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WO2001059446A2 - Analyse amelioree - Google Patents

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Publication number
WO2001059446A2
WO2001059446A2 PCT/GB2001/000516 GB0100516W WO0159446A2 WO 2001059446 A2 WO2001059446 A2 WO 2001059446A2 GB 0100516 W GB0100516 W GB 0100516W WO 0159446 A2 WO0159446 A2 WO 0159446A2
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ion transport
membrane
transport activity
electrogenic ion
activity
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WO2001059446A3 (fr
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Neil Castle
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Biofocus Discovery Ltd
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Biofocus Discovery Ltd
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Publication of WO2001059446A3 publication Critical patent/WO2001059446A3/fr
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5076—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving cell organelles, e.g. Golgi complex, endoplasmic reticulum

Definitions

  • This invention relates to improved methods for detecting changes in the activity of an electrogenic ion transport activity in a membrane.
  • An electrogenic ion transport activity is an activity which causes or allows movement of an ion across a membrane and is thereby capable of causing a change in the transmembrane potential of the membrane.
  • the invention also relates to improved methods for identifying inhibitors, activators, or enhancers of electrogenic ion transport activity.
  • the plasma membrane of a cell typically has a transmembrane potential which is negative with respect to the outside, due to K + , Na + and CI" concentration gradients that are maintained by active transport processes. Increases and decreases in membrane potential play a central role in many physiological processes, including nerve-impulse propagation, muscle contraction, cell signalling and ion-channel gating.
  • Ion channels are proteins that span cellular and intracellular membranes and function as conduits for the movement of electrolytes into and out of cells. Ion channels comprise a diverse family of proteins that are most often subdivided on the basis of which physiological ion is preferentially translocated. Ion channels have been characterised which preferentially translocate cations such as potassium, sodium and calcium or anions such as chloride.
  • ion channels are important contributors to a multitude of physiological processes. These include the electrical impulses that underlie sensory and motor function in the brain and control contractile activity of the heart, skeletal, vascular and visceral smooth muscle. Even in non- excitable cells and tissues, ion channels are known to play critical roles in nutrient uptake, hormone secretion as well as cell replication and development. Consequently, there is much interest in detecting changes in ion channel activity and in identifying modulators of ion channel activity.
  • Ion flow through an ion channel in a cell membrane may contribute to setting the resting membrane potential (RMP) of the cell.
  • RMP resting membrane potential
  • the RMP of the cell can be altered. If ion flow through the ion channel is inhibited, the RMP of the cell will be altered to a value similar to that of the RMP of the cell in the absence of the channel. If ion flow through the ion channel is enhanced, the RMP of the cell will be altered to a value closer to the equilibrium potential for the ion.
  • CHO cells which are commonly used as expression hosts for recombinant proteins can have RMPs of 0 to -30 mV (Felipe et al . Am J " Physiol (1993) 265:C1230).
  • CHO cells heterologously expressing recombinant potassium channels can have RMPs of as low as -80 mV.
  • pharmacological inhibition of an expressed recombinant potassium channel is expected to cause a change in the RMP of the cell from -80mV to 0 to -30 mV (the RMP of a CHO cell not expressing the recombinant potassium channel) .
  • Perozo and Hubbell Biochemistry 1993, 32, 10471- 104778 describe activation of purified eel sodium channels using the light induced proton pump, bacteriorhodopsin (bR) , as a current source.
  • Reconstituted vesicles containing purified sodium channels (a first electrogenic ion transport activity) and bacteriorhodopsin, bR, (a second electrogenic ion transport activity) were prepared and held at a negative-inside transmembrane potential (of approximately - 65mV) generated using n-methylglucamine (NMG) as an impermeable cation and N0 3 " as an anion.
  • the negative holding potential serves to bring the purified sodium channels from an inactivated to a closed state.
  • the vesicles were then mixed with 22 Na + in the dark and subsequently illuminated to induce proton pumping by bR.
  • the resulting depolarisation opened the sodium channels thereby driving the transmembrane potential to approximately OmV within a few seconds.
  • a method for enhancing the change in transmembrane potential of a membrane when the activity of an electrogenic ion transport activity in the membrane is reduced which comprises : a) providing a membrane with a first and a second electrogenic ion transport activity, the membrane having a transmembrane potential predominantly set by the first or the second electrogenic ion transport activity, the first electrogenic ion transport activity having an opposite effect on the transmembrane potential of the membrane to the second electrogenic ion transport activity compared to the transmembrane potential of the membrane in the absence of the first and second electrogenic ion transport activities, wherein the first ion electrogenic ion transport activity predominantly sets the transmembrane potential of the membrane unless the activity of the first electrogenic ion transport activity is reduced in which case the second electrogenic ion transport activity predominantly sets the transmembrane potential; and b) reducing the activity of the first electrogenic ion transport activity.
  • This first aspect of the invention can be illustrated by a hypothetical example in which the transmembrane potential of the membrane is OmV in the absence of the first and second electrogenic ion transport activities.
