The Identification and Characterization of a Noncontinuous Calmodulin-binding Site in Noninactivating Voltage-dependent KCNQ Potassium Channels

Abstract

We show here that in a yeast two-hybrid assay calmodulin (CaM) interacts with the intracellular C-terminal region of several members of the KCNQ family of potassium channels. CaM co-immunoprecipitates with KCNQ2, KCNQ3, or KCNQ5 subunits better in the absence than in the presence of Ca2+. Moreover, in two-hybrid assays where it is possible to detect interactions with apo-CaM but not with Ca2+-bound calmodulin, we localized the CaM-binding site to a region that is predicted to contain two α-helices (A and B). These two helices encompass ∼85 amino acids, and in KCNQ2 they are separated by a dispensable stretch of ∼130 amino acids. Within this CaM-binding domain, we found an IQ-like CaM-binding motif in helix A and two overlapping consensus 1–5-10 CaM-binding motifs in helix B. Point mutations in helix A or B were capable of abolishing CaM binding in the two-hybrid assay. Moreover, glutathione S-transferase fusion proteins containing helices A and B were capable of binding to CaM, indicating that the interaction with KCNQ channels is direct. Full-length CaM (both N and C lobes) and a functional EF-1 hand were required for these interactions to occur. These observations suggest that apo-CaM is bound to neuronal KCNQ channels at low resting Ca2+ levels and that this interaction is disturbed when the [Ca2+] is raised. Thus, we propose that CaM acts as a mediator in the Ca2+-dependent modulation of KCNQ channels. We show here that in a yeast two-hybrid assay calmodulin (CaM) interacts with the intracellular C-terminal region of several members of the KCNQ family of potassium channels. CaM co-immunoprecipitates with KCNQ2, KCNQ3, or KCNQ5 subunits better in the absence than in the presence of Ca2+. Moreover, in two-hybrid assays where it is possible to detect interactions with apo-CaM but not with Ca2+-bound calmodulin, we localized the CaM-binding site to a region that is predicted to contain two α-helices (A and B). These two helices encompass ∼85 amino acids, and in KCNQ2 they are separated by a dispensable stretch of ∼130 amino acids. Within this CaM-binding domain, we found an IQ-like CaM-binding motif in helix A and two overlapping consensus 1–5-10 CaM-binding motifs in helix B. Point mutations in helix A or B were capable of abolishing CaM binding in the two-hybrid assay. Moreover, glutathione S-transferase fusion proteins containing helices A and B were capable of binding to CaM, indicating that the interaction with KCNQ channels is direct. Full-length CaM (both N and C lobes) and a functional EF-1 hand were required for these interactions to occur. These observations suggest that apo-CaM is bound to neuronal KCNQ channels at low resting Ca2+ levels and that this interaction is disturbed when the [Ca2+] is raised. Thus, we propose that CaM acts as a mediator in the Ca2+-dependent modulation of KCNQ channels. calmodulin apo-calmodulin, Ca2+-free calmodulin Ca2+-bound calmodulin calmodulin binding motif glutathione S-transferase 5-dimethylaminonaphthalene-1-sulfonyl amino acid(s) The KCNQ transmembrane proteins are members of a family of voltage-dependent potassium selective channels that are involved in the control of cellular excitability. Remarkably, mutations in four of the five known members of this family have been associated with different hereditary human disorders. While mutations in the KCNQ1 subunit (KvQT1) lead to arrhythmia in the human long QT syndrome, mutations in KCNQ2 or KCNQ3 are associated with a benign form of epilepsy. It has also been shown that KCNQ4 is mutated in a dominant form of progressive hearing loss (1Jentsch T.J. Nat. Rev. Neurosci. 2000; 1: 21-30Crossref PubMed Scopus (680) Google Scholar).With regards to the normal physiology of this protein family, the KCNQ2 and KCNQ3 subunits have been shown to form M-type potassium channels whose expression is restricted to neuronal tissue (2Wang H.S. Pan Z. Shi W. Brown B.S. Wymore R.S. Cohen I.S. Dixon J.E. McKinnon D. Science. 1998; 282: 1890-1893Crossref PubMed Scopus (1015) Google Scholar). Moreover, in some brain areas and neuronal tissues, KCNQ4 and KCNQ5 also contribute to the formation of M channels, suggesting that the different combinations of KCNQ subunits may be in part responsible for the diversity of M channel properties (1Jentsch T.J. Nat. Rev. Neurosci. 