol. (Lond.), 339:395-417; Lingle, C., 1983b, J. Physiol. (Lond.), 339:419-437), some agents that block cholinergic currents by an open-channel block mechanism appear to become trapped within the channel when it subsequently closes. It is unknown whether trapping of some open-channel blockers might also occur at the neuromuscular nicotinic acetylcholine channel. Here we show that the long-lived cholinergic blocking action of chlorisondamine, a ganglionic nicotinic blocker, can in part be most simply explained by an open-channel block mechanism followed by a subsequent trapping of the blocking molecule within the closed ion channel. Unique structural characteristics of the chlorisondamine molecule place several provocative constraints on the mechanism by which trapping may be occurring.     

Voltage-dependent gap junction channels are formed by connexin32, the major gap junction protein of rat liver.

We report here experiments undertaken in pairs of hepatocytes that demonstrate a marked voltage sensivity of junctional conductance and, thus, contradict earlier findings reported by this laboratory (Spray, D.C., R.D.ginzberg, E.A., E. A. Morales, Z. Gatmaitan and I.M. Arias, 1986, J. Cell Biol. 101:135-144; Spray C.D. R.L. White, A.C. Campos de Carvalho, and M.V.L. Bennett. 1984. Biophys. J. 45:219-230) and by others (Dahl, G., T. Moller, D. Paul, R. Voellmy, and R. Werner. 1987. Science [Wash. DC] 236:1290-1293; Riverdin, E.C., and R. Weingart. 1988. Am. J. Physiol. 254:C226-C234). Expression in exogenous systems, lipid bilayers in which fragments of isolated gap junction membranes were incorporated (Young, J.D.-E., Z. Cohn, and N.B. Gilula. 1987. Cell. 48:733-743.) and noncommunicating cells transfected with connexin32 cDNA (Eghbali, B., J.A. Kessler, and D.C. Spray. 1990. Proc. Natl. Acad. Sci. USA. 87:1328-1331), support these findings and indicate that the voltage-dependent channel is composed of connexin32, the major gap junction protein of rat liver (Paul, D. 1986. J. Cell Biol. 103:123-134).     

Grayanotoxin-I-modified eel electroplax sodium channels. Correlation with batrachotoxin and veratridine modifications.

To probe the structure-function relationships of voltage-dependent sodium channels, we have been examining the mechanisms of channel modification by batrachotoxin (BTX), veratridine (VTD), and grayanotoxin-I (GTX), investigating the unifying mechanisms that underlie the diverse modifications of this class of neurotoxins. In this paper, highly purified sodium channel polypeptides from the electric organ of the electric eel were incorporated into planar lipid bilayers in the presence of GTX for comparison with our previous studies of BTX (Recio-Pinto, E., D. S. Duch, S. R. Levinson, and B. W. Urban. 1987. J. Gen. Physiol. 90:375-395) and VTD (Duch, D. S., E. Recio-Pinto, C. Frenkel, S. R. Levinson, and B. W. Urban. 1989. J. Gen. Physiol. 94:813-831) modifications. GTX-modified channels had a single channel conductance of 16 pS. An additional large GTX-modified open state (40-55 pS) was found which occurred in bursts correlated with channel openings and closings. Two voltage-dependent processes controlling the open time of these modified channels were characterized: (a) a concentration-dependent removal of inactivation analogous to VTD-modified channels, and (b) activation gating similar to BTX-modified channels, but occurring at more hyperpolarized potentials. The voltage dependence of removal of inactivation correlated with parallel voltage-dependent changes in the estimated K1/2 of VTD and GTX modifications. Ranking either the single channel conductances or the depolarization required for 50% activation, the same sequence is obtained: unmodified > BTX > GTX > VTD. The efficacy of the toxins as activators follows the same ranking (Catterall, W. A. 1977. J. Biol. Chem. 252:8669-8676).     

