The inactivating K+ current in GH3 pituitary cells and its modification by chemical reagents

G. S. Oxford, P. K. Wagoner

Research output: Contribution to journalArticle

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Abstract

Whole-cell and single-channel recording techniques were applied to the study of the permeability and gating of inactivating K+ channels from clonal pituitary cells. The cation selectivity sequence (measured from reversal potentials) for the channels underlying the inactivating K+ current was Tl+ > K+ > Rb+ > NH4+. The conductance sequence (determined from current amplitudes) was K+ = Tl+ > Rb+ > NH4+. The inactivating current (I(K(i))) which was blocked by 4-aminopyridine (4-AP), activated at voltages more positive than -40 mV and half-inactivated at that voltage. Inactivation proceeded as the sum of two exponentials with mean time constants of 21 and 82 ms. Deactivation followed a single-exponential time course. Recovery from inactivation was slow, voltage dependent and multi-exponential, taking more than 50 s near the cell's resting potential. The magnitudes of outward current and of slope conductance increased as the concentration of external K+ was increased. On-cell and outside-out membrane patches revealed minicurrents with gating and pharmacological properties identical to whole-cell currents. Single channels with inactivating characteristics, while rarely observed, had an average slope conductance of 6-8 pS. Intracellular application of the disulphonic stilbene derivative, SITS, and the protein-modifying reagent, N-bromoacetamide (NBA), at concentrations of 0.2-1 mM for several tens of minutes dramatically slowed the decay (inactivation) of K+ currents and caused coincident increases in the magnitude of outward K(KI(i)). Extracellular application of NBA at much lower concentrations (1-100 μM) and much shorter exposure times (1-30 s) also slowed inactivation. This effect was reversible for brief applications at low doses, but became irreversible after longer exposures. Both internal and external NBA shifted the steady-state inactivation-voltage relation by +10 mV and reduced inactivation at voltages more positive than 0 mV. The efficacy of external NBA was independent of holding potential between -80 and 0 mV. Potassium minicurrents and single channels recorded from on-cell membrane patches were not affected by application of NBA to the extrapatch membrane. In contrast, NBA reversibly slowed the decay, increased the magnitude of minicurrents and prolonged the open times of single K+ channels recorded from outside-out patches. The single-channel conductance was unchanged by NBA. External NBA effects were mimicked by N-bromosuccinimide and N-chlorosuccinimide, but not by 2,4,6-trinitrobenzenesulphonic acid (TNBS), dithiothreitol (DTT), N-ethylmaleimide (NEM), N-methylacetamide (NMA), iodoacetamide (IAA) or acetamide suggesting that a halogenation reaction underlies the effects on I(K(i)).

Original languageEnglish (US)
Pages (from-to)587-612
Number of pages26
JournalJournal of Physiology
Volume410
StatePublished - 1989
Externally publishedYes

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4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid
Trinitrobenzenesulfonic Acid
Bromosuccinimide
Iodoacetamide
Stilbenes
4-Aminopyridine
Ethylmaleimide
Membranes
N-bromoacetamide
Dithiothreitol
Halogenation
Membrane Potentials
Cations
Permeability
Potassium
Cell Membrane
Pharmacology
Proteins
acetamide
N-methylacetamide

ASJC Scopus subject areas

  • Physiology

Cite this

The inactivating K+ current in GH3 pituitary cells and its modification by chemical reagents. / Oxford, G. S.; Wagoner, P. K.

In: Journal of Physiology, Vol. 410, 1989, p. 587-612.

Research output: Contribution to journalArticle

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abstract = "Whole-cell and single-channel recording techniques were applied to the study of the permeability and gating of inactivating K+ channels from clonal pituitary cells. The cation selectivity sequence (measured from reversal potentials) for the channels underlying the inactivating K+ current was Tl+ > K+ > Rb+ > NH4+. The conductance sequence (determined from current amplitudes) was K+ = Tl+ > Rb+ > NH4+. The inactivating current (I(K(i))) which was blocked by 4-aminopyridine (4-AP), activated at voltages more positive than -40 mV and half-inactivated at that voltage. Inactivation proceeded as the sum of two exponentials with mean time constants of 21 and 82 ms. Deactivation followed a single-exponential time course. Recovery from inactivation was slow, voltage dependent and multi-exponential, taking more than 50 s near the cell's resting potential. The magnitudes of outward current and of slope conductance increased as the concentration of external K+ was increased. On-cell and outside-out membrane patches revealed minicurrents with gating and pharmacological properties identical to whole-cell currents. Single channels with inactivating characteristics, while rarely observed, had an average slope conductance of 6-8 pS. Intracellular application of the disulphonic stilbene derivative, SITS, and the protein-modifying reagent, N-bromoacetamide (NBA), at concentrations of 0.2-1 mM for several tens of minutes dramatically slowed the decay (inactivation) of K+ currents and caused coincident increases in the magnitude of outward K(KI(i)). Extracellular application of NBA at much lower concentrations (1-100 μM) and much shorter exposure times (1-30 s) also slowed inactivation. This effect was reversible for brief applications at low doses, but became irreversible after longer exposures. Both internal and external NBA shifted the steady-state inactivation-voltage relation by +10 mV and reduced inactivation at voltages more positive than 0 mV. The efficacy of external NBA was independent of holding potential between -80 and 0 mV. Potassium minicurrents and single channels recorded from on-cell membrane patches were not affected by application of NBA to the extrapatch membrane. In contrast, NBA reversibly slowed the decay, increased the magnitude of minicurrents and prolonged the open times of single K+ channels recorded from outside-out patches. The single-channel conductance was unchanged by NBA. External NBA effects were mimicked by N-bromosuccinimide and N-chlorosuccinimide, but not by 2,4,6-trinitrobenzenesulphonic acid (TNBS), dithiothreitol (DTT), N-ethylmaleimide (NEM), N-methylacetamide (NMA), iodoacetamide (IAA) or acetamide suggesting that a halogenation reaction underlies the effects on I(K(i)).",
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