DNA encoding small and intermediate conductance...

Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...

Reexamination Certificate

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C435S320100, C435S325000, C536S023500

Reexamination Certificate




The present invention relates to compositions relating to, and methods for identifying, small conductance (SK) and intermediate conductance (IK), calcium-activated potassium channels. The invention further provides a method to assay for compounds that increase or decrease potassium ion flux through calcium-activated potassium channels.
Calcium-activated potassium currents are found in a wide variety of animal cells such as nervous, muscular, glandular or epithelial tissue and from the immune system. The channels regulating these currents open and allow the escape of potassium as the internal calcium concentration increases. This outward flow of potassium ions makes the interior of the cell more negative, counteracting depolarizing voltages applied to the cell.
Two distinct classes of calcium-activated K
channels (K
channels) have been described. Large conductance calcium-activated K
channels (BK channels) are gated by the concerted actions of internal calcium ions and membrane potential, and have a unit conductance between 100 and 220 pS. Small (SK) and intermediate (IK) conductance calcium-activated K
channels are gated solely by internal calcium ions, with a unit conductance of 2-20 and 20-85 pS, respectively, and are more sensitive to calcium than are BK channels (for review see Latorre et al., 1989
, Ann Rev Phys
, 51, 385-399.). In addition, each type of K
channel shows a distinct pharmacological profile. All three classes are widely expressed, and their activity hyperpolarizes the membrane potential. Members of the BK (Atkinson et al., 1991
, Science
, 253, 551-555. ; Adelman et al., 1992
, 9, 209-216.; Butler, 1993
, Science
, 261, 221-224) and SK (Kohler et al., 1996
, Science
, 273, 1709-1714.) subfamilies have been cloned and expressed in heterologous cell types where they recapitulate the fundamental properties of their native counterparts.
In vertebrate neurons action potentials are followed by an afterhyperpolarization (AHP) that may persist for several seconds and have profound consequences for the firing pattern of the neuron. Alterations in the AHP have been implicated in seizure activity (Alger et al.,
J. Physiol
. 399:191-205 (1988)) and learning and memory (de Jonge et al,
Exp. Br. Res
. 80:456-462 (1990)). The AHP is composed of two prominent components, a fast component (fAHP) which mediates spike frequency at the onset of a burst, and a subsequent slow component (sAHP) which is responsible for spike-frequency adaptation (Nicoll,
241:545-551 (1988)).
Each component of the AHP is kinetically distinct and is due to activation of different calcium-activated potassium channels. Activation of large-conductance (100-200 picoSiemens (pS)), voltage- and calcium-activated potassium channels (BK channels) underlies the fAHP (Lancaster et al.,
J. Physiol
. 389:187-203 (1987); Viana et al,
J. Neurophysiol
. 69:2150-2163 (1993)) which develops rapidly (1-2 ms) and decays within tens of milliseconds. The channels underlying the sAHP are small conductance. calcium activated, potassium channels (SK channels) which differ from BK channels, being more calcium-sensitive, are not voltage-gated, and possessing a smaller unit conductance (Lancaster et al.,
J. Neurosci
. 11:23-30 (1991); Sah.
J. Neurophysiol
. 74:1772-1776 (1995)).
The fAHP and the sAHP also differ in their pharmacology. The fAHP is blocked by low concentrations of external tetraethylammonium (TEA) and charybdotoxin (CTX), in accord with the pharmacology of the BK channels. Lancaster et al,
J. Physiol
. 389:187-203 (1987); Viana et al.,
J. Neurophysiol
. 69:2150-2163 (1993); Butler et al.,
261:221-224 (1993). In contrast, the sAHP is insensitive to CTX, but fall into two classes regarding sensitivity to the bee venom peptide toxin, apamin. For example, in hippocampal pyramidal neurons, the sAHP is insensitive to apamin (Lancaster et al.,