  • the first and second electrogenic ion transport activities each contributes to setting the transmembrane potential of the membrane, but the contribution of the first electrogenic ion transport activity is dominant to the contribution of the second electrogenic ion transport activity.
  • the transmembrane potential is -5OmV. If the first electrogenic ion transport activity is inhibited, its contribution to the transmembrane potential is no longer dominant to that of the second electrogenic ion transport activity and the transmembrane potential of the membrane is then predominantly set by the second electrogenic ion transport activity.
  • the transmembrane potential of the membrane is +5OmV.
  • the resulting change in the transmembrane potential of the membrane is 10OmV. This is compared to a maximum change in transmembrane potential of only 50mV if the first electrogenic ion transport activity is inhibited in the absence of the second electrogenic ion transport activity, as in a conventional method.
  • the enhanced change in transmembrane potential can be more readily detected thereby facilitating detection of a reduction in the activity of the first electrogenic ion transport activity. This, in turn, facilitates identification of inhibitors of the first electrogenic ion transport activity.
  • a second aspect of the invention provides a method for identifying an inhibitor of electrogenic ion transport activity which comprises: a) providing a membrane with a first and a second electrogenic ion transport activity, the membrane having a transmembrane potential predominantly set by the first or the second electrogenic ion transport activity, the first electrogenic ion transport activity having an opposite effect on the transmembrane potential of the membrane to the second electrogenic ion transport activity compared to the transmembrane potential of the membrane in the absence of the first and second electrogenic ion transport activities, wherein the first electrogenic ion transport activity predominantly sets the transmembrane potential of the membrane unless the first electrogenic ion transport activity is inhibited in which case the second electrogenic ion transport activity predominantly sets the transmembrane potential; b) adding a candidate inhibitor of the first electrogenic ion transport activity; and c) monitoring any change in the transmembrane potential of the membrane caused by inhibition of the first electrogenic ion transport activity by the
  • the invention also provides methods for enhancing the change in transmembrane potential of a membrane when the activity of an electrogenic ion transport activity in the membrane is enhanced, or when an inactive electrogenic ion transport activity in the membrane is activated, and for identifying enhancers or activators of electrogenic ion transport activity.
  • a method for enhancing the change in transmembrane potential of a membrane when the activity of an electrogenic ion transport activity in the membrane is enhanced which comprises : a) providing a membrane with a first and a second electrogenic ion transport activity, the membrane having a transmembrane potential predominantly set by the first or the second electrogenic ion transport activity, the first electrogenic ion transport activity having an opposite effect on the transmembrane potential of the membrane to the second electrogenic ion transport activity compared to the transmembrane potential of the membrane in the absence of the first and second electrogenic ion transport activities, wherein the second electrogenic ion transport activity predominantly sets the transmembrane potential of the membrane unless the activity of the first electrogenic ion transport activity is enhanced in which case the transmembrane potential is predominantly set by the enhanced activity of the first electrogenic ion transport activity; and b) enhancing the activity of the first electrogenic ion transport activity.
  • a method for identifying an enhancer of electrogenic ion transport activity which comprises: a) providing a membrane with a first and a second electrogenic ion transport activity, the membrane having a transmembrane potential predominantly set by the first or the second electrogenic ion transport activity, the first electrogenic ion transport activity having an opposite effect on the transmembrane potential of the membrane to the second electrogenic ion transport activity compared to the transmembrane potential of the membrane in the absence of the first and second electrogenic ion transport activities, wherein the second electrogenic ion transport activity predominantly sets the transmembrane potential of the membrane unless the activity of the first electrogenic ion transport activity is enhanced in which case the transmembrane potential is predominantly set by the enhanced activity of the first electrogenic ion transport activity; b) adding a candidate enhancer of the first electrogenic ion transport activity; and c) monitoring any change in the transmembrane potential of the membrane caused by enhancement of the
  • a method for enhancing the change in transmembrane potential of a membrane when an inactive electrogenic ion transport activity in the membrane is activated which comprises : a) providing a membrane with an inactive first electrogenic ion transport activity and a second electrogenic ion transport activity, the membrane having a transmembrane potential predominantly set by the active first or the second electrogenic ion transport activity, the active first electrogenic ion transport activity having an opposite effect on the transmembrane potential of the membrane to the second electrogenic ion transport activity compared to the transmembrane potential of the membrane in the absence of the first and second electrogenic ion transport activities, wherein the transmembrane potential of the membrane is predominantly set by the second electrogenic ion transport activity until the first electrogenic ion transport activity is activated; and b) activating the first electrogenic ion transport activity.
  • a method for identifying an activator of electrogenic ion transport activity which comprises: a) providing a membrane with an inactive first electrogenic ion transport activity and a second electrogenic ion transport activity, the membrane having a transmembrane potential predominantly set by the active first or the second electrogenic ion transport activity, the active first electrogenic ion transport activity having an opposite effect on the transmembrane potential of the membrane to the second electrogenic ion transport activity compared to the transmembrane potential of the membrane in the absence of the first and second electrogenic ion transport activities, wherein the transmembrane potential of the membrane is predominantly set by the second electrogenic ion transport activity until the first electrogenic ion transport activity is activated; b) adding a candidate activator of the inactive first electrogenic ion transport activity; and c) monitoring any change in the transmembrane potential of the membrane caused by activation of the first electrogenic ion transport activity by the candidate activator.
