Subunit stoichiometry of a mammalian K+ channel determined by construction of multimeric cDNAs

Liman, Emily R.; Tytgat, Jan; Hess, Peter

doi:10.1016/0896-6273(92)90239-a

Cited by 1,047 publications

(847 citation statements)

References 20 publications

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“…In other K ϩ channels, external TEA ions block in the pore at a site coordinated by the four subunits, with a blocking affinity in heteromeric channels that can be predicted by adding up the energy of interaction with each subunit (Heginbotham and MacKinnon, 1992;Kavanaugh et al, 1992;Liman et al, 1992). Thus, the dose-response relation for KCNQ2/KCNQ3 was nicely fit by the sum of five Hill equations, assuming the binomial distribution and energy additivity (Fig.…”

Section: Expression and Modulation Of Homomeric Kcnq2 And Kcnq3 Channelsmentioning

confidence: 98%

Reconstitution of Muscarinic Modulation of the KCNQ2/KCNQ3 K⁺Channels That Underlie the Neuronal M Current

Shapiro¹,

Roche²,

Kaftan³

et al. 2000

J. Neurosci.

199

244

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Channels from KCNQ2 and KCNQ3 genes have been suggested to underlie the neuronal M-type K ϩ current. The M current is modulated by muscarinic agonists via G-proteins and an unidentified diffusible cytoplasmic messenger. Using wholecell clamp, we studied tsA-201 cells in which cloned KCNQ2/ KCNQ3 channels were coexpressed with M 1 muscarinic receptors. Heteromeric KCNQ2/KCNQ3 currents were modulated by the muscarinic agonist oxotremorine-M (oxo-M) in a manner having all of the characteristics of modulation of native M current in sympathetic neurons. Oxo-M also produced obvious intracellular Ca 2ϩ transients, observed by using indo-1 fluorescence. However, modulation of the current remained strong even when Ca 2ϩ signals were abolished by the combined use of strong intracellular Ca 2ϩ buffers, an inhibitor of IP 3 receptors, and thapsigargin to deplete Ca 2ϩ stores. Muscarinic modulation was not blocked by staurosporine, a broad-spectrum protein kinase inhibitor, arguing against involvement of protein kinases. The modulation was not associated with a shift in the voltage dependence of channel activation. Homomeric KCNQ2 and KCNQ3 channels also expressed well and were modulated individually by oxo-M, suggesting that the motifs for modulation are present on both channel subtypes. Homomeric KCNQ2 and KCNQ3 currents were blocked, respectively, at very low and at high concentrations of tetraethylammonium ion. Finally, when KCNQ2 subunits were overexpressed by intranuclear DNA injection in sympathetic neurons, total M current was fully modulated by the endogenous neuronal muscarinic signaling mechanism. Our data further rule out Ca 2ϩ as the diffusible messenger. The reconstitution of muscarinic modulation of the M current that uses cloned components should facilitate the elucidation of the muscarinic signaling mechanism. Key words: K ϩ channel; muscarinic receptor; G-protein; calcium; patch clamp; M current A diverse family of neurotransmitters and hormones regulates Ca 2ϩ and K ϩ channels via G-protein-mediated signaling pathways (Wickman and Clapham, 1995;Brown et al., 1997;Dolphin, 1998). Nearly 20 years ago, several investigators gave the name M current to a noninactivating K ϩ current with slow kinetics in sympathetic neurons that is strongly suppressed by muscarinic acetylcholine receptor (mAChR) agonists (Brown and Adams, 1980;Constanti and Brown, 1981). The M current is thought to play an important role in neuronal excitability, and its suppression increases responses to excitatory synaptic inputs (Jones et al., 1995;Wang and McKinnon, 1995). Modulation of the M current by muscarinic receptor agonists and by angiotensin (Constanti and Brown, 1981; Marrion, 1997;Shapiro et al., 1994a) is mediated by a G-protein of the G q /11 class (Delmas et al., 1998; Haley et al., 1998) via a diffusible cytoplasmic second messenger (Selyanko et al., 1992) that is yet to be identified.Although the buffering of intracellular free Ca 2ϩ ([Ca 2ϩ ] i ) to very low levels prevents muscarinic suppression of the M current in rat sym...

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Section: Expression and Modulation Of Homomeric Kcnq2 And Kcnq3 Channelsmentioning

confidence: 98%

Reconstitution of Muscarinic Modulation of the KCNQ2/KCNQ3 K⁺Channels That Underlie the Neuronal M Current

Shapiro¹,

Roche²,

Kaftan³

et al. 2000

J. Neurosci.

199

244

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“…No. X68302) were obtained using an overlap polymerase chain reaction (40) and were cloned into the pGEM vector (41). For the different chimeras, fragments containing the nucleotides given in Table 1 were fused together.…”

Section: In Vitro Mutagenesis and Rna Synthesismentioning

confidence: 99%

Extracellular Linkers Completely Transplant the Voltage Dependence from Kv1.2 Ion Channels to Kv2.1