J. Neurophysiol
. 55:1268-1282 (1986)), while in hippocampal interneurons and vagal neurons it is blocked by nanomolar concentrations of the toxin (Sah,
J. Neurophysiol
. 74:1772-1776 (1995); Zhang et al.,
J. Physiol
. 488:661-672 (1995)).
In addition to its role in neuronal cells, non-voltage gated, apamin-sensitive potassium channels activated by submicromolar concentrations of calcium have also been described from peripheral cell types, including skeletal muscle (Blatz et al.,
323:718-720 (1986)), gland cells (Tse et al., Science 255:462464 (1992); Park,
J. Physiol
. 481:555-570 (1994)) and T-lymphocytes (Grissmer et al.,
J. Gen. Physiol
. 99:63-84 (1992)).
For example, SK channels have been suggested to represent the apamin receptor found in muscle membrane of patients with myotonic muscular dystrophy. Renaud et al.,
319:678-680 (1986)). Also, Grissmer et al. (
J. Gen. Physiol
. 99:63-84 (1992)) report that CTX insensitive, apamin sensitive calcium-activated potassium channels were identified in a human leukemic T cell line and suggest that calcium-activated potassium channels play a supporting role during T-cell activation by sustaining dynamic patterns of calcium signaling. And in many cells, SK channels are activated as a result of neurotransmitter or hormone action. Haylett et al., in
Potassium Channels: Structure, Classification, Function and Therapeutic Potential
(Cook, N. S., ed.), pp.71-95, John Wiley and Sons, 1990). Intermediate channels play a role in the physiology of red blood cells.
Intermediate conductance, calcium activated potassium channels have been previously described in the literature by their electrophysiology. The Gardos channel is opened by submicromolar concentrations of internal calcium and has a rectifying unit conductance, ranging from 50 pS at −120 mV to 13 pS at 120 mV (symmetrical 120 mM K
; Christophersen, 1991
, J. Membrane Biol
., 119, 75-83. ). It is blocked by charybdotoxin (CTX) but not the structurally related peptide iberiotoxin (IBX), both of which block BK channels (Brugnara et al., 1995a,
J. Membr. Biol
., 147, 71-82). Apamin, a potent blocker of certain native (Vincent et al., 1975
, J. Biochem
., 14, 2521.; Blatz and Magleby, 1986
, Nature
, 323, 718-720. ) and cloned SK channels do not block IK channels (de-Allie et al.,1996
, Br. J. Pharm
., 117,479-487). The Gardos channel is also blocked by some imidazole compounds, such as clotrimazole, but not ketoconazole (Brugnara et al., 1993
, J. Clin. Invest
., 92,520-526). The electrophysiological and pharmacological properties of the Gardos channel show that it belongs to the IK subfamily of this invention.
IK channels have been described in a variety of other cell types. Principle cells of the rat cortical collecting duct segregate different classes of K
channels to the luminal and basolateral membranes. IK channels are present in the basolateral membrane where they promote the recirculation of K
across this membrane, elevating the activity of the Na
-ATPase and thereby Na
reabsorption into the blood (Hirsch and Schlatter, 1995
, Pflügers Arch
Eur.J. Physiol
., 449, 338-344. ) IK channels have also been implicated in the microvasculature of the kidney where they may be responsible for the vasodilatory effects of bradykinin (Rapacon et al., 1996). In brain capillary endothelial cells, IK channels are activated by endothelin, produced by neurons and glia, shunting excess K
into the blood (Renterghem et al., 1995
, J. Neurochem
., 65, 1274-1281). Neutrophil granulocytes, mobile phagocytic cells which defend against microbial invaders, undergo a large depolarization subsequent to agonist stimulation, and IK channels have been implicated in repolarizing the stimulated granulocyte (Varnai et al., 1993
, J. Physiol
., 472, 373-390. ). IK channels have also been identified in both resting and activated human T-lymphocytes. Grissmer et al. 1993
, J. Gen. Physiol
. 102,601-630 reported that IK channels were blocked by low nanomolar concentrations of charybdotoxin, showed little or


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