2000; 1: 21-30Crossref PubMed Scopus (680) Google Scholar). The M current (I M) is a subthreshold noninactivating voltage-dependent potassium current that is found in many neuronal cell types. The M current controls membrane excitability, and it has been shown to be modulated by a variety of intracellular signals that in turn dramatically affect the firing rate of neurons. Among those intracellular signals, Ca2+ has been shown to mediate the inhibition of I M by B2bradykinin receptors in sympathetic neurons (3Cruzblanca H. Koh D.S. Hille B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7151-7156Crossref PubMed Scopus (157) Google Scholar). Indeed, intracellular Ca2+ can suppress the activity of M channels under conditions that do not support enzymatic activities such as phosphorylation (4Selyanko A.A. Brown D.A. Neuron. 1996; 16: 151-162Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). This phenomenon suggests that an intermediary might be involved in this Ca2+-dependent modulation.In a search for candidates that might mediate the effects of Ca2+ in modulating I M, we screened a human brain cDNA library using the yeast two-hybrid system. We found that calmodulin (CaM)1bound to the C-terminal region of KCNQ channels. CaM is a small Ca2+-binding protein that acts as a ubiquitous intracellular Ca2+ sensor in the regulation of a growing diverse array of ion channels (5Saimi Y. Kung C. Annu. Rev. Physiol. 2002; 64: 289-311Crossref PubMed Scopus (290) Google Scholar). Efforts to define common characteristics of CaM binding have indicated that it associates with short α-helical sequences within its targets (6O'Neil K.T. DeGrado W.F. Trends Biochem. Sci. 1990; 15: 59-64Abstract Full Text PDF PubMed Scopus (712) Google Scholar, 7Rhoads A.R. Friedberg F. FASEB J. 1997; 11: 331-340Crossref PubMed Scopus (734) Google Scholar, 8Jurado L.A. Chockalingam P.S. Jarrett H.W. Physiol. Rev. 1999; 79: 661-682Crossref PubMed Scopus (276) Google Scholar). However, it appears that the interaction of CaM with KCNQ channels does not conform to this simple model. Rather, our data suggest that the CaM-binding site in KCNQ channels is formed by two α-helices that are separated by a stretch of ∼130 amino acids. We hypothesize that those two helices come into close proximity in the tertiary structure, facilitating CaM binding.DISCUSSIONWe have demonstrated here that CaM binds to the intracellular C-terminal domain of neuronal KCNQ2, KCNQ3, and KCNQ5 transmembrane channels. Two-hybrid experiments suggest that CaM also binds to KCNQ1 and KCNQ4, but more experiments are necessary to unequivocally confirm this interaction. The voltage-dependent channels to which members of this family of proteins contribute have been implicated in a variety of physiological processes and pathologies. As a the modulation of the activity of these channels intracellular is in the physiological of tissue Annu. Rev. Physiol. 1997; PubMed Scopus Google Scholar). of this can be in the Ca2+-dependent regulation of M channels of KCNQ that the firing rate of the sympathetic in which they are that its with these CaM may mediate the Ca2+-dependent modulation of channels that KCNQ to here that CaM associates with members of the KCNQ family, we have also its binding The binding site in this is in that it is of two helix A and helix B. A an IQ-like binding a motif that apo-CaM binding in a variety of proteins A. 2002; PubMed Scopus Google and that at and with in the helix A of the KCNQ channels the is by a or amino As a this domain the motif the region to which the to A of apo-CaM binding this and bound to motifs that the of this motif is the Moreover, it is this region that is by the and and that a A. Cohen C. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). with this the interaction of KCNQ2 with CaM is when mutations are in this part of the many the C of CaM has been shown to be that which interacts with as also with proteins that with Moreover, the bound the to the C-terminal hand domain C. D. A. Nat. PubMed Scopus Google Scholar). The that the C is to to channels J.E. A. A. B. J. J. Neurosci. 1999; PubMed Google Ca2+ channels, and this However, in it appears that to to KCNQ2 or KCNQ3 channels, the C and N are we found that when using the C-terminal region of as mutations that to or also the interaction with and to that Ca2+ is not bound to or when CaM interacts with it has been shown that the Ca2+-free C-terminal is that which the binding of CaM to the CaM-binding domain J.E. A. A. B. J. J. Neurosci. 