The role of the lipid matrix for structure and function of the GPCR rhodopsin

Photoactivation of rhodopsin in lipid bilayers results within milliseconds in a metarhodopsin I (MI)-metarhodopsin II (MII) equilibrium that is very sensitive to the lipid composition. It has been well established that lipid bilayers that are under negative curvature elastic stress from incorporation of lipids like phosphatidylethanolamines (PE) favor formation of MII, the rhodopsin photointermediate that is capable of activating G protein. Furthermore, formation of the MII state is favored by negatively charged lipids like phosphatidylserine and by lipids with longer hydrocarbon chains that yield bilayers with larger membrane hydrophobic thickness. Cholesterol and rhodopsin-rhodopsin interactions from crowding of rhodopsin molecules in lipid bilayers shift the MI-MII equilibrium towards MI. A variety of mechanisms seems to be responsible for the large, lipid-induced shifts between MI and MII: adjustment of the thickness of lipid bilayers to rhodopsin and adjustment of rhodopsin helicity to the thickness of bilayers, curvature elastic deformations in the lipid matrix surrounding the protein, direct interactions of PE headgroups and polyunsaturated hydrocarbon chains with rhodopsin, and direct or lipid-mediated interactions between rhodopsin molecules. This article is part of a Special Issue entitled: Membrane protein structure and function.     

Diffusion coefficient of cyclic GMP in salamander rod outer segments estimated with two fluorescent probes.

Experiments have demonstrated that single photoisomerizations in amphibian and primate rods can cause the suppression of 3-5% of the dark circulating current at the response peak (Baylor, D. A., T. D. Lamb, and K. W. Yau. 1979. J. Physiol. (Lond.). 288:613-634; Baylor, D. A., B. J. Nunn, and J. L. Schnapf. 1984. J. Physiol. (Lond.). 357:575-607). These results indicate that the change in [cGMP] effected by a single isomerization must spread longitudinally over at least the corresponding fractional length of the outer segment. The effective longitudinal diffusion coefficient, Dx, of cGMP is thus an important determinant of rod sensitivity. We report here measurements of the effective longitudinal diffusion coefficients, Dx, of two fluorescently labeled molecules: 5/6-carboxyfluorescein and 8-(fluoresceinyl)thioguanosine 3',5'-cyclic monophosphate, introduced into detached outer segments via whole-cell patch electrodes. For these compounds, the average time for equilibration of the entire outer segment with the patch pipette was approximately 6 min. Fluorescence images of rods were analyzed with a one-dimensional diffusion model that included limitations on transfer between the electrode and outer segment and the effects of intracellular binding of the dyes. The analyses yielded estimates of Dx of 1.9 and 1.0 microns 2.s-1 for the two dyes. It is shown that these results place an upper limit on Dx for cGMP of 11 microns2.s-1. The actual value of Dx for cGMP in the rod will depend on the degree of intracellular binding of cGMP. Estimates of the effective buffering power for cGMP in the rod at rest range from two to six (Lamb and Pugh, 1992; Cote and Brunnock, 1993). When combined with these estimates, our results predict that for cGMP itself, Dx falls within the range of 1.4-5.5 microns 2.s-1.     

Modulation of gramicidin channel conformation and organization by hydrophobic mismatch in saturated phosphatidylcholine bilayers

The matching of hydrophobic lengths of integral membrane proteins and the surrounding lipid bilayer is an important factor that influences both structure and function of integral membrane proteins. The ion channel gramicidin is known to be uniquely sensitive to membrane properties such as bilayer thickness and membrane mechanical properties. The functionally important carboxy terminal tryptophan residues of gramicidin display conformation-dependent fluorescence which can be used to monitor gramicidin conformations in membranes [S.S. Rawat, D.A. Kelkar, A. Chattopadhyay, Monitoring gramicidin conformations in membranes: a fluorescence approach, Biophys. J. 87 (2004) 831-843]. We have examined the effect of hydrophobic mismatch on the conformation and organization of gramicidin in saturated phosphatidylcholine bilayers of varying thickness utilizing the intrinsic conformation-dependent tryptophan fluorescence. Our results utilizing steady state and time-resolved fluorescence spectroscopic approaches, in combination with circular dichroism spectroscopy, show that gramicidin remains predominantly in the channel conformation and gramicidin tryptophans are at the membrane interfacial region over a range of mismatch conditions. Interestingly, gramicidin conformation shifts toward non-channel conformations in extremely thick gel phase membranes although it is not excluded from the membrane. In addition, experiments utilizing self quenching of tryptophan fluorescence indicate peptide aggregation in thicker gel phase membranes.     