  • Methods according to the second, fourth or sixth aspect of the invention may further comprise identifying an inhibitor, enhancer or activator of electrogenic ion transport activity, respectively.
  • the methods of the invention differ from the method disclosed by Perozo and Hubbell.
  • the purified sodium channels and the bacteriorhodopsin do not have opposite effects on the transmembrane potential of the membrane, but instead both act to depolarise the transmembrane potential from a negative potential towards OmV.
  • a screening assay for screening for an inhibitor of electrogenic ion transport activity which comprises the steps of the second aspect of the invention.
  • a screening assay for screening for an enhancer of electrogenic ion transport activity which comprises the steps of the fourth aspect of the invention.
  • a screening assay for screening for an activator of electrogenic ion transport activity which comprises the steps of the sixth aspect of the invention.
  • the first and second electrogenic ion transport activities have opposite effects on the transmembrane potential of the membrane compared to the transmembrane potential of the membrane in the absence of the first and second electrogeni ⁇ ion transport activities.
  • the change in transmembrane potential can be enhanced if the first and second electrogenic ion transport activities have opposite effects on the transmembrane potential of the membrane compared to the transmembrane potential that would result if the first electrogenic ion transport activity was partially inhibited in the absence of the second electrogenic ion transport activity.
  • transmembrane potential of the membrane in the absence of the first and second electrogenic ion transport activities is OmV but is -80mV in the presence of the first electrogenic ion transport activity. Partial inhibition of the first electrogenic ion transport activity in the absence of the second electrogenic ion transport activity may only cause the transmembrane potential of the membrane to increase to -60mV.
  • Such a change in transmembrane potential could be enhanced if the second electrogenic ion transport activity causes the transmembrane potential to increase to over OmV when the first electrogenic ion transport activity is inhibited, as according to the first and second aspects of the invention. However the change is also enhanced if the second electrogenic ion transport activity causes the transmembrane potential to increase to over -6OmV when the first electrogenic ion transport activity is inhibited.
  • the first and second electrogenic ion transport activities have opposite effects on the transmembrane potential of the membrane compared to the transmembrane potential of the membrane in the absence of the first and second electrogenic ion transport activities.
  • the first and second electrogenic ion transport activities have opposite effects on the transmembrane potential of the membrane compared to the transmembrane potential of the membrane when the first electrogenic ion transport activity is inhibited in the absence of the second electrogenc ion transport activity. Both types of methods are within the scope of the invention.
  • a seventh aspect of the invention provides a method for enhancing the change in transmembrane potential of a membrane when the activity of an electrogenic ion transport activity in the membrane is reduced which comprises : a) providing a membrane with a first and a second electrogenic ion transport activity, the membrane having a transmembrane potential predominantly set by the first or the second electrogenic ion transport activity, the first electrogenic ion transport activity having an opposite effect on the transmembrane potential of the membrane to the second electrogenic ion transport activity compared to the transmembrane potential of the membrane when the first electrogenic ion transport activity is inhibited in the absence of the second electrogenic ion transport activity, wherein the first ion electrogenic ion transport activity predominantly sets the transmembrane potential of the membrane unless the activity of the first electrogenic ion transport activity is reduced in which case the second electrogenic ion transport activity predominantly sets the transmembrane potential; and b) reducing the activity of the first electrogenic ion
  • a method for identifying an inhibitor of electrogenic ion transport activity which comprises: a) providing a membrane with a first and a second electrogenic ion transport activity, the membrane having a transmembrane potential predominantly set by the first or the second electrogenic ion transport activity, the first electrogenic ion transport activity having an opposite effect on the transmembrane potential of the membrane to the second electrogenic ion transport activity compared to the transmembrane potential of the membrane when the first electrogenic ion transport activity is inhibited in the absence of the second electrogenic ion transport activity, wherein the first electrogenic ion transport activity predominantly sets the transmembrane potential of the membrane unless the first electrogenic ion transport activity is inhibited in which case the second electrogenic ion transport activity predominantly sets the transmembrane potential ; b) adding a candidate inhibitor of the first electrogenic ion transport activity; and c) monitoring any change in the transmembrane potential of the membrane caused by
  • enhancement of the change in transmembrane potential is possible in methods corresponding to the third and fourth aspects of the invention in which the first and second electrogenic ion transport activities have opposite effects on the transmembrane potential compared to the transmembrane potential that would result in the absence of the second electrogenic ion transport activity without enhancement of the activity of the first electrogenic ion transport activity.