1999; PubMed Google Scholar, PubMed Scopus Google Scholar). that the to in the that mediate the interaction of apo-CaM with the a in the binding with the CaM-binding domain, the that the interaction of CaM with KCNQ2 or KCNQ3 does not mutations at hand and a suggests that binding to the is by the and hand is to hand L.A. Chockalingam P.S. Jarrett H.W. Physiol. Rev. 1999; 79: 661-682Crossref PubMed Scopus (276) Google suggesting that a in the in apo-CaM interactions have been by Neuron. Full Text Full Text PDF PubMed Scopus Google Scholar). this a has been that the of CaM with channels in Neuron. Full Text Full Text PDF PubMed Scopus Google Scholar). The yeast two-hybrid is for this that the two-hybrid assay is capable of interactions the Ca2+-free form of CaM and proteins such as the channels J.E. A. A. B. J. J. Neurosci. 1999; PubMed Google Scholar). It be in that a of the two-hybrid is that the transmembrane of proteins be to of the to the 1998; PubMed Scopus Google Scholar). However, the is that this is a simple assay and that it does the to interactions in a cell J. 1999; Full Text Full Text PDF PubMed Scopus Google of the of CaM associated with the domain has shown that the binding domain is of two α-helices by a short PubMed Scopus Google Scholar). The that the two KCNQ contain α-helices suggests that a to the CaM-binding domain may also in However, some are in channels the is in KCNQ channels this to the C of CaM and is for binding to channels J.E. A. A. B. J. J. Neurosci. 1999; PubMed Google the CaM is necessary for binding to KCNQ2 or KCNQ3 channels. our data suggests that the C-terminal domain of KCNQ channels in such a that helix A and helix B form a that can be the and C-terminal of apo-CaM is the of CaM in KCNQ The of apo-CaM to a protein whose activity may be by CaM a and selective to in Ca2+ L.A. Chockalingam P.S. Jarrett H.W. Physiol. Rev. 1999; 79: 661-682Crossref PubMed Scopus (276) Google Scholar, Neuron. Full Text Full Text PDF PubMed Scopus Google Scholar). Indeed, the of the M current by in sympathetic neurons is by this (3Cruzblanca H. Koh D.S. Hille B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7151-7156Crossref PubMed Scopus (157) Google and apo-CaM has been shown to the Ca2+-dependent of many channels Neuron. Full Text Full Text PDF PubMed Scopus Google Neuron. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). The Ca2+-dependent modulation of M channels can also be in these such as phosphorylation are not and the effects are it that the modulation of I Ca2+ is to of the M channel or of an associated the absence of a and the to in the is suggests that the activity of Ca2+ does not the of the of the channel (4Selyanko A.A. Brown D.A. Neuron. 1996; 16: 151-162Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). These observations that the mediator in in and that it is the of Ca2+. These properties suggest the of a Ca2+ sensor such as CaM, as we have interacts with the C-terminal intracellular region of KCNQ channels. this it is to the that helix A is to the of the transmembrane domain channels with a transmembrane such as channels and channels, is modulated by a that to the of PubMed Scopus Google Scholar, PubMed Scopus Google Scholar). However, we also in that the binding of CaM may also be in processes such as or H. J. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, J. Scholar). The functional of to CaM to the of CaM in KCNQ channel The KCNQ transmembrane proteins are members of a family of voltage-dependent potassium selective channels that are involved in the control of cellular excitability. Remarkably, mutations in four of the five known members of this family have been associated with different hereditary human disorders. While mutations in the KCNQ1 subunit (KvQT1) lead to arrhythmia in the human long QT syndrome, mutations in KCNQ2 or KCNQ3 are associated with a benign form of epilepsy. It has also been shown that KCNQ4 is mutated in a dominant form of progressive hearing loss (1Jentsch T.J. Nat. Rev. Neurosci. 2000; 1: 21-30Crossref PubMed Scopus (680) Google Scholar). regards to the normal physiology of this protein family, the KCNQ2 and KCNQ3 subunits have been shown to form M-type potassium channels whose expression is restricted to neuronal tissue (2Wang H.S. Pan Z. Shi W. Brown B.S. Wymore R.S. Cohen I.S. Dixon J.E. McKinnon D. Science. 1998; 282: 1890-1893Crossref PubMed Scopus (1015) Google Scholar). Moreover, in some brain areas and neuronal tissues, KCNQ4 and KCNQ5 also contribute to the formation of M channels, suggesting that the different combinations of KCNQ subunits may be in part responsible for the diversity of M channel properties (1Jentsch T.J. Nat. Rev. Neurosci. 