Modulation of gramicidin channel conformation and organization by hydrophobic mismatch in saturated phosphatidylcholine bilayers

The matching of hydrophobic lengths of integral membrane proteins and the surrounding lipid bilayer is an important factor that influences both structure and function of integral membrane proteins. The ion channel gramicidin is known to be uniquely sensitive to membrane properties such as bilayer thickness and membrane mechanical properties. The functionally important carboxy terminal tryptophan residues of gramicidin display conformation-dependent fluorescence which can be used to monitor gramicidin conformations in membranes [S.S. Rawat, D.A. Kelkar, A. Chattopadhyay, Monitoring gramicidin conformations in membranes: a fluorescence approach, Biophys. J. 87 (2004) 831-843]. We have examined the effect of hydrophobic mismatch on the conformation and organization of gramicidin in saturated phosphatidylcholine bilayers of varying thickness utilizing the intrinsic conformation-dependent tryptophan fluorescence. Our results utilizing steady state and time-resolved fluorescence spectroscopic approaches, in combination with circular dichroism spectroscopy, show that gramicidin remains predominantly in the channel conformation and gramicidin tryptophans are at the membrane interfacial region over a range of mismatch conditions. Interestingly, gramicidin conformation shifts toward non-channel conformations in extremely thick gel phase membranes although it is not excluded from the membrane. In addition, experiments utilizing self quenching of tryptophan fluorescence indicate peptide aggregation in thicker gel phase membranes.     

Diverse K+ channels in primary human T lymphocytes

We used patch clamp techniques to identify and characterize a variety of K+ channels in primary human peripheral T lymphocytes. The most common channel observed in cell-attached configuration was voltage gated and inactivating. In ensemble averages, the kinetics of its activation and inactivation were similar to those of the whole-cell, voltage-gated K+ current described previously (Cahalan, M. D., K. G. Chandy, T. E. DeCoursey, and S. Gupta. 1985. J. Physiol. [Lond.]. 358:197-237; Deutsch, C., D. Krause, and S. C. Lee. 1986. J. Physiol. [Lond.]. 372:405-423), suggesting that this channel underlies the major portion of the outward current in lymphocytes. A small fraction of the time, this or another very similar channel was observed to inactivate significantly more slowly. Another channel type observed in cell-attached recording was seen less frequently and was transient in its appearance. This channel has a unitary conductance of approximately 10 pS, similar to the voltage-gated channel, but its voltage-independent gating, lack of inactivation, and different kinetic parameters showed it to be distinct. In whole-cell recording there is often a significant plateau current during sustained depolarization. Experiments using whole-cell and excised outside-out configurations indicate that at least part of this residual current is carried by K+ and, as opposed to the predominant voltage-gated current, is charybdotoxin insensitive. These findings are consistent with evidence that implicates charybdotoxin-sensitive and -insensitive components in T lymphocyte proliferation and volume regulation.     

Modification of the erythrocyte membrane dielectric constant by alcohols

Summary Aliphatic alcohols are found to stimulate the transmembrane fluxes of a hydrophobic cation (tetraphenylarsonium, TPA) and anion (AN-12) 5–20 times in red blood cells. The results are analyzed using the Born-Parsegian equation (Parsegian, A., 1969,Nature (London) 221:844–846), together with the Clausius-Mossotti equation to calculate membrane dielectric energy barriers. Using established literature values of membrane thickness, native membrane dielectric constant, TPA ionic radius, and alcohol properties (partition coefficient, molar volume, dielectric constant), the TPA permeability data is predicted remarkably well by theory. If the radius of AN-12 is taken as 1.9 Å, its permeability in the presence of butanol is also described by our analysis. Further, the theory quantitatively accounts for the data of Gutknecht and Tosteson (Gutknecht, J., Tosteson, D.C., 1970,J. Gen. Physiol. 55:359–374) covering alcohol-induced conductivity changes of 3 orders of magnitude in artificial bilayers. Other explanations including perturbations of membrane fluidity, surface charge, membrane thickness, and dipole potential are discussed. However, the large magnitude of the stimulation, the more pronounced effect on smaller ions, and the acceleration of both anions and cations suggest membrane dielectric constant change as the primary basis of alcohol effects.     

