Delineation of the Clotrimazole/TRAM-34 Binding Site on the Intermediate Conductance Calcium-activated Potassium Channel, IKCa1

Wulff, Heike; Gutman, George A.; Cahalan, Michael D.; Chandy, K. George

doi:10.1074/jbc.m105231200

Cited by 138 publications

(158 citation statements)

References 23 publications

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“…As shown in Fig. 2, mutation of Leu 18 to Ala (L18A) had no significant effect on surface expression as assessed by both IF (Fig. 2A) and CS-IP (Fig.…”

Section: Role Of the Nh 2 Terminus In The Expression Trafficking Andmentioning

confidence: 83%

“…Role of the NH 2 -terminal Dileucine Motif-Mutation of the dileucine motif at amino acids Leu 18 and Leu 19 results in a channel that is degraded 3-fold faster than wild type channel (Fig. 3B).…”

Section: Discussionmentioning

confidence: 99%

“…We (17) and others (2,3) have demonstrated that clotrimazole is an inhibitor of IK channels. Recently Chandy and colleagues (18) mapped the molecular binding site for this blocker to the pore of hIK1.…”

mentioning

confidence: 99%

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Role of the NH2 Terminus in the Assembly and Trafficking of the Intermediate Conductance Ca2+-activated K+ Channel hIK1

Jones

Hamilton

Papworth

et al. 2004

Journal of Biological Chemistry

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The role of the NH 2 -terminal leucine zipper and dileucine motifs of hIK1 in the assembly, trafficking, and function of the channel was investigated using cell surface immunoprecipitation, co-immunoprecipitation (Co-IP), immunoblot, and whole-cell patch clamp techniques. Mutation of the NH 2 -terminal leucine zipper at amino acid positions 18 and 25 (L18A/L25A) resulted in a complete loss of steady-state protein expression, cell surface expression, and whole-cell current density. Inhibition of proteasomal degradation with lactacystin restored L18A/L25A protein expression, although this channel was not expressed at the cell surface as assessed by cell surface immunoprecipitation and wholecell patch clamp. In contrast, inhibitors of lysosomal degradation (leupeptin/pepstatin) and endocytosis (chloroquine) had little effect on L18A/L25A protein expression or localization. Further studies confirmed the rapid degradation of this channel, having a time constant of 19.0 ؎ 1.3 min compared with 3.2 ؎ 0.8 h for wild type hIK1. Co-expression studies demonstrated that the L18A/L25A channel associates with wild type channel, thereby attenuating its expression at the cell surface. Co-IP studies confirmed this association. However, L18A/L25A channels failed to form homotetrameric channels, as assessed by Co-IP, suggesting the NH 2 terminus plays a role in tetrameric channel assembly. As with the leucine zipper, mutation of the dileucine motif to alanines, L18A/L19A, resulted in a near complete loss in steady-state protein expression with the protein being similarly targeted to the proteasome for degradation. In contrast to our results on the leucine zipper, however, both chloroquine and growing the cells at the permissive temperature of 27°C restored expression of L18A/L19A at the cell surface, suggesting that the defect in the channel trafficking is the result of a subtle folding error. In conclusion, we demonstrate that the NH 2 terminus of hIK1 contains overlapping leucine zipper and dileucine motifs essential for channel assembly and trafficking to the plasma membrane.The KCNN gene family is composed of four members, including the small conductance Ca 2ϩ -activated K ϩ channels (SK1, SK2, and SK3) 1 and the intermediate conductance Ca 2ϩ -activated K ϩ channel (IK1 or SK4). While the IK1 and SK channels share roughly 40% homology, their expression patterns and pharmacology are widely disparate, consistent with their unique physiological functions (1). The human IK1 channel (hIK1) is widely expressed, being found in salivary gland, colon, bladder, stomach, lung, smooth muscle, red blood cells, T-cells, and placenta (2, 3) where it plays a crucial role in a variety of physiological functions. Indeed hIK1 plays a seminal role in modulating Ca 2ϩ -dependent transepithelial ion transport (4, 5), has recently been confirmed to be the Gardos channel of red blood cells (6), and has been proposed to play a role in both vascular remodeling (7) and in modulating vascular tone (8). Because of these critical physiological roles, the ...

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“…As shown in Fig. 2, mutation of Leu 18 to Ala (L18A) had no significant effect on surface expression as assessed by both IF (Fig. 2A) and CS-IP (Fig.…”

Section: Role Of the Nh 2 Terminus In The Expression Trafficking Andmentioning

confidence: 83%

Section: Discussionmentioning

confidence: 99%

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Role of the NH2 Terminus in the Assembly and Trafficking of the Intermediate Conductance Ca2+-activated K+ Channel hIK1

Jones

Hamilton

Papworth

et al. 2004

Journal of Biological Chemistry

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“…ChTx is known to block Ca 2+ -activated KCa1.1 and KCa3.1 channels and specific Kv1.x channels (7,20). KCa3.1 channels are apamin-insensitive but have specific binding sites for ChTx and the clotrimazole-related compound TRAM-34 (9,21). TRAM-34 has been established as a selective KCa3.1 blocker with no effects on KCa1.1, KCa2.2, or a wide array of Kv channels (9,10) or Cav3 channels (Fig.…”

Section: Resultsmentioning

confidence: 99%

Intermediate conductance calcium-activated potassium channels modulate summation of parallel fiber input in cerebellar Purkinje cells

