Molecular and functional identification of cyclic AMP-sensitive BKCa potassium channels (ZERO variant) and L-type voltage-dependent calcium channels in single rat juxtaglomerular cells

Ulla G Friis, Finn Jørgensen, Ditte Andreasen, Boye L Jensen, Ole Skøtt

Research output: Contribution to journalJournal articleResearchpeer-review

Abstract

This study aimed at identifying the type and functional significance of potassium channels and voltage-dependent calcium channels (Ca(v)) in single rat JG cells using whole-cell patch clamp. Single JG cells displayed outward rectification at positive membrane potentials and limited net currents between -60 and -10 mV. Blockade of K+ channels with TEA inhibited 83% of the current at +105 mV. Inhibition of KV channels with 4-AP inhibited 21% of the current. Blockade of calcium-sensitive voltage-gated K+ channels (BKCa) with charybdotoxin or iberiotoxin inhibited 89% and 82% of the current, respectively. Double immunofluorescence confirmed the presence of BKCa and renin in the same cell. cAMP increased the outward current by 1.6-fold, and this was inhibited by 74% with iberiotoxin. Expression of the cAMP-sensitive splice variant (ZERO) of BKCa was confirmed in single-sampled JG cells by RT-PCR. The resting membrane potential of JG cells was -32 mV and activation of BKCa with cAMP hyperpolarized cells on average 16 mV, and inhibition with TEA depolarized cells by 17 mV. The cells displayed typical high-voltage activated calcium currents sensitive to the L-type Ca(v) blocker calciseptine. RT-PCR analysis and double-immunofluorescence labeling showed coexpression of renin and L-type Ca(v) 1.2. The cAMP-mediated increase in exocytosis (measured as membrane capacitance) was inhibited by depolarization to +10 mV, and this inhibitory effect was blocked with calciseptine, whereas K+-blockers had no effect. We conclude that JG cells express functional cAMP-sensitive BKCa channels (the ZERO splice variant) and voltage-dependent L-type Ca2+ channels.

Original languageEnglish
JournalCirculation
Volume93
Issue number3
Pages (from-to)213-20
Number of pages8
ISSN0009-7322
DOIs
Publication statusPublished - 8. Aug 2003

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L-Type Calcium Channels
Renin
Charybdotoxin
Polymerase Chain Reaction
Exocytosis

Keywords

  • Alternative Splicing
  • Animals
  • Calcium
  • Calcium Channels, L-Type
  • Cell Membrane
  • Cells, Cultured
  • Cyclic AMP
  • Electric Capacitance
  • Ion Channel Gating
  • Juxtaglomerular Apparatus
  • Large-Conductance Calcium-Activated Potassium Channels
  • Male
  • Patch-Clamp Techniques
  • Potassium
  • Potassium Channel Blockers
  • Potassium Channels, Calcium-Activated
  • Rats
  • Rats, Sprague-Dawley
  • Renin
  • Sodium
  • Journal Article
  • Research Support, Non-U.S. Gov't

Cite this

@article{7438f8d796324a28bc23fe02d754ed43,
title = "Molecular and functional identification of cyclic AMP-sensitive BKCa potassium channels (ZERO variant) and L-type voltage-dependent calcium channels in single rat juxtaglomerular cells",
abstract = "This study aimed at identifying the type and functional significance of potassium channels and voltage-dependent calcium channels (Ca(v)) in single rat JG cells using whole-cell patch clamp. Single JG cells displayed outward rectification at positive membrane potentials and limited net currents between -60 and -10 mV. Blockade of K+ channels with TEA inhibited 83{\%} of the current at +105 mV. Inhibition of KV channels with 4-AP inhibited 21{\%} of the current. Blockade of calcium-sensitive voltage-gated K+ channels (BKCa) with charybdotoxin or iberiotoxin inhibited 89{\%} and 82{\%} of the current, respectively. Double immunofluorescence confirmed the presence of BKCa and renin in the same cell. cAMP increased the outward current by 1.6-fold, and this was inhibited by 74{\%} with iberiotoxin. Expression of the cAMP-sensitive splice variant (ZERO) of BKCa was confirmed in single-sampled JG cells by RT-PCR. The resting membrane potential of JG cells was -32 mV and activation of BKCa with cAMP hyperpolarized cells on average 16 mV, and inhibition with TEA depolarized cells by 17 mV. The cells displayed typical high-voltage activated calcium currents sensitive to the L-type Ca(v) blocker calciseptine. RT-PCR analysis and double-immunofluorescence labeling showed coexpression of renin and L-type Ca(v) 1.2. The cAMP-mediated increase in exocytosis (measured as membrane capacitance) was inhibited by depolarization to +10 mV, and this inhibitory effect was blocked with calciseptine, whereas K+-blockers had no effect. We conclude that JG cells express functional cAMP-sensitive BKCa channels (the ZERO splice variant) and voltage-dependent L-type Ca2+ channels.",
keywords = "Alternative Splicing, Animals, Calcium, Calcium Channels, L-Type, Cell Membrane, Cells, Cultured, Cyclic AMP, Electric Capacitance, Ion Channel Gating, Juxtaglomerular Apparatus, Large-Conductance Calcium-Activated Potassium Channels, Male, Patch-Clamp Techniques, Potassium, Potassium Channel Blockers, Potassium Channels, Calcium-Activated, Rats, Rats, Sprague-Dawley, Renin, Sodium, Journal Article, Research Support, Non-U.S. Gov't",
author = "Friis, {Ulla G} and Finn J{\o}rgensen and Ditte Andreasen and Jensen, {Boye L} and Ole Sk{\o}tt",
year = "2003",
month = "8",
day = "8",
doi = "10.1161/01.RES.0000085041.70276.3D",
language = "English",
volume = "93",
pages = "213--20",
journal = "Circulation",
issn = "0009-7322",
publisher = "Lippincott Williams & Wilkins",
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Molecular and functional identification of cyclic AMP-sensitive BKCa potassium channels (ZERO variant) and L-type voltage-dependent calcium channels in single rat juxtaglomerular cells. / Friis, Ulla G; Jørgensen, Finn; Andreasen, Ditte; Jensen, Boye L; Skøtt, Ole.