Formation of cation channels in planar lipid bilayers by brefeldin A.

Brefeldin A (BFA) is a novel agent with the unique property of effecting a rapid increase of Golgi cisternae volume and subsequent loss of a recognizable Golgi apparatus in treated cells. Although a receptor-mediated mechanism has been proposed, the molecular basis of BFA action remains unknown (Lippincott-Schwartz, J., Glickman, J., Donaldson, J. G., Robbins, J., Kreis, T. E., Seamon, K. B., Sheetz, M. P., and Klausner, R. D. (1991) J. Cell Biol. 112, 567-577). Since a variety of ionophores distort Golgi architecture by initially causing osmotic swelling of the cisternae (Mollenhauer, H. H., Morre, D. J., and Rowe, L. D. (1990) Biochim. Biophys. Acta 1031, 225-246), Golgi membrane permeabilization by BFA seemed possible. We examined the effects of BFA on the conductance of planar lipid bilayers bathed in several aqueous salt solutions. Addition of BFA (1 microgram/ml) quickly augmented alkali cation conductance (K+ greater than Na+ much greater than Li+) but not anion conductance of the bilayer. Lower concentrations (1 ng/ml) indicated that BFA formed discrete, cation-selective channels in these bilayers. Given that Golgi cisternae volume increases immediately upon treatment with BFA, these findings suggest that alteration of ion gradients or Golgi membrane potential followed by an influx of water may be the mechanism by which BFA initiates disruption of Golgi structural integrity. Subsequent functional perturbations may then ensue either as a consequence of these initial structural changes or by a combination of several distinct mechanisms.     

Stimulation of cardiac alpha receptors increases Na/K pump current and decreases gK via a pertussis toxin-sensitive pathway.

Alpha-adrenergic amines exert concentration-dependent actions on the automaticity of cardiac Purkinje fibers (Posner, P., E. L. Farrar, and C. R. Lambert. 1976. Am. J. Physiol. 231:1415-1420; Rosen, M. R., A. J. Hordof, J. P. Ilvento, and P. Danilo, Jr. 1977. Circ. Res. 40:390-400; Rosen, M. R., R. M. Weiss, and P. Danilo, Jr. 1984. J. Pharmacol. Exp. Ther. 231:1415-1420). At high concentrations they induce a largely beta adrenergic increase in the spontaneous firing rate of adult canine Purkinje fibers, whereas at concentrations less than 10(-6) M, their effect is mediated through alpha-adrenergic receptors and is seen predominantly as a decrease in the fibers' spontaneous firing rate. The mechanism for this decrease in spontaneous firing rate remains unexplained. We report here that phenylephrine (10(-7) M) increases the activity of the Na/K pump and decreases background gK in Purkinje myocytes. Both effects appear to be alpha-1 adrenergic and, in addition, are abolished on pretreatment with pertussis toxin. These results suggest that like the atrial muscarinic receptor (Pffafinger, P. J., J. M. Martin, D. D. Hunter, N. M. Nathanson, and B. Hille. 1985. Nature [Lond.]. 317:536-538; Breitwieser, G. E., and G. Szabo. 1985. Nature [Lond.]. 317:538-540) the Purkinje fiber alpha-1 receptor is coupled to background gK via a GTP-regulatory protein. Further, they suggest that the phenylephrine-induced decrease in spontaneous firing rate is due to stimulation of the Na/K pump via a novel coupling of the Na/K pump to a pertussis toxin-sensitive GTP regulatory protein.     

Reconstitution of channels from preparations enriched in lens gap junction protein MP70

Summary Detergent-solubilized ovine lens membrane proteins, enriched in the 70-kDa gap junction component (MP70), were reconstituted into planar lipid bilayers and analyzed for channel activities. Three distinct activities were found. Those showing conductance steps of 290 pS (symmetrical 150-mM KCl solutions) had properties similar to those reported earlier for MIP26 (Ehring, G.R., Zampighi, G., Horwitz, J., Bok, D., Hall, J.E. 1990. J. Gen. Physiol. 96:631–664.) of which minor amounts were normally present in the detergent-solubilized preparations. Two novel channel activities had unitary conductances of 90 and 45 pS, were halothane sensitive and did not discriminate between sodium and potassium ions. The 90-pS channel was asymmetrically voltage dependent, and its properties would be consistent with the expected properties of junctional hemichannels.This work was supported by grants from the Health Research Council of New Zealand, from the New Zealand Lottery Grants Board and from the New Zealand Vice Chancellor's Committee Fund.     