  • a ninth aspect of the invention provides a method for enhancing the change in transmembrane potential of a membrane when the activity of an electrogenic ion transport activity in the membrane is enhanced which comprises: a) providing a membrane with a first and a second electrogenic ion transport activity, the membrane having a transmembrane potential predominantly set by the first or the second electrogenic ion transport activity, the first electrogenic ion transport activity having an opposite effect on the transmembrane potential of the membrane to the second electrogenic ion transport activity compared to the transmembrane potential of the membrane when the activity of the first electrogenic ion transport activity is not enhanced in the absence of the second electrogenic ion transport activity, wherein the second electrogenic ion transport activity predominantly sets the transmembrane potential of the membrane unless the activity of the first electrogenic ion transport activity is enhanced in which case the transmembrane potential is predominantly set by the enhanced activity of the first electrogenic ion transport activity; and b) enhancing the activity of the first
  • a method for identifying an enhancer of electrogenic ion transport activity which comprises: a) providing a membrane with a first and a second electrogenic ion transport activity, the membrane having a transmembrane potential predominantly set by the first or the second electrogenic ion transport activity, the first electrogenic ion transport activity having an opposite effect on the transmembrane potential of the membrane to the second electrogenic ion transport activity compared to the transmembrane potential of the membrane when the activity of the first electrogenic ion transport activity is not enhanced in the absence of the second electrogenic ion transport activity, wherein the second electrogenic ion transport activity predominantly sets the transmembrane potential of the membrane unless the activity of the first electrogenic ion transport activity is enhanced in which case the transmembrane potential is predominantly set by the enhanced activity of the first electrogenic ion transport activity; b) adding a candidate enhancer of the first electrogenic ion transport activity; and c) monitoring any change
  • a method for detecting a change in the activity of an electrogenic ion transport activity which comprises : a) providing a membrane having a transmembrane potential and a first electrogenic ion transport activity; b) causing the transmembrane potential of the membrane to change, wherein the rate at which the transmembrane potential changes is altered if the activity of the first electrogenic ion transport activity is modulated; and c) comparing the change in transmembrane potential of the membrane when the activity of the first electrogenic ion transport activity is modulated with the change when the activity of the first electrogenic ion transport activity is not modulated.
  • the transmembrane potential of the membrane is caused to change by introducing a second electrogenic ion transport activity to the membrane.
  • the change of magnitude in transmembrane potential occurs at a different rate than the change of magnitude when the activity of the first electrogenic ion transport activity is not modulated.
  • this difference in rate of change of transmembrane potential causes the transmembrane potential of the membrane when the first electrogenic ion transport activity has been modulated to be different in magnitude to the transmembrane potential of the membrane when the first electrogenic ion transport activity has not been modulated.
  • (c) includes comparing the rate of change in transmembrane potential caused by the second electrogenic ion transport activity when the activity of the first electrogenic ion transport activity is modulated with the rate of change in transmembrane potential caused by the second electrogenic ion transport activity when the activity of the first electrogenic ion transport activity is not modulated. A difference between the rates indicates that the activity of the first electrogenic ion transport activity has been modulated.
  • the activity of the first electrogenic ion transport activity may be modulated by contacting the first electrogenic ion transport activity with a modulator.
  • the modulator may be an inhibitor, enhancer or activator of the first electrogenic ion transport activity.
  • the activity of the first electrogenic ion transport activity may be modulated before or after, but preferably at substantially the same time as the transmembrane potential of the membrane is caused to change.
  • a change in the activity of the first electrogenic ion transport activity may be more readily detectable using a method of the eleventh aspect of the invention than a conventional method.
  • a change in the activity of the first electrogenic ion transport activity which is not detectable using a conventional method may be detectable using a method of the eleventh aspect of the invention.
  • FIG. 1(a) shows a conventional method for detecting a change in the activity of the electrogenic ion transport activity when an inhibitor is added.
  • the electrogenic ion transport activity consists of potassium ion channels which keep the transmembrane potential of the membrane at -20mV.
  • the transmembrane potential of the membrane rises to OmV. Consequently, the maximum change in the transmembrane potential of the membrane caused by the inhibitor is 20mV.
  • a 20mV difference and, consequently, a change in the activity of the potassium ion channels can be hard to detect.
  • Figure 1 (b) shows the change in transmembrane potential when the potassium ion channels of the membrane are inhibited in accordance with a method of the eleventh aspect of the invention.
  • the transmembrane potential of the membrane is again -20mV.
  • a sodium ionophore is introduced to the membrane in the presence and absence of the inhibitor. This causes the transmembrane potential of the membrane to change towards +50mV.
  • the rate at which the transmembrane potential of the membrane changes is increased by the presence of the inhibitor of the potassium ion channels. This is because the tendency of the potassium ion channels to keep the transmembrane potential at -20mV is inhibited.
  • the difference between the transmembrane potential of the membrane in the presence of the inhibitor and the transmembrane potential of the membrane in the absence of the inhibitor is 3OmV.
  • modulators of electrogenic ion transport activity may be more readily identified.