Engbers

Anderson²,

Asmara³

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

100

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Encoding sensory input requires the expression of postsynaptic ion channels to transform key features of afferent input to an appropriate pattern of spike output. Although Ca 2+ -activated K + channels are known to control spike frequency in central neurons, Ca 2+ -activated K + channels of intermediate conductance (KCa3.1) are believed to be restricted to peripheral neurons. We now report that cerebellar Purkinje cells express KCa3.1 channels, as evidenced through single-cell RT-PCR, immunocytochemistry, pharmacology, and single-channel recordings. Furthermore, KCa3.1 channels coimmunoprecipitate and interact with low voltage-activated Cav3.2 Ca 2+ channels at the nanodomain level to support a previously undescribed transient voltage-and Ca 2+ -dependent current. As a result, subthreshold parallel fiber excitatory postsynaptic potentials (EPSPs) activate Cav3 Ca 2+ influx to trigger a KCa3.1-mediated regulation of the EPSP and subsequent after-hyperpolarization. The Cav3-KCa3.1 complex provides powerful control over temporal summation of EPSPs, effectively suppressing low frequencies of parallel fiber input. KCa3.1 channels thus contribute to a high-pass filter that allows Purkinje cells to respond preferentially to high-frequency parallel fiber bursts characteristic of sensory input.C entral neurons receive an enormous number of spontaneously active synaptic inputs, but exhibit the capacity to differentiate features of sensory input from background noise. Cerebellar Purkinje cells are contacted by up to ∼150,000 parallel fibers from granule cells, of which only a subset will convey sensory information at any given time. The activation of a peripheral receptive field is transmitted to the cerebellar cortex by mossy fibers in the form of high-frequency spike bursts (1). The resulting temporal summation of excitatory postsynaptic potentials (EPSPs) generates a similar high-frequency burst in granule cells (2). Purkinje cells should then also possess the means to respond effectively to bursts of parallel fiber input that convey sensory information compared with background activity.Postsynaptic membrane excitability can be controlled by activation of K + channels. There are two established types of Ca 2+ -activated K + (KCa) channels in CNS neurons: small conductance (SK, KCa2.x) and big conductance (BK, KCa1.1) (3, 4). A third class of intermediate conductance (KCa3.1, SK4, IK1) KCa channel is thought to be expressed only in microglia and endothelial cells in the CNS (3, 5, 6). KCa3.1 channels are gated by calmodulin in a similar manner to KCa2.x channels but are insensitive to block by apamin and tetraethylammonium (TEA) (6-8). Instead, the KCa3.1 α-subunit, encoded by the gene KCNN4, has specific residues that bind charybdotoxin and 1-[(2-chlorophenyl) diphenylmethyl]-1H-pyrazole (TRAM-34) (5-7, 9, 10).In cerebellar Purkinje cells, KCa1.1 and KCa2.2 channels are activated during a spike by high voltage-activated (HVA) P-type Ca 2+ channels (11). In contrast, low voltage-activated (LVA) Cav3 (T-type) Ca 2+ channe...

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“…It is noteworthy that K Ca 3.1 inhibitors like TRAM-34 act at the equivalently positioned residues in K Ca 3.1, which questions whether these also act by "shutting the gate" or -as hitherto assumed -by "blocking ion flow." 78 However, the inhibition by TRAM-34 shows no clear dependence on intracellular Ca 2C (or degree of activation) and the effect cannot be "reversed" by positive modulators. 76 In contrast to NS8593, Bu-TPMF, the K Ca 2.1 selective negative gating modulator, interacts with a site lower down in the inner pore vestibule in TM5 (Fig.…”

Section: Negative Modulation Of K Ca 2 Channelsmentioning

confidence: 99%

Pharmacological gating modulation of small- and intermediate-conductance Ca²⁺-activated K⁺channels (K_Ca2.x and K_Ca3.1)

Christophersen

Wulff

2015

Channels

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Delineation of the Clotrimazole/TRAM-34 Binding Site on the Intermediate Conductance Calcium-activated Potassium Channel, IKCa1

Cited by 138 publications

References 23 publications

Role of the NH2 Terminus in the Assembly and Trafficking of the Intermediate Conductance Ca2+-activated K+ Channel hIK1

Role of the NH2 Terminus in the Assembly and Trafficking of the Intermediate Conductance Ca2+-activated K+ Channel hIK1

Intermediate conductance calcium-activated potassium channels modulate summation of parallel fiber input in cerebellar Purkinje cells

Pharmacological gating modulation of small- and intermediate-conductance Ca²⁺-activated K⁺channels (K_Ca2.x and K_Ca3.1)

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Delineation of the Clotrimazole/TRAM-34 Binding Site on the Intermediate Conductance Calcium-activated Potassium Channel, IKCa1

Cited by 138 publications

References 23 publications

Role of the NH2 Terminus in the Assembly and Trafficking of the Intermediate Conductance Ca2+-activated K+ Channel hIK1

Role of the NH2 Terminus in the Assembly and Trafficking of the Intermediate Conductance Ca2+-activated K+ Channel hIK1

Intermediate conductance calcium-activated potassium channels modulate summation of parallel fiber input in cerebellar Purkinje cells

Pharmacological gating modulation of small- and intermediate-conductance Ca2+-activated K+channels (KCa2.x and KCa3.1)

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Pharmacological gating modulation of small- and intermediate-conductance Ca²⁺-activated K⁺channels (K_Ca2.x and K_Ca3.1)