Elevated Potassium Shortens Action Potential Duration by Altering Outward Currents in Chick Dorsal Root Ganglia Neurons Jay Yangl9**and Charles F. Z~rurnski*’~ Departments of ‘Anesthesiology, *Anatomy & Neurobiology, and 3Psychiatry, Washington University School of Medicine, St. Louis, MO 631 10, USA
SUMMARY Dissociated embryonic chick dorsal root ganglion (DRG) neurons maintained in culture exhibit a mixed N a + / C a 2 + action potential. T h e characteristic “shoulder” on the repolarizing phase is due to the relatively prolonged inward Ca” current. DRG neurons grown in an elevated K + medium (25 versus. 5 m M ) lack the plateau phase of the action potential. Voltage-clamp analysis showed that this plastic change in action poten-
tial duration is not due to the loss of the inward CaZ+ current but is partly due to the appearance of a Ca2+-dependent, 4-aminopyridine-(4-AP)-sensitive transient outward current. Faster activation of the purely voltagedependent delayed rectifier outward current also contributes to the rapid repolarization observed in neurons cultured in elevated K + medium.
INTRODUCTION
Similar modulation of mouse and chick DRG action potential duration has been well documented also. Murine DRG maintained in vitrn with low doses of nerve growth factor (NGF) exhibit marked shortening of action potential duration (Chalazonitis, Peterson, and Crain, I987), and cultured chick DRG neurons show a dramatic shortening of the action potential duration in response to elevated K + in the culture medium (Chalazonitis & Fischbach, 1980). The biophysical basis for the K+-induced change in action potential duration is not known. In this paper, we use the whole-cell patch-clamp technique to investigate the mechanisms of K’induced action potential shortening in cultured chick DRG neurons. A preliminary report of this work has been published (Yang, Zorumski, and Fischbach, 1986).
Early studies by Spitzer & Baccaglini ( 1976j demonstrated a developmental regulation of voltagegated ionic channels. In Xenopus Rohon beard cells, the early and largely Ca2+-dependentaction potential initially exhibits a characteristically slow upstroke. Over time this gives way to a predominantly Na’ -dependent action potential with a typical rapid upstroke. This initial observation of developmental regulation in the timing of neuronal voltage-gated ionic channel appearance has been extended to in vitro preparations of cultured neurons (Spitzer, 1983). Recent voltage-clamp studies of cultured amphibian neurons suggest both an increase in the density and a faster kinetic of K’ -dependent repolarizing currents as the mechanism underlying the developmental switch in action potential duration (Barish, 1986; O’Dowd, Ribera, and Spitzer, 1988j . Received November 2, 1989; accepted January 24, 1990 Journal of Neurobiology, Vol. 2 1. No. 4. pp. 66 1-67 1 (1990) 0 1990 John Wiley & Sons, Inc. CCC 0022-3034/90/04066 1-1 1$04.00 * To whom correspondence should be addressed.
METHODS Cell Culture DRG were dissected from lumbar regions of 9- 1O-dayold chick embryos. Isolated DRG were incubated in
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TABLE 1 Composition of External and Internal Solutions lised to Record Action Potentials in Current Clamp and to Separate Ionic Currents Under Voltage Clamp
NaCl KCI CSCl TEA-CI CaCI* MgCh EGTA HEPES Glucose pH titration to 7.4 with (1 M solution)
Std Ext
Std Int
Cs-TEA Int
K-EGTA Int
143 4.8 0 0 5 .O 0 0
4.0 0 120 20 1 .o 2.0 10 10 0
4.0 0 0 0 1.o 2.0 140
10 10
4.0 140 0 0 1 .0 2.0 10 10 0
NaOH
KOH
CsOH
KOH
10
0
Note: All concentrations in mM unless otherwise noted. Ext = external; Int = internal; Std = standard. Throughout thc text, the specific compositions of solutions for any particular experiment are described as externaljinternal with modifications from the above standard solutions noted: Action potential (std ext/std int), total transmembrane current (std ext/std int). CaZ+current (std ext + 1 pMTTX/Cs-TEA int), total outward current (std ext 1 pMTTX/std int), and Cazc-independent outward current (std ext + 1 pM TTX 0.5 mM Cd2+/stdint).