  • a twelfth aspect of the invention provides a method for identifying a modulator of the activity of an electrogenic ion transport activity which comprises : a) providing a membrane having a transmembrane potential and a first electrogenic ion transport activity; b) causing the transmembrane potential of the membrane to change, wherein the rate at which the transmembrane potential changes is altered if the activity of the first electrogenic ion transport activity is modulated; and c) comparing the change in transmembrane potential of the membrane in the presence and absence of a candidate modulator of the activity of the first electrogenic ion transport activity.
  • the transmembrane potential of the membrane is caused to change by introducing a second electrogenic ion transport activity to the membrane.
  • the candidate modulator may be added before or after, but preferably at substantially the same time as the transmembrane potential of the membrane is caused to change.
  • the second electrogenic ion transport activity may be introduced to the membrane by adding it to the membrane, or by activating it in the membrane.
  • the second electrogenic ion transport activity may be activated in the membrane by adding an activator to, or removing an inhibitor from, the membrane .
  • Methods of the twelfth aspect of the invention may further comprise identifying a modulator of the activity of the first electrogenic ion transport activity.
  • a screening assay for screening for a modulator of electrogenic ion transport activity which comprises the steps of the twelfth aspect of the invention.
  • An advantage of methods of the eleventh and twelfth aspects of the invention over conventional methods is that modulators of the activity of the first electrogenic ion transport activity can be identified even if the transmembrane potential of the membrane is not dependent on the activity of the first electrogenic ion transport activity.
  • the transmembrane potential of the membrane comprising the first electrogenic ion transport activity is OmV
  • the first eletrogenic ion transport activity is only activated when the transmembrane potential of the membrane is increased to +10mV and then acts to drive the transmembrane potential of the membrane to +3 OmV
  • a conventional method will not detect an inhibitor of the first electrogenic ion transport activity.
  • the transmembrane potential of the membrane is caused to increase to +80mV
  • the first electrogenic ion transport activity once activated when the transmembrane potential is at +10mV, may reduce the rate at which the transmembrane potential increases to +80mV.
  • an inhibitor of the first electrogenic ion transport activity would be expected to increase the rate at which the transmembrane potential rises to +80mV. This could be detected by a difference between the change in transmembrane potential of the membrane in the presence and absence of the inhibitor.
  • the membrane may be any membrane capable of exhibiting a transmembrane potential.
  • the membrane comprises a biological membrane such as a cell plasma membrane or an intracellular organelle membrane.
  • suitable biological membranes include the cell membrane of a Xenopus oocyte, Chinese hamster ovary, HEK-293, C0S-7, or fibroblast cell.
  • the membrane may comprise an extracted biological membrane or a synthetic membrane, such as a synthetic phospholipid bilayer.
  • the first or the second electrogenic ion transport activity may be any type of electrogenic ion transport activity which causes or allows movement of an ion across a membrane, but does not include an ion gradient.
  • an ion gradient may of course be required in order for an electrogenic ion transport activity to cause or allow movement of an ion across the membrane .
  • an electrogenic ion transport activity will be a membrane-associated activity.
  • An electrogenic ion transport activity may cause or allow movement of ions across the membrane by allowing ions to pass through it (for example, an ion channel) or by receiving an ion on one side of the membrane and releasing it on the other side of the membrane, for example by changing orientation in the membrane, or undergoing a conformational change, after the ion has been received.
  • electrogenic ion transport activities are ion channels such as those gated by voltage, electrogenic ion cotransporters or symporters, or ion selective ionophores . It is preferred that the first or second electrogenic ion transport activity causes or allows selective movement of potassium, sodium, or chloride ions across the membrane .
  • the first or the second electrogenic ion transport activity may comprise synthetic, naturally occurring or recombinant material.
  • the first or second electrogenic ion transport activity may comprise a synthetic ionophore, a natural protein or a recombinant protein.
  • part or all of the first or second electrogenic ion transport activity may be expressed in the membrane by the cell to which the membrane belongs or from which the membrane was extracted.
  • Components of the first or second electrogenic ion transport activity which are expressed by the cell to which the membrane belongs or from which the membrane was extracted may be expressed from nucleic acid which is endogenous or exogenous to the cell.
  • Part or all of the first or second electrogenic ion transport activity may be expressed by a different cell, or expression system, than the cell to which the membrane belongs or from which the membrane was extracted.
  • first or second electrogenic ion transport activity it may be undesirable for the first or second electrogenic ion transport activity to be active or fully active until it is required to be active in order for a method of the invention to be performed.
  • the activity of the first or the second electrogenic ion transport activity may disrupt or have an adverse affect on the normal functioning of the cell. It may be desirable, therefore, for the first or the second electrogenic ion transport activity to be inactive or of reduced activity until it is required to be active in order to carry out the method of the invention which is being performed on the cell.
  • the first or the second electrogenic ion transport activity may be an activatable activity or an inhibitable activity.
  • the activatable activity may then be activated before its activity is required.
  • the inhibitable activity may be inhibited and inhibition relieved before its activity is required.
  • activatable electrogenic ion transport activities include ion channels, electrogenic ion cotransporters, and electrogenic ion symporters for which activators are known.