2000; 1: 21-30Crossref PubMed Scopus (680) Google Scholar). The M current (I M) is a subthreshold noninactivating voltage-dependent potassium current that is found in many neuronal cell types. The M current controls membrane excitability, and it has been shown to be modulated by a variety of intracellular signals that in turn dramatically affect the firing rate of neurons. Among those intracellular signals, Ca2+ has been shown to mediate the inhibition of I M by B2bradykinin receptors in sympathetic neurons (3Cruzblanca H. Koh D.S. Hille B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7151-7156Crossref PubMed Scopus (157) Google Scholar). Indeed, intracellular Ca2+ can suppress the activity of M channels under conditions that do not support enzymatic activities such as phosphorylation (4Selyanko A.A. Brown D.A. Neuron. 1996; 16: 151-162Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). This phenomenon suggests that an intermediary might be involved in this Ca2+-dependent a search for candidates that might mediate the effects of Ca2+ in modulating I M, we screened a human brain cDNA library using the yeast two-hybrid system. We found that calmodulin (CaM)1bound to the C-terminal region of KCNQ channels. CaM is a small Ca2+-binding protein that acts as a ubiquitous intracellular Ca2+ sensor in the regulation of a growing diverse array of ion channels (5Saimi Y. Kung C. Annu. Rev. Physiol. 2002; 64: 289-311Crossref PubMed Scopus (290) Google Scholar). Efforts to define common characteristics of CaM binding have indicated that it associates with short α-helical sequences within its targets (6O'Neil K.T. DeGrado W.F. Trends Biochem. Sci. 1990; 15: 59-64Abstract Full Text PDF PubMed Scopus (712) Google Scholar, 7Rhoads A.R. Friedberg F. FASEB J. 1997; 11: 331-340Crossref PubMed Scopus (734) Google Scholar, 8Jurado L.A. Chockalingam P.S. Jarrett H.W. Physiol. Rev. 1999; 79: 661-682Crossref PubMed Scopus (276) Google Scholar). However, it appears that the interaction of CaM with KCNQ channels does not conform to this simple model. Rather, our data suggest that the CaM-binding site in KCNQ channels is formed by two α-helices that are separated by a stretch of ∼130 amino acids. We hypothesize that those two helices come into close proximity in the tertiary structure, facilitating CaM have demonstrated here that CaM binds to the intracellular C-terminal domain of neuronal KCNQ2, KCNQ3, and KCNQ5 transmembrane channels. Two-hybrid experiments suggest that CaM also binds to KCNQ1 and KCNQ4, but more experiments are necessary to unequivocally confirm this interaction. The voltage-dependent channels to which members of this family of proteins contribute have been implicated in a variety of physiological processes and pathologies. As a the modulation of the activity of these channels intracellular is in the physiological of tissue Annu. Rev. Physiol. 1997; PubMed Scopus Google Scholar). of this can be in the Ca2+-dependent regulation of M channels of KCNQ that the firing rate of the sympathetic in which they are that its with these CaM may mediate the Ca2+-dependent modulation of channels that KCNQ to here that CaM associates with members of the KCNQ family, we have also its binding The binding site in this is in that it is of two helix A and helix B. A an IQ-like binding a motif that apo-CaM binding in a variety of proteins A. 2002; PubMed Scopus Google and that at and with in the helix A of the KCNQ channels the is by a or amino As a this domain the motif the region to which the to A of apo-CaM binding this and bound to motifs that the of this motif is the Moreover, it is this region that is by the and and that a A. Cohen C. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). with this the interaction of KCNQ2 with CaM is when mutations are in this part of the many the C of CaM has been shown to be that which interacts with as also with proteins that with Moreover, the bound the to the C-terminal hand domain C. D. A. Nat. PubMed Scopus Google Scholar). The that the C is to to channels J.E. A. A. B. J. J. Neurosci. 1999; PubMed Google Ca2+ channels, and this However, in it appears that to to KCNQ2 or KCNQ3 channels, the C and N are we found that when using the C-terminal region of as mutations that to or also the interaction with and to that Ca2+ is not bound to or when CaM interacts with it has been shown that the Ca2+-free C-terminal is that which the binding of CaM to the CaM-binding domain J.E. A. A. B. J. J. Neurosci. 