In: Circulation, Vol. 93, No. 3, 08.08.2003, p. 213-20.

Research output: Contribution to journalJournal articleResearchpeer-review

TY - JOUR

T1 - Molecular and functional identification of cyclic AMP-sensitive BKCa potassium channels (ZERO variant) and L-type voltage-dependent calcium channels in single rat juxtaglomerular cells

AU - Friis, Ulla G

AU - Jørgensen, Finn

AU - Andreasen, Ditte

AU - Jensen, Boye L

AU - Skøtt, Ole

PY - 2003/8/8

Y1 - 2003/8/8

N2 - This study aimed at identifying the type and functional significance of potassium channels and voltage-dependent calcium channels (Ca(v)) in single rat JG cells using whole-cell patch clamp. Single JG cells displayed outward rectification at positive membrane potentials and limited net currents between -60 and -10 mV. Blockade of K+ channels with TEA inhibited 83% of the current at +105 mV. Inhibition of KV channels with 4-AP inhibited 21% of the current. Blockade of calcium-sensitive voltage-gated K+ channels (BKCa) with charybdotoxin or iberiotoxin inhibited 89% and 82% of the current, respectively. Double immunofluorescence confirmed the presence of BKCa and renin in the same cell. cAMP increased the outward current by 1.6-fold, and this was inhibited by 74% with iberiotoxin. Expression of the cAMP-sensitive splice variant (ZERO) of BKCa was confirmed in single-sampled JG cells by RT-PCR. The resting membrane potential of JG cells was -32 mV and activation of BKCa with cAMP hyperpolarized cells on average 16 mV, and inhibition with TEA depolarized cells by 17 mV. The cells displayed typical high-voltage activated calcium currents sensitive to the L-type Ca(v) blocker calciseptine. RT-PCR analysis and double-immunofluorescence labeling showed coexpression of renin and L-type Ca(v) 1.2. The cAMP-mediated increase in exocytosis (measured as membrane capacitance) was inhibited by depolarization to +10 mV, and this inhibitory effect was blocked with calciseptine, whereas K+-blockers had no effect. We conclude that JG cells express functional cAMP-sensitive BKCa channels (the ZERO splice variant) and voltage-dependent L-type Ca2+ channels.

AB - This study aimed at identifying the type and functional significance of potassium channels and voltage-dependent calcium channels (Ca(v)) in single rat JG cells using whole-cell patch clamp. Single JG cells displayed outward rectification at positive membrane potentials and limited net currents between -60 and -10 mV. Blockade of K+ channels with TEA inhibited 83% of the current at +105 mV. Inhibition of KV channels with 4-AP inhibited 21% of the current. Blockade of calcium-sensitive voltage-gated K+ channels (BKCa) with charybdotoxin or iberiotoxin inhibited 89% and 82% of the current, respectively. Double immunofluorescence confirmed the presence of BKCa and renin in the same cell. cAMP increased the outward current by 1.6-fold, and this was inhibited by 74% with iberiotoxin. Expression of the cAMP-sensitive splice variant (ZERO) of BKCa was confirmed in single-sampled JG cells by RT-PCR. The resting membrane potential of JG cells was -32 mV and activation of BKCa with cAMP hyperpolarized cells on average 16 mV, and inhibition with TEA depolarized cells by 17 mV. The cells displayed typical high-voltage activated calcium currents sensitive to the L-type Ca(v) blocker calciseptine. RT-PCR analysis and double-immunofluorescence labeling showed coexpression of renin and L-type Ca(v) 1.2. The cAMP-mediated increase in exocytosis (measured as membrane capacitance) was inhibited by depolarization to +10 mV, and this inhibitory effect was blocked with calciseptine, whereas K+-blockers had no effect. We conclude that JG cells express functional cAMP-sensitive BKCa channels (the ZERO splice variant) and voltage-dependent L-type Ca2+ channels.

KW - Alternative Splicing

KW - Animals

KW - Calcium

KW - Calcium Channels, L-Type

KW - Cell Membrane

KW - Cells, Cultured

KW - Cyclic AMP

KW - Electric Capacitance

KW - Ion Channel Gating

KW - Juxtaglomerular Apparatus

KW - Large-Conductance Calcium-Activated Potassium Channels

KW - Male

KW - Patch-Clamp Techniques

KW - Potassium

KW - Potassium Channel Blockers

KW - Potassium Channels, Calcium-Activated

KW - Rats

KW - Rats, Sprague-Dawley

KW - Renin

KW - Sodium

KW - Journal Article

KW - Research Support, Non-U.S. Gov't

U2 - 10.1161/01.RES.0000085041.70276.3D

DO - 10.1161/01.RES.0000085041.70276.3D

M3 - Journal article

VL - 93

SP - 213

EP - 220

JO - Circulation

JF - Circulation

SN - 0009-7322

IS - 3

ER -