  • Cotransporters and symporters are only fully active in the presence of the ion and the moiety which they cotransport or symport .
  • An example of a cotransporter is the sodium-glucose cotransporter.
  • An example of a symporter is the sodium-iodide symporter.
  • Veratridine and batrachotoxin are examples of known activators for voltage dependant potassium channels .
  • inhibitable electrogenic ion transport activities include barium sensitive inwardly rectifying potassium channels or any of the activatable electrogenic ion transport activities which can be inhibited by removal of their activator.
  • activatable or inhibitable electrogenic ion transport activities in methods of the invention is not limited to methods in which the electrogenic ion transport activity is activated or inhibited during the method. It will be appreciated that an activatable or inhibitable electrogenic ion transport activity may also be used in accordance with a method of the invention in which the electrogenic ion transport activity is active throughout the method, unless it is inhibited by an inhibitor or a candidate inhibitor.
  • the first electrogenic ion transport activity may cause the transmembrane potential of the membrane to be increased or decreased, but preferably decreased, compared to the transmembrane potential of the membrane in the absence of the first and second electrogenic ion transport activities.
  • the first electrogenic ion transport activity may cause the transmembrane potential of the membrane to be increased or decreased, but preferably decreased, compared to the transmembrane potential of the membrane when the first electrogenic ion transport activity is inhibited in the absence of the second electrogenic ion transport activity.
  • the first electrogenic ion transport activity may cause the transmembrane potential of the membrane to be increased or decreased, but preferably decreased, compared to the transmembrane potential of the membrane when the activity of the first electrogenic ion transport activity is not enhanced in the absence of the second electrogenic ion transport activity.
  • the first electrogenic ion transport activity may increase or decrease the transmembrane potential of the membrane, but preferably decrease, compared to the transmembrane potential of the membrane in the absence of the first electrogenic ion transport activity.
  • the transmembrane potential of the membrane may be caused to increase or decrease.
  • the transmembrane potential of the membrane is caused to increase when the first electrogenic ion transport activity decreases the transmembrane potential of the membrane.
  • the transmembrane potential of the membrane is caused to decrease when the first electrogenic ion transport activity increases the transmembrane potential of the membrane .
  • Methods of the first ten aspects of the invention are found to be particularly advantageous compared to conventional methods for identifying changes in, or modulators of, electrogenic ion transport activity when the difference between the transmembrane potential of the membrane when the first electrogenic ion transport activity predominantly sets the transmembrane potential and the second electrogenic ion transport activity predominantly sets the transmembrane potential is greater than about 20mV.
  • Methods of the eleventh and twelfth aspects of the invention are found to be particularly advantageous compared to conventional methods when the difference between the transmembrane potential of the membrane when the first electrogenic ion transport activity has been modulated and the transmembrane potential of the membrane when the first electrogenic ion transport activity has not been modulated is greater than the change in transmembrane potential that would occur if the method was performed omitting step (b) .
  • Electrogenic ion transport activities which are particularly suitable as the first electrogenic ion transport activity in methods of the first ten aspects of the invention are those which predominantly set the transmembrane potential of the membrane to a value which is about 0-50mV above or below the transmembrane potential of the membrane in the absence of the first and second electrogenic ion transport activity.
  • Electrogenic ion transport activities which are particularly suitable as the first electrogenic ion transport activity in methods of the eleventh and twelfth aspects of the invention are those which decrease or increase the transmembrane potential of the membrane by about 0-50mV.
  • the second electrogenic ion transport activity predominantly sets the transmembrane potential of the membrane to a value which is more than 15mV above or below the transmembrane potential of the membrane in the absence of the fist and second electrogenic ion transport activity.
  • the first electrogenic ion transport activity causes or allows selective movement of potassium ions across the membrane .
  • the second electrogenic ion transport activity causes or allows selective movement of sodium ions across the membrane .
  • the second electrogenic ion transport activity may be, for example, an electrogenic sodium transport protein or synthetic sodium selective ionophore that can incorporate into a biological membrane or a synthetic phospholipid bilayer.
  • electrogenic sodium transport proteins include voltage-dependent sodium channels, amiloride- sensitive epithelial sodium channels, degenerin-related sodium channels and electrogenic sodium carrier proteins such as the sodium-iodide symporter (NIS) , and sodium- glucose cotransporter.
  • NIS sodium-iodide symporter
  • sodium- glucose cotransporter sodium-iodide symporter
  • sodium selective ionophores include hemisodium, SQI-Pr.
  • a change in transmembrane potential caused by a change in the activity of an electrogenic ion transport activity in a method according to the invention may be monitored by any suitable method. For example, by direct measurement of the change in transmembrane potential, or by indirect measurement using a voltage sensitive reporter.
  • a preferred method is indirect measurement using a voltage sensitive reporter which allows or causes a fluorescent or luminescent signal to be generated. Generation of a fluorescent or luminescent signal is particularly preferred when methods of the invention are used for high throughput screening of candidate inhibitors, activators, or enhancers of electrogenic ion transport activity as large numbers of different samples can be most rapidly and efficiently monitored using these reporter signals.
