Tetraethylammonium block of Slowpoke calcium-activated potassium channels expressed in Xenopus oocytes: Evidence for tetrameric channel formation

Ke-Zhong Shen, A. Lagrutta, N. W. Davies, N. B. Standen, John Adelman, R. A. North

Research output: Contribution to journalArticle

152 Citations (Scopus)

Abstract

Unitary currents were recorded from insideout membrane patches pulled from Xenopus oocytes that had been injected with RNA transcribed from a cDNA encoding the Drosophila maxi-K channel (Slowpoke). Site-directed mutagenesis was used to make cDNAs encoding channel subunits with single amino acid substitutions (Y308V and C309P). The extracellular side of the patch was exposed to tetraethylammonium (TEA) in the pipette solution; unitary currents in the presence of TEA were compared with currents in the absence of TEA to compute the inhibition. Amplitude distributions were fit by β functions to estimate the blocking and unblocking rate constants. For wild-type channels, TEA blocked with an apparent Kd of 80 μM at 0 mV and sensed 0.18 of the membrane electric field; the voltage dependence lay entirely in the blocking rate constant. TEA blocked currents through C309P channels with a similar affinity to wild-type at 0 mV, but this was not voltage-dependent. Currents through Y308V channels were very insensitive to any block by TEA; the apparent Kd at 0 mV was 26 mM and the blockade sensed 0.18 of the electric field. Oocytes injected with a mixture of RNAs encoding wild-type and Y308V channels showed unitary currents of four discrete amplitudes in the presence of 3 mM TEA; at 40 mV these corresponded to inhibitions of approximately 80%, 55%, 25% and 10%. These values agreed well with those expected for inhibition by TEA of currents through channels containing 3, 2, 1 and 0 tyrosine residues at the channel mouth, assuming that a tyrosine residue from each of four subunits contributes equally to the binding of the TEA ion. This indicates that Slowpoke channels form as tetramers.

Original languageEnglish (US)
Pages (from-to)440-445
Number of pages6
JournalPflügers Archiv European Journal of Physiology
Volume426
Issue number5
DOIs
StatePublished - Mar 1994

Fingerprint

Calcium-Activated Potassium Channels
Tetraethylammonium
Xenopus
Oocytes
Tyrosine
Rate constants
Complementary DNA
Electric fields
RNA
Large-Conductance Calcium-Activated Potassium Channels
Membranes
Mutagenesis
Electric potential
Amino Acid Substitution
Site-Directed Mutagenesis
Drosophila
Mouth
Substitution reactions
Amino Acids

Keywords

  • Calcium-activated channel
  • Channel blockers
  • Maxi-K channels
  • Potassium
  • Potassium channel
  • Slowpoke
  • Tetraethylammonium

ASJC Scopus subject areas

  • Physiology

Cite this

Tetraethylammonium block of Slowpoke calcium-activated potassium channels expressed in Xenopus oocytes : Evidence for tetrameric channel formation. / Shen, Ke-Zhong; Lagrutta, A.; Davies, N. W.; Standen, N. B.; Adelman, John; North, R. A.

In: Pflügers Archiv European Journal of Physiology, Vol. 426, No. 5, 03.1994, p. 440-445.

Research output: Contribution to journalArticle

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title = "Tetraethylammonium block of Slowpoke calcium-activated potassium channels expressed in Xenopus oocytes: Evidence for tetrameric channel formation",
abstract = "Unitary currents were recorded from insideout membrane patches pulled from Xenopus oocytes that had been injected with RNA transcribed from a cDNA encoding the Drosophila maxi-K channel (Slowpoke). Site-directed mutagenesis was used to make cDNAs encoding channel subunits with single amino acid substitutions (Y308V and C309P). The extracellular side of the patch was exposed to tetraethylammonium (TEA) in the pipette solution; unitary currents in the presence of TEA were compared with currents in the absence of TEA to compute the inhibition. Amplitude distributions were fit by β functions to estimate the blocking and unblocking rate constants. For wild-type channels, TEA blocked with an apparent Kd of 80 μM at 0 mV and sensed 0.18 of the membrane electric field; the voltage dependence lay entirely in the blocking rate constant. TEA blocked currents through C309P channels with a similar affinity to wild-type at 0 mV, but this was not voltage-dependent. Currents through Y308V channels were very insensitive to any block by TEA; the apparent Kd at 0 mV was 26 mM and the blockade sensed 0.18 of the electric field. Oocytes injected with a mixture of RNAs encoding wild-type and Y308V channels showed unitary currents of four discrete amplitudes in the presence of 3 mM TEA; at 40 mV these corresponded to inhibitions of approximately 80{\%}, 55{\%}, 25{\%} and 10{\%}. These values agreed well with those expected for inhibition by TEA of currents through channels containing 3, 2, 1 and 0 tyrosine residues at the channel mouth, assuming that a tyrosine residue from each of four subunits contributes equally to the binding of the TEA ion. This indicates that Slowpoke channels form as tetramers.",
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T2 - Evidence for tetrameric channel formation

AU - Shen, Ke-Zhong

AU - Lagrutta, A.

AU - Davies, N. W.

AU - Standen, N. B.

AU - Adelman, John

AU - North, R. A.

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