Elinder

Madeja

Zeberg

et al. 2016

Biophysical Journal

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The transmembrane voltage needed to open different voltage-gated K (Kv) channels differs by up to 50 mV from each other. In this study we test the hypothesis that the channels' voltage dependences to a large extent are set by charged amino-acid residues of the extracellular linkers of the Kv channels, which electrostatically affect the charged amino-acid residues of the voltage sensor S4. Extracellular cations shift the conductance-versus-voltage curve, G(V), by interfering with these extracellular charges. We have explored these issues by analyzing the effects of the divalent strontium ion (Sr 2þ ) on the voltage dependence of the G(V) curves of wild-type and chimeric Kv channels expressed in Xenopus oocytes, using the voltage-clamp technique. Out of seven Kv channels, Kv1.2 was found to be most sensitive to Sr 2þ (50 mM shifted G(V) by þ21.7 mV), and Kv2.1 to be the least sensitive (þ7.8 mV). Experiments on 25 chimeras, constructed from Kv1.2 and Kv2.1, showed that the large Sr 2þ -induced G(V) shift of Kv1.2 can be transferred to Kv2.1 by exchanging the extracellular linker between S3 and S4 (L3/4) in combination with either the extracellular linker between S5 and the pore (L5/P) or that between the pore and S6 (LP/6). The effects of the linker substitutions were nonadditive, suggesting specific structural interactions. The free energy of these interactions was~20 kJ/mol, suggesting involvement of hydrophobic interactions and/or hydrogen bonds. Using principles from double-layer theory we derived an approximate linear equation (relating the voltage shifts to altered ionic strength), which proved to well match experimental data, suggesting that Sr 2þ acts on these channels mainly by screening surface charges. Taken together, these results highlight the extracellular surface potential at the voltage sensor as an important determinant of the channels' voltage dependence, making the extracellular linkers essential targets for evolutionary selection.

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“…For expression studies, a9 and a10 rat nAChR subunits were subcloned into a modified pGEMHE vector (Liman et al 1992). Capped cRNAs were in vitro transcribed from linearized plasmid DNA templates using the mMessage mMachine T7 Transcription Kit (Ambion Corporation, Austin, TX).…”

Section: Expression Of Recombinant A9a10 Receptors In Xenopus Laevismentioning

confidence: 99%

Linopirdine Blocks α9α10-Containing Nicotinic Cholinergic Receptors of Cochlear Hair Cells

Gómez-Casati

Katz

Glowatzki

et al. 2004

JARO

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Studies of the electrophysiological response to acetylcholine (ACh) in mammalian outer hair cells (OHCs) are hindered by the presence of a large potassium current, I K,n , most likely mediated by channels containing the KCNQ4 subunit. Since I K,n can be blocked by linopirdine, cholinergic effects might be better revealed in the presence of this compound. The aim of the present work was to study the effects of linopirdine on the ACh-evoked responses through a9a10-containing native and recombinant nicotinic cholinergic receptors. Responses to ACh were blocked by linopirdine in both OHCs and inner hair cells (IHCs) of rats at postnatal days 21-27 (OHCs) and 9-11 (IHCs). In addition, linopirdine blocked responses of recombinant a9a10 nicotinic cholinergic receptors (nAChRs) in a concentrationdependent manner with an IC 50 of 5.2 lM. Block by linopirdine was readily reversible, voltage independent, and surmountable at high concentrations of ACh, thus suggestive of a competitive type of interaction with the receptor. The present results contribute to the pharmacological characterization of a9a10-containing nicotinic receptors and indicate that linopirdine should be used with caution when analyzing the cholinergic sensitivity of cochlear hair cells.

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Subunit stoichiometry of a mammalian K+ channel determined by construction of multimeric cDNAs

Cited by 1,047 publications

References 20 publications

Reconstitution of Muscarinic Modulation of the KCNQ2/KCNQ3 K⁺Channels That Underlie the Neuronal M Current

Reconstitution of Muscarinic Modulation of the KCNQ2/KCNQ3 K⁺Channels That Underlie the Neuronal M Current

Extracellular Linkers Completely Transplant the Voltage Dependence from Kv1.2 Ion Channels to Kv2.1

Linopirdine Blocks α9α10-Containing Nicotinic Cholinergic Receptors of Cochlear Hair Cells

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Subunit stoichiometry of a mammalian K+ channel determined by construction of multimeric cDNAs

Cited by 1,047 publications

References 20 publications

Reconstitution of Muscarinic Modulation of the KCNQ2/KCNQ3 K+Channels That Underlie the Neuronal M Current

Reconstitution of Muscarinic Modulation of the KCNQ2/KCNQ3 K+Channels That Underlie the Neuronal M Current

Extracellular Linkers Completely Transplant the Voltage Dependence from Kv1.2 Ion Channels to Kv2.1

Linopirdine Blocks α9α10-Containing Nicotinic Cholinergic Receptors of Cochlear Hair Cells

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Product

Resources

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Reconstitution of Muscarinic Modulation of the KCNQ2/KCNQ3 K⁺Channels That Underlie the Neuronal M Current

Reconstitution of Muscarinic Modulation of the KCNQ2/KCNQ3 K⁺Channels That Underlie the Neuronal M Current