1999; PubMed Google Scholar, PubMed Scopus Google Scholar). that the to in the that mediate the interaction of apo-CaM with the a in the binding with the CaM-binding domain, the that the interaction of CaM with KCNQ2 or KCNQ3 does not mutations at hand and a suggests that binding to the is by the and hand is to hand L.A. Chockalingam P.S. Jarrett H.W. Physiol. Rev. 1999; 79: 661-682Crossref PubMed Scopus (276) Google suggesting that a in the in apo-CaM interactions have been by Neuron. Full Text Full Text PDF PubMed Scopus Google Scholar). this a has been that the of CaM with channels in Neuron. Full Text Full Text PDF PubMed Scopus Google Scholar). The yeast two-hybrid is for this that the two-hybrid assay is capable of interactions the Ca2+-free form of CaM and proteins such as the channels J.E. A. A. B. J. J. Neurosci. 1999; PubMed Google Scholar). It be in that a of the two-hybrid is that the transmembrane of proteins be to of the to the 1998; PubMed Scopus Google Scholar). However, the is that this is a simple assay and that it does the to interactions in a cell J. 1999; Full Text Full Text PDF PubMed Scopus Google of the of CaM associated with the domain has shown that the binding domain is of two α-helices by a short PubMed Scopus Google Scholar). The that the two KCNQ contain α-helices suggests that a to the CaM-binding domain may also in However, some are in channels the is in KCNQ channels this to the C of CaM and is for binding to channels J.E. A. A. B. J. J. Neurosci. 1999; PubMed Google the CaM is necessary for binding to KCNQ2 or KCNQ3 channels. our data suggests that the C-terminal domain of KCNQ channels in such a that helix A and helix B form a that can be the and C-terminal of apo-CaM is the of CaM in KCNQ The of apo-CaM to a protein whose activity may be by CaM a and selective to in Ca2+ L.A. Chockalingam P.S. Jarrett H.W. Physiol. Rev. 1999; 79: 661-682Crossref PubMed Scopus (276) Google Scholar, Neuron. Full Text Full Text PDF PubMed Scopus Google Scholar). Indeed, the of the M current by in sympathetic neurons is by this (3Cruzblanca H. Koh D.S. Hille B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7151-7156Crossref PubMed Scopus (157) Google and apo-CaM has been shown to the Ca2+-dependent of many channels Neuron. Full Text Full Text PDF PubMed Scopus Google Neuron. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). The Ca2+-dependent modulation of M channels can also be in these such as phosphorylation are not and the effects are it that the modulation of I Ca2+ is to of the M channel or of an associated the absence of a and the to in the is suggests that the activity of Ca2+ does not the of the of the channel (4Selyanko A.A. Brown D.A. Neuron. 1996; 16: 151-162Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). These observations that the mediator in in and that it is the of Ca2+. These properties suggest the of a Ca2+ sensor such as CaM, as we have interacts with the C-terminal intracellular region of KCNQ channels. this it is to the that helix A is to the of the transmembrane domain channels with a transmembrane such as channels and channels, is modulated by a that to the of PubMed Scopus Google Scholar, PubMed Scopus Google Scholar). However, we also in that the binding of CaM may also be in processes such as or H. J. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, J. Scholar). The functional of to CaM to the of CaM in KCNQ channel We have demonstrated here that CaM binds to the intracellular C-terminal domain of neuronal KCNQ2, KCNQ3, and KCNQ5 transmembrane channels. Two-hybrid experiments suggest that CaM also binds to KCNQ1 and KCNQ4, but more experiments are necessary to unequivocally confirm this interaction. The voltage-dependent channels to which members of this family of proteins contribute have been implicated in a variety of physiological processes and pathologies. As a the modulation of the activity of these channels intracellular is in the physiological of tissue Annu. Rev. Physiol. 1997; PubMed Scopus Google Scholar). of this can be in the Ca2+-dependent regulation of M channels of KCNQ that the firing rate of the sympathetic in which they are that its with these CaM may mediate the Ca2+-dependent modulation of channels that KCNQ to here that CaM associates with members of the KCNQ family, we have also its binding The binding site in this is in that it is of two helix A and helix B. A an IQ-like binding a motif that apo-CaM binding in a variety of proteins A. 2002; PubMed Scopus Google and that at and with in the helix A of the KCNQ channels the is by a or amino As a this domain the motif the region to which the to A of apo-CaM binding this and bound to motifs that the of this motif is the Moreover, it is this region that is by the and and that a A. Cohen C. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). with this the interaction of KCNQ2 with CaM is when mutations are in this part of the many the C of CaM has been shown to be that which interacts with as also with proteins that with Moreover, the bound the to the C-terminal hand domain C. D. A. Nat. PubMed Scopus Google Scholar). The that the C is to to channels J.E. A. A. B. J. J. Neurosci. 1999; PubMed Google Ca2+ channels, and this However, in it appears that to to KCNQ2 or KCNQ3 channels, the C and N are we found that when using the C-terminal region of as mutations that to or also the interaction with and to that Ca2+ is not bound to or when CaM interacts with it has been shown that the Ca2+-free C-terminal is that which the binding of CaM to the CaM-binding domain J.E. A. A. B. J. J. Neurosci. 1999; PubMed Google Scholar, PubMed Scopus Google Scholar). that the to in the that mediate the interaction of apo-CaM with the a in the binding with the CaM-binding domain, the that the interaction of CaM with KCNQ2 or KCNQ3 does not mutations at hand and a suggests that binding to the is by the and hand is to hand L.A. Chockalingam P.S. Jarrett H.W. Physiol. Rev. 1999; 79: 661-682Crossref PubMed Scopus (276) Google suggesting that a in the The in apo-CaM interactions have been by Neuron. Full Text Full Text PDF PubMed Scopus Google Scholar). this a has been that the of CaM with channels in Neuron. Full Text Full Text PDF PubMed Scopus Google Scholar). The yeast two-hybrid is for this that the two-hybrid assay is capable of interactions the Ca2+-free form of CaM and proteins such as the channels J.E. A. A. B. J. J. Neurosci. 1999; PubMed Google Scholar). It be in that a of the two-hybrid is that the transmembrane of proteins be to of the to the 1998; PubMed Scopus Google Scholar). However, the is that this is a simple assay and that it does the to interactions in a cell J. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). The of the of CaM associated with the domain has shown that the binding domain is of two α-helices by a short PubMed Scopus Google Scholar). The that the two KCNQ contain α-helices suggests that a to the CaM-binding domain may also in However, some are in channels the is in KCNQ channels this to the C of CaM and is for binding to channels J.E. A. A. B. J. J. Neurosci. 1999; PubMed Google the CaM is necessary for binding to KCNQ2 or KCNQ3 channels. our data suggests that the C-terminal domain of KCNQ channels in such a that helix A and helix B form a that can be the and C-terminal of apo-CaM is the of CaM in KCNQ The of apo-CaM to a protein whose activity may be by CaM a and selective to in Ca2+ L.A. Chockalingam P.S. Jarrett H.W. Physiol. Rev. 1999; 79: 661-682Crossref PubMed Scopus (276) Google Scholar, Neuron. Full Text Full Text PDF PubMed Scopus Google Scholar). Indeed, the of the M current by in sympathetic neurons is by this (3Cruzblanca H. Koh D.S. Hille B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7151-7156Crossref PubMed Scopus (157) Google and apo-CaM has been shown to the Ca2+-dependent of many channels Neuron. Full Text Full Text PDF PubMed Scopus Google Neuron. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). The Ca2+-dependent modulation of M channels can also be in these such as phosphorylation are not and the effects are it that the modulation of I Ca2+ is to of the M channel or of an associated the absence of a and the to in the is suggests that the activity of Ca2+ does not the of the of the channel (4Selyanko A.A. Brown D.A. Neuron. 1996; 16: 151-162Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). These observations that the mediator in in and that it is the of Ca2+. These properties suggest the of a Ca2+ sensor such as CaM, as we have interacts with the C-terminal intracellular region of KCNQ channels. this it is to the that helix A is to the of the transmembrane domain channels with a transmembrane such as channels and channels, is modulated by a that to the of PubMed Scopus Google Scholar, PubMed Scopus Google Scholar). However, we also in that the binding of CaM may also be in processes such as or H. J. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, J. Scholar). The functional of to CaM to the of CaM in KCNQ channel We are to J. and W. A. and J. W. A. of B. J. of J. of and S. (I for with and in this We and for and in the We the of and in some of the for the and and and for