Korean J Physiol Pharmacol.  2017 Mar;21(2):259-265. 10.4196/kjpp.2017.21.2.259.

Chronic Ca²⁺ influx through voltage-dependent Ca²⁺ channels enhance delayed rectifier K⁺ currents via activating Src family tyrosine kinase in rat hippocampal neurons

• Yang YS
• Jeon SC
• Kim DK
• Eun SY
• Jung SC

• KMID: 2371045
• DOI: http://doi.org/10.4196/kjpp.2017.21.2.259

Abstract

Excessive influx and the subsequent rapid cytosolic elevation of Ca²âº in neurons is the major cause to induce hyperexcitability and irreversible cell damage although it is an essential ion for cellular signalings. Therefore, most neurons exhibit several cellular mechanisms to homeostatically regulate cytosolic Ca²âº level in normal as well as pathological conditions. Delayed rectifier K⁺ channels (I(DR) channels) play a role to suppress membrane excitability by inducing K⁺ outflow in various conditions, indicating their potential role in preventing pathogenic conditions and cell damage under Ca²âº-mediated excitotoxic conditions. In the present study, we electrophysiologically evaluated the response of IDR channels to hyperexcitable conditions induced by high Ca²âº pretreatment (3.6 mM, for 24 hours) in cultured hippocampal neurons. In results, high Ca²âº-treatment significantly increased the amplitude of IDR without changes of gating kinetics. Nimodipine but not APV blocked Ca²âº-induced IDR enhancement, confirming that the change of I(DR) might be targeted by Ca²âº influx through voltage-dependent Ca²âº channels (VDCCs) rather than NMDA receptors (NMDARs). The VDCC-mediated I(DR) enhancement was not affected by either Ca²âº-induced Ca²âº release (CICR) or small conductance Ca²âº-activated K⁺ channels (SK channels). Furthermore, PP2 but not H89 completely abolished I(DR) enhancement under high Ca²âº condition, indicating that the activation of Src family tyrosine kinases (SFKs) is required for Ca²âº-mediated I(DR) enhancement. Thus, SFKs may be sensitive to excessive Ca²âº influx through VDCCs and enhance I(DR) to activate a neuroprotective mechanism against Ca²âº-mediated hyperexcitability in neurons.

Keyword

A-type K+ channels; Delayed rectifier K⁺ channel; NMDA receptors; Src family tyrosine kinase; Voltage-dependent Ca²⁺ channel

MeSH Terms

Animals
Calcium Channels
Cytosol
Humans
Kinetics
Membranes
Neurons*
Nimodipine
Protein-Tyrosine Kinases*
Rats*
Receptors, N-Methyl-D-Aspartate
src-Family Kinases
Tyrosine*
Calcium Channels
Nimodipine
Protein-Tyrosine Kinases
Receptors, N-Methyl-D-Aspartate
Tyrosine
src-Family Kinases

Figure

• 

  Fig. 1 IDR is enhanced under high Ca2+ condition in cultured hippocampal neurons, showing the dependence on VDCCs but not NMDARs.(A) The experimental protocol of voltage clamping to record IDR in hippocampal neurons and example traces of IDR. High Ca2+ condition was induced by doubling Ca2+ concentration (CaCl2, 3.6 mM) of culture media for 24 hours (High Ca2+) and then neurons were washed in normal ACSF during recording. Either APV (100 µM, APV+High Ca2+) or nimodipine (10 µM, Nimodipine + High Ca2+) was added to culture media for 24 hours under high Ca2+ condition. Scale bars 500 pA, 100 ms. (B) Individuals (circles) and averaged (square bars) densities of IDR. Currents were adjusted with whole cell capacitance for measuring current densities of each group. Error bars represent SEM.*p<0.05.

• 

  Fig. 2 The enhancement of IDR under high Ca2+ condition does not involve the significant alteration of activation properties of IDR channels.The activation property was measured at −60 to 80 mV with 20 steps after prepulse injection (−20 mV, 200 ms duration). (A) The experimental protocol of voltage clamping to measure the activation kinetics of IDR channels and example traces of IDR. The amplitude of IDR is increased by steps of voltage clamping. However, the constant rate of increment indicates no changes of IDR activating kinetics. Scale bars 500 pA, 100 ms. (B) Averaged densities of IDR at each clamping voltage (−40 to 80 mV). (C) and (D) Boltzmann fitted curves of gating kinetics of IDR activation and averaged values of voltage half (Vh, dotted line in C). The significant change of activation curves was not observed. Error bars represent SEM.

• 

  Fig. 3 SFKs are participated in IDR enhancement under high Ca2+ condition.(A) Example traces of IDR in each group. To confirm the contribution of SK channels, apamin (100 nM, Ca2++Apamin) was added to culture media. PP2 (1 µM) abolished the Ca2+-induced enhancement of IDR, indicating the involvement of SFK in IDR upregulation. (B) Individual (open circles) and averaged values (square bars) of IDR densities. Error bars represent SEM. *p<0.05 compared with control and †p<0.05 compared with High Ca2+. (C) Averaged densities of IDR at each clamping voltage (−40 to 80 mV). Error bars represent SEM.

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