Korean J Physiol Pharmacol.  2012 Oct;16(5):343-348. 10.4196/kjpp.2012.16.5.343.

Sustained K+ Outward Currents are Sensitive to Intracellular Heteropodatoxin2 in CA1 Neurons of Organotypic Cultured Hippocampi of Rats

• Jung SC
• Eun SY

• KMID: 1493968
• DOI: http://doi.org/10.4196/kjpp.2012.16.5.343

Abstract

Blocking or regulating K+ channels is important for investigating neuronal functions in mammalian brains, because voltage-dependent K+ channels (Kv channels) play roles to regulate membrane excitabilities for synaptic and somatic processings in neurons. Although a number of toxins and chemicals are useful to change gating properties of Kv channels, specific effects of each toxin on a particular Kv subunit have not been sufficiently demonstrated in neurons yet. In this study, we tested electrophysiologically if heteropodatoxin2 (HpTX2), known as one of Kv4-specific toxins, might be effective on various K+ outward currents in CA1 neurons of organotypic hippocampal slices of rats. Using a nucleated-patch technique and a pre-pulse protocol in voltage-clamp mode, total K+ outward currents recorded in the soma of CA1 neