Showing items related by title, author, creator and subject.

• Rat supraoptic magnocellular neurones show distinct large conductance, Ca2+-activated K+ channel subtypes in cell bodies versus nerve endings

  Dopico, Alejandro M.; Widmer, Helene; Wang, Gang; Lemos, Jose R.; Treistman, Steven N. (1999-08-05)

  1. Large conductance, Ca2+-activated K+ (BK) channels were identified in freshly dissociated rat supraoptic neurones using patch clamp techniques. 2. The single channel conductance of cell body BK channels, recorded from inside-out patches in symmetric 145 mM K+, was 246.1 pS, compared with 213 pS in nerve ending BK channels (P1.53 microM for the neurohypophysial channel, indicating the higher Ca2+ sensitivity of the cell body isochannel. 5. Cell body BK channels showed fast kinetics (open time constant, 8.5 ms; fast closed time constant, 1.6 and slow closed time constant, 12.7 ms), identifying them as 'type I' isochannels, as opposed to the slow gating (type II) of neurohypophysial BK channels. 6. Cell body BK activity was reduced by 10 nM charybdotoxin (NPo, 37% of control), or 10 nM iberiotoxin (NPo, 5% of control), whereas neurohypophysial BK channels are insensitive to charybdotoxin at concentrations as high as 360 nM. 7. Whilst blockade of nerve ending BK channels markedly slowed the repolarization of evoked single spikes, blockade of cell body channels was without effect on repolarization of evoked single spikes. 8. Ethanol reversibly increased neurohypophysial BK channel activity (EC50, 22 mM; maximal effect, 100 mM). In contrast, ethanol (up to 100 mM) failed to increase cell body BK channel activity. 9. In conclusion, we have characterized BK channels in supraoptic neuronal cell bodies, and demonstrated that they display different electrophysiological and pharmacological properties from their counterparts in the nerve endings.
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  Dynamic Regulation at the Neuronal Plasma Membrane: Novel Endocytic Mechanisms Control Anesthetic-Activated Potassium Channels and Amphetamine-Sensitive Dopamine Transporters: A Dissertation

  Gabriel, Luke R. (2013-06-13)

  Endocytic trafficking dynamically regulates neuronal plasma membrane protein presentation and activity, and plays a central role in excitability and plasticity. Over the course of my dissertation research I investigated endocytic mechanisms regulating two neuronal membrane proteins: the anesthetic-activated potassium leak channel, KCNK3, as well as the psychostimulant-sensitive dopamine transporter (DAT). My results indicate that KCNK3 internalizes in response to Protein Kinase C (PKC) activation, using a novel pathway that requires the phosphoserine binding protein, 14-3-3β, and demonstrates for the first time regulated KCNK3 channel trafficking in neurons. Additionally, PKC-mediated KCNK3 trafficking requires a non-canonical endocytic motif, which is shared exclusively between KCNK3 and sodium-dependent neurotransmitter transporters, such as DAT. DAT trafficking studies in intact ex vivo adult striatal slices indicate that DAT endocytic trafficking has both dynamin-dependent and –independent components. Moreover, DAT segregates into two populations at the neuronal plasma membrane: trafficking-competent and -incompetent. Taken together, these results demonstrate that novel, non-classical endocytic mechanisms dynamically control the plasma membrane presentation of these two important neuronal proteins.
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  Structural and Functional Studies of the KCNQ1-KCNE K⁺ Channel Complex: A Dissertation

  Gage, Steven D. (2008-09-09)

  KCNQ1 is a homotetrameric voltage-gated potassium channel expressed in cardiomyocytes and epithelial tissues. However, currents arising from KCNQ1 have never been physiologically observed. KCNQ1 is able to provide the diverse potassium conductances required by these distinct cell types through coassembly with and modulation by type I transmembrane β-subunits of the KCNE gene family. KCNQ1-KCNE K+ channels play important physiological roles. In cardiac tissues the association of KCNQ1 with KCNE1 gives rise to IKs, the slow delayed outwardly rectifying potassium current. IKs is in part responsible for repolarizing heart muscle, and is therefore crucial in maintaining normal heart rhymicity. IKschannels help terminate each action potential and provide cardiac repolarization reserve. As such, mutations in either subunit can lead to Romano-Ward Syndrome or Jervell and Lange-Nielsen Syndrome, two forms of Q-T prolongation. In epithelial cells, KCNQ1-KCNE1, KCNQ1-KCNE2 and KCNQ1-KCNE3 give rise to potassium currents required for potassium recycling and secretion. These functions arise because the biophysical properties of KCNQ1 are always dramatically altered by KCNE co-expression. We wanted to understand how KCNE peptides are able to modulate KCNQ1. In Chapter II, we produce partial truncations of KCNE3 and demonstrate the transmembrane domain is necessary and sufficient for both assembly with and modulation of KCNQ1. Comparing these results with published results obtained from chimeric KCNE peptides and partial deletion mutants of KCNE1, we propose a bipartite modulation residing in KCNE peptides. Transmembrane modulation is either active (KCNE3) or permissive (KCNE1). Active transmembrane KCNE modulation masks juxtamembranous C-terminal modulation of KCNQ1, while permissive modulation allows C-terminal modulation of KCNQ1 to express. We test our hypothesis, and demonstrate C-terminal Long QT point mutants in KCNE1 can be masked by active trasnsmembrane modulation. Having confirmed the importance the C-terminus of KCNE1, we continue with two projects designed to elucidate KCNE1 C-terminal structure. In Chapter III we conduct an alanine-perturbation scan within the C-terminus. C-terminal KCNE1 alanine point mutations result in changes in the free energy for the KCNQ1-KCNE1 channel complex. High-impact point mutants cluster in an arrangement consistent with an alphahelical secondary structure, "kinked" by a single proline residue. In Chapter IV, we use oxidant-mediated disulfide bond formation between non-native cysteine residues to demonstrate amino acid side chains residing within the C-terminal domain of KCNE1 are close and juxtaposed to amino acid side chains on the cytoplasmic face of the KCNQ1 pore domain. Many of the amino acids identified as high impact through alanine perturbation correspond with residues identified as able to form disulfide bonds with KCNQ1. Taken together, we demonstrate that the interaction between the C-terminus of KCNE1 and the pore domain of KCNQ1 is required for the proper modulation of KCNQ1 by KCNE1, and by extension, normal IKs function and heart rhymicity.