  • voltage sensitive fluorescent dyes are preferred in methods according to the invention.
  • suitable voltage sensitive fluorescent dyes include anionic oxonols and bisoxonols which exhibit voltage-dependent changes in their transmembrane distribution that are accompanied by a fluorescence change.
  • Voltage-sensitive fluorescent dyes can be used to perform membrane potential measurements in subcellular organelles, cells or tissues (Plasek and Sigler, J Photochemistry and PhotobiologyB : Biology (1996) 33:101) .
  • Figure 2 illustrates an embodiment of the first aspect of the invention
  • Figure 3 illustrates examples of introduced electrogenic sodium ion transport activities which can be used to enhance the change in transmembrane potential of a cell membrane in accordance with the invention
  • Figure 4 illustrates an embodiment of the eleventh aspect of the invention.
  • a potassium ion channel KvA or KvB
  • KvA or KvB is expressed in a CHO cell membrane.
  • Expression of the KvA channel causes the transmembrane potential of the CHO cell to decrease to about -60mV relative to the transmembrane potential of the CHO cell in the absence of the KvA channel .
  • Expression of the KvB channel causes the transmembrane potential of the CHO cell to decrease to about -5mV relative to the transmembrane potential of the CHO cell in the absence of the KvB channel .
  • a sodium ion premeability is introduced into the CHO cell membrane.
  • the expressed potassium ion channel competes with the introduced sodium ion permeability to be the dominant contributor to setting the transmembrane potential of the CHO cell.
  • the expressed potassium ion channel When the expressed potassium ion channel is active, it is the dominant contributor to setting the transmembrane potential of the CHO cell.
  • the activity of the expressed potassium ion channel is reduced, for example when an inhibitor of that activity is added, the introduced sodium ion permeability becomes the dominant contributor to setting the transmembrane potential.
  • the introduced sodium ion permeability then causes the transmembrane potential of the CHO cell membrane to increase towards +50mV relative to the transmembrane potential of the CHO cell in the absence of the KvA or KvB channel. This is approximately the Nernst equilibrium potential for sodium ions .
  • the transmembrane potential of the CHO cell changes from about -6OmV or -5mV (depending on whether the CHO cell is expressing the KvA or KvB channel) to about +50mV when the activity of the expressed potassium ion channel is reduced.
  • This is in contrast to a change in transmembrane potential of from about -60mV, or -5mV, to about OmV when the activity of the expressed potassium ion channel is reduced in the absence of the introduced sodium ion permeability.
  • the change in transmembrane potential caused by a reduction in the activity of the expressed potassium ion channel is greatly enhanced and is, therefore, more readily detectable according to the invention. Consequently, inhibitors of the expressed potassium ion channel activity can be more readily identified.
  • the expressed potassium ion channel is inactive and the introduced sodium ion permeability is the dominant contributor to setting the transmembrane potential of the CHO cell membrane.
  • the expressed potassium ion channel When the expressed potassium ion channel is activated, it becomes the dominant contributor to setting the transmembrane potential of the membrane. Consequently, the transmembrane potential of the CHO cell membrane changes from about +50mV to about -60mV or -5mV when the expressed potassium ion channel is activated (depending on whether the CHO cell is expressing the KvA or KvB channel) , compared to the transmembrane potential in the absence of the expressed potassium ion channel and the introduced sodium ion permeability.
  • the change in transmembrane potential caused by activation of the expressed potassium ion channel is greatly enhanced and is, therefore, more readily detectable according to the invention. Consequently, activators of the expressed potassium channel activity can be more readily identified. In other embodiments, the activity of the expressed potassium ion channel could be enhanced rather than activated from a state of complete inhibition.
  • the membrane may comprise an electrogenic potassium ion transport activity and an activatable electrogenic sodium ion transport activity such as a voltage-dependent sodium ion channel activated by chemical activators such as veratridine or batrachotoxin, a sodium- iodide symporter (NIS) (activated by elevated iodide levels) , or a sodium-glucose cotransporter (SGLT) (activated by elevated glucose levels) .
  • a voltage-dependent sodium ion channel activated by chemical activators such as veratridine or batrachotoxin
  • NIS sodium- iodide symporter
  • SGLT sodium-glucose cotransporter
  • the activated electrogenic sodium ion transport activity is empirically set to be the dominant contributor to setting the transmembrane potential of the membrane only when the activity of the electrogenic potassium ion transport activity is reduced, for example by pharmacological inhibition.
  • the activatable electrogenic sodium ion transport activity has little or no contribution to the transmembrane potential of the membrane in the absence of the appropriate activator.
  • the transmembrane potential of the membrane is increased.
  • the magnitude of the change in transmembrane potential of the membrane is determined by the net flow of sodium ions into the cell compared to the net flow of potassium ions out of the cell through the potassium channel being examined.
  • the maximum change in transmembrane potential will be achieved when potassium ion flow is completely inhibited.
  • the change in transmembrane potential will be smallest when the potassium channel function is maximal.
  • Selective sodium ionophores such as hemisodium or SQI-Pr, can also be used to introduce sodium ion permeability into a membrane.
  • hemisodium or SQI-Pr can also be used to introduce sodium ion permeability into a membrane.
  • FIG. 4 An embodiment of the eleventh aspect of the invention is illustrated by figure 4.
  • the figure shows a graph of the change in transmembrane potential of a membrane comprising potassium ion channels after addition of a sodium ion ionophore to the membrane in the presence and absence of an inhibitor of the potassium ion channels.
  • the potassium ion channels act to decrease the transmembrane potential of the membrane.
  • the sodium ion ionophore acts to increase the transmembrane potential of the membrane and its effect is dominant to that of the potassium ion channels.
  • the sodium ion ionophore is added.
  • the potassium ion channels slow the rate at which the sodium ionophore causes the transmembrane potential of the membrane to change.
  • the potassium ion channels can no longer slow the rate at which the transmembrane potential of the membrane changes.
  • ⁇ V m there is a difference, between the transmembrane potential of the membrane in the presence of the inhibitor and the transmembrane potential of the membrane in the absence of the inhibitor. This difference is caused by the difference in the rate of change of the transmembrane potential and shows that the potassium ion channels have been inhibited.
  • Methods of the invention facilitate identification of changes in electrogenic ion transport activity and of modulators of electrogenic ion transport activity.
  • references in the specification to inhibition includes partial or complete inhibition. Similarly, reference to an inhibitor includes a partial or complete inhibitor.

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Abstract

L'invention concerne des méthodes améliorées permettant de détecter des modifications de l'activité des canaux ioniques. Une membrane comprend un premier et un second canal ionique. Le potentiel transmembranaire de cette membrane dépend essentiellement du premier ou du second canal ionique, ces canaux présentant une relation dominant/récessif et produisant des effets opposés sur ce potentiel transmembranaire. Lorsque le premier canal ionique est dominant, l'inhibition de ce canal ionique permet au second canal ionique de déterminer l'essentiel du potentiel transmembranaire. Etant donné que lesdits canaux ioniques produisent des effets opposés sur le potentiel transmembranaire, l'ampleur du changement de potentiel transmembranaire lorsque le premier canal ionique est inhibé est plus important que l'ampleur de ce changement lorsque le premier canal ionique est inhibé en l'absence du second canal ionique. Ces changements accentués de potentiel transmembranaire sont plus facilement détectables. Lesdites méthodes peuvent être utilisées comme analyses de criblage afin d'identifier des inhibiteurs de l'activité des canaux ioniques. L'invention concerne également des méthodes correspondantes permettant d'identifier des activateurs et des amplificateurs de l'activité des canaux ioniques.
PCT/GB2001/000516 2000-02-11 2001-02-09 Analyse amelioree Ceased WO2001059446A2 (fr)

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US5661035A (en) * 1995-06-07 1997-08-26 The Regents Of The University Of California Voltage sensing by fluorescence resonance energy transfer
AU7689298A (en) * 1997-05-20 1998-12-11 Yale University (nbc), a gene that encodes a member of the bicarbonate transporter family of proteins
CA2312477A1 (fr) * 1997-12-05 1999-06-17 Loyola University Of Chicago Canaux calciques potentiel-dependants de type t et leurs methodes d'utilisation
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US8290714B2 (en) 2005-03-08 2012-10-16 Life Technologies Corporation Monitoring and manipulating cellular transmembrane potentials using nanostructures
US9506930B2 (en) 2005-03-08 2016-11-29 Life Technologies Corporation Monitoring and manipulating cellular transmembrane potentials using nanostructures
US8952041B2 (en) 2005-06-15 2015-02-10 Hydra Biosciences, Inc. Modulators of sperm hypermobility and uses thereof
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US9732050B2 (en) 2005-06-15 2017-08-15 Hydra Biosciences, Inc. Modulators of sperm hypermotility and uses thereof
WO2010002540A3 (fr) * 2008-06-05 2010-03-25 Life Technologies Corporation Activation et contrôle de potentiels cellulaires transmembranaires
US9012204B2 (en) 2008-06-05 2015-04-21 Life Technologies Corporation Activation and monitoring of cellular transmembrane potentials
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US9702874B2 (en) 2010-08-23 2017-07-11 President And Fellows Of Harvard College Systems, methods, and workflows for optogenetics analysis
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CN102393451A (zh) * 2011-08-01 2012-03-28 重庆大学 基于能斯特电势荧光染料的细胞钾电极特性的检测方法
US9518103B2 (en) 2014-06-18 2016-12-13 President And Fellows Of Harvard College Optogenetic probes for measuring membrane potential
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US10800829B2 (en) 2014-06-18 2020-10-13 President And Fellows Of Harvard College Optogenetic probes for measuring membrane potential
US10077463B2 (en) 2015-01-15 2018-09-18 President And Fellows Of Harvard College Optical selection of cells

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