Increased voltage-gated potassium conductance during interleukin 2- stimulated proliferation of a mouse helper T lymphocyte clone

Recent work has demonstrated the presence of voltage-gated potassium channels in human peripheral blood T lymphocytes (Matteson, R., and C. Deutsch, 1984, Nature (Lond.), 307:468-471; DeCoursey T. E., T. G. Chandy, S. Gupta, and M. D. Cahalan, 1984, Nature (Lond.), 307:465-468) and a murine cytolytic T-cell clone (Fukushima, Y., S. Hagiwara, and M. Henkart, 1984, J. Physiol., 351:645-656). Using the whole cell patch clamp, we have found a potassium conductance with similar properties in a murine noncytolytic T lymphocyte clone, L2. Under voltage clamp, a step from a holding potential of -70 mV to +50 mV produces an average outward current of 100-150 pA in "quiescent" L2 cells at the end of their weekly maintenance cycle. When these cells are stimulated with human recombinant interleukin 2 (rIL2, 100 U/ml), they grow in size and initiate DNA synthesis at approximately 24 h. Potassium conductance is increased as early as 8 h after stimulation with rIL2 and rises to a level 3-4 times that of excipient controls by 24 h. The level remains elevated through 72 h, but as the cells begin to leave the cell cycle at 72-96 h, the conductance decreases quickly to a value only slightly higher than the initial one. Quinine, a blocker of this conductance, markedly reduces the rate at which L2 cells traverse the cell cycle, while also reducing the rate of stimulated protein synthesis. The regulation of potassium conductance in L2 cells during rIL2-stimulated proliferation suggests that potassium channel function may play a role in support of the proliferative response.     

Combined influence of cholesterol and synthetic amphiphillic peptides upon bilayer thickness in model membranes.

Deuterium (2H) NMR was used to study bilayer hydrophobic thickness and mechanical properties when cholesterol and/or synthetic amphiphillic polypeptides were added to deuterated POPC lipid bilayer membranes in the liquid-crystalline (fluid) phase. Smoothed acyl chain orientational order profiles were used to calculate bilayer hydrophobic thickness. Addition of 30 mol% cholesterol to POPC at 25 degrees C increased the bilayer thickness from 2.58 to 2.99 nm. The peptides were chosen to span the bilayers with more or less mismatch between the hydrophobic peptide length and membrane hydrophobic thickness. The average thickness of the pure lipid bilayers was significantly perturbed upon addition of peptide only in cases of large mismatch, being increased (decreased) when the peptide hydrophobic length was greater (less) than that of the pure bilayer, consistent with the "mattress" model of protein lipid interactions (Mouritsen, O.G., and M. Bloom. 1984. Biophys. J. 46:141-153). The experimental results were also used to examine the combined influence of the polypeptides and cholesterol on the orientational order profile and thickness expansivity of the membranes. A detailed model for the spatial distribution of POPC and cholesterol molecules in the bilayers was proposed to reconcile the general features of these measurements with micromechanical measurements of area expansivity in closely related systems. Experiments to test the model were proposed.     

Area per molecule and distribution of water in fully hydrated dilauroylphosphatidylethanolamine bilayers