+
Ca2' /Mg2+-free Pucks solution with 0.01% trypsin for 30 min at 37"C, centrifuged, and resuspended in culture media. Neurons were dissociated by trituration, filtered through a double layer of course lens paper, and plated on a collagen-coated tissue culture dish at an approximate density of 4 ganglia/35 m m dish (20,000 neurons/ml). Culture medium consisted of Eagles Minimal Essential Media supplemented with ( v / v ) 10% heat-inactivated horse serum, 5% chick embryo extract, 50 units/ml penicillin and 50 pg/ml streptomycin. Exogenous nerve growth factor other than that in chick extract, was not necessary for short-term (
SUMMARY Dissociated embryonic chick dorsal root ganglion (DRG) neurons maintained in culture exhibit a mixed N a + / C a 2 + action potential. T h e characteristic “shoulder” on the repolarizing phase is due to the relatively prolonged inward Ca” current. DRG neurons grown in an elevated K + medium (25 versus. 5 m M ) lack the plateau phase of the action potential. Voltage-clamp analysis showed that this plastic change in action poten-
tial duration is not due to the loss of the inward CaZ+ current but is partly due to the appearance of a Ca2+-dependent, 4-aminopyridine-(4-AP)-sensitive transient outward current. Faster activation of the purely voltagedependent delayed rectifier outward current also contributes to the rapid repolarization observed in neurons cultured in elevated K + medium.
INTRODUCTION
Similar modulation of mouse and chick DRG action potential duration has been well documented also. Murine DRG maintained in vitrn with low doses of nerve growth factor (NGF) exhibit marked shortening of action potential duration (Chalazonitis, Peterson, and Crain, I987), and cultured chick DRG neurons show a dramatic shortening of the action potential duration in response to elevated K + in the culture medium (Chalazonitis & Fischbach, 1980). The biophysical basis for the K+-induced change in action potential duration is not known. In this paper, we use the whole-cell patch-clamp technique to investigate the mechanisms of K’induced action potential shortening in cultured chick DRG neurons. A preliminary report of this work has been published (Yang, Zorumski, and Fischbach, 1986).
Early studies by Spitzer & Baccaglini ( 1976j demonstrated a developmental regulation of voltagegated ionic channels. In Xenopus Rohon beard cells, the early and largely Ca2+-dependentaction potential initially exhibits a characteristically slow upstroke. Over time this gives way to a predominantly Na’ -dependent action potential with a typical rapid upstroke. This initial observation of developmental regulation in the timing of neuronal voltage-gated ionic channel appearance has been extended to in vitro preparations of cultured neurons (Spitzer, 1983). Recent voltage-clamp studies of cultured amphibian neurons suggest both an increase in the density and a faster kinetic of K’ -dependent repolarizing currents as the mechanism underlying the developmental switch in action potential duration (Barish, 1986; O’Dowd, Ribera, and Spitzer, 1988j . Received November 2, 1989; accepted January 24, 1990 Journal of Neurobiology, Vol. 2 1. No. 4. pp. 66 1-67 1 (1990) 0 1990 John Wiley & Sons, Inc. CCC 0022-3034/90/04066 1-1 1$04.00 * To whom correspondence should be addressed.
METHODS Cell Culture DRG were dissected from lumbar regions of 9- 1O-dayold chick embryos. Isolated DRG were incubated in
661
662
Yang and Zormitski
TABLE 1 Composition of External and Internal Solutions lised to Record Action Potentials in Current Clamp and to Separate Ionic Currents Under Voltage Clamp
NaCl KCI CSCl TEA-CI CaCI* MgCh EGTA HEPES Glucose pH titration to 7.4 with (1 M solution)
Std Ext
Std Int
Cs-TEA Int
K-EGTA Int
143 4.8 0 0 5 .O 0 0
4.0 0 120 20 1 .o 2.0 10 10 0
4.0 0 0 0 1.o 2.0 140
10 10
4.0 140 0 0 1 .0 2.0 10 10 0
NaOH
KOH
CsOH
KOH
10
0
Note: All concentrations in mM unless otherwise noted. Ext = external; Int = internal; Std = standard. Throughout thc text, the specific compositions of solutions for any particular experiment are described as externaljinternal with modifications from the above standard solutions noted: Action potential (std ext/std int), total transmembrane current (std ext/std int). CaZ+current (std ext + 1 pMTTX/Cs-TEA int), total outward current (std ext 1 pMTTX/std int), and Cazc-independent outward current (std ext + 1 pM TTX 0.5 mM Cd2+/stdint).
+
Ca2' /Mg2+-free Pucks solution with 0.01% trypsin for 30 min at 37"C, centrifuged, and resuspended in culture media. Neurons were dissociated by trituration, filtered through a double layer of course lens paper, and plated on a collagen-coated tissue culture dish at an approximate density of 4 ganglia/35 m m dish (20,000 neurons/ml). Culture medium consisted of Eagles Minimal Essential Media supplemented with ( v / v ) 10% heat-inactivated horse serum, 5% chick embryo extract, 50 units/ml penicillin and 50 pg/ml streptomycin. Exogenous nerve growth factor other than that in chick extract, was not necessary for short-term (