The area per lipid molecule for fully hydrated dilauroylphosphatidylethanolamine (DLPE) has been obtained in both the gel and liquid-crystalline states by combining wide-angle X-ray diffraction, electron density profiles, and previously published dilatometry results [Wilkinson, D. A., & Nagle, J. F. (1981) Biochemistry 20, 187-192]. The molecular area increases from 41.0 +/- 0.2 to 49.1 +/- 1.2 A2 upon melting from the gel to liquid-crystalline phase. The thickness of the bilayer, as measured from the electron density profiles, decreases about 4 A upon melting, from 45.2 +/- 0.3 to 41.0 +/- 0.6 A. A somewhat unexpected result is that the fluid layer between fully hydrated bilayers is the same in both gel and liquid-crystalline phases and is only about 5 A thick. From these data, plus the volume of the anhydrous DLPE molecule, it is possible to determine the number of water molecules per lipid and their approximate distribution relative to the lipid molecule. Our analysis shows that there are about 7 and 9 waters per DLPE molecule in the gel and liquid-crystalline phases, respectively. About half of the water is located in the fluid space between adjacent bilayers, and the remaining waters are intercalated into the bilayer, presumably in the head group region. There are significantly fewer water molecules in the fluid spaces between DLPE bilayers than in the fluid spaces in gel- or liquid-crystalline-phase phosphatidylcholine bilayers. This small fluid space in PE bilayers could arise from interbilayer hydrogen bond formation through the water molecules or electrostatic interactions between the amine and phosphate groups on apposing bilayers.     

Phosphate burst in permeable muscle fibers of the rabbit.

The transient kinetics of ATP hydrolysis in chemically skinned psoas muscle fibers of the rabbit have been measured. Muscles fibers in the rigor state (absence of nucleotide) were relaxed rapidly by the photochemical release of [2-3H]ATP from caged-ATP (P3-1-(2-nitro)phenylethyl[2-3H]adenosine 5'-triphosphate) in the absence of calcium ions. Rapid freezing of the fiber to stop hydrolysis, followed by analysis of the tritiated nucleotide content allowed the course of the hydrolysis to be determined. The timecourse of ATP hydrolysis was biphasic, with an initial rapid phase occurring at a rate of approximately 60 s-1 at 12 degrees C for fibers exposed to greater than 0.7 mM ATP. The amplitude of the rapid phase was as previously reported (Ferenczi, M. A., E. Homsher, and D. R. Trentham, 1984, J. Physiol. (Lond.)., 352:575-599).     

Acute alcohol tolerance is intrinsic to the BKCa protein, but is modulated by the lipid environment

Ethanol tolerance, in which exposure leads to reduced sensitivity, is an important component of alcohol abuse and addiction. The molecular mechanisms underlying this process remain poorly understood. The BKCa channel plays a central role in the behavioral response to ethanol in Caenorhabditis elegans (Davies, A. G., Pierce-Shimomura, J. T., Kim, H., VanHoven, M. K., Thiele, T. R., Bonci, A., Bargmann, C. I., and McIntire, S. L. (2003) Cell 115, 655-666) and Drosophila (Cowmeadow, R. B., Krishnan, H. R., and Atkinson, N. S. (2005) Alcohol. Clin. Exp. Res. 29, 1777-1786) . In neurons, ethanol tolerance in BKCa channels has two components: a reduced number of membrane channels and decreased potentiation of the remaining channels (Pietrzykowski, A. Z., Martin, G. E., Puig, S. I., Knott, T. K., Lemos, J. R., and Treistman, S. N. (2004) J. Neurosci. 24, 8322-8332) . Here, heterologous expression coupled with planar bilayer techniques examines two additional aspects of tolerance in human BKCa channels. 1) Is acute tolerance observed in a single channel protein complex within a lipid environment reduced to only two lipids? 2) Does lipid bilayer composition affect the appearance of acute tolerance? We found that tolerance was observable in BKCa channels in membrane patches pulled from HEK cells and when they are placed into reconstituted 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylethanolamine/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylserine membranes. Furthermore, altering bilayer thickness by incorporating the channel into lipid mixtures of 1,2-dioleoyl-3-phosphatidylethanolamine with phosphatidylcholines of increasing chain length, or with sphingomyelin, strongly affected the sensitivity of the channel, as well as the time course of the acute response. Ethanol sensitivity changed from a strong potentiation in thin bilayers to inhibition in thick sphingomyelin/1,2-dioleoyl-3-phosphatidylethanolamine bilayers. Thus, tolerance can be an intrinsic property of the channel protein-lipid complex, and bilayer thickness plays an important role in shaping the pattern of response to ethanol. As a consequence of these findings the protein-lipid complex should be treated as a unit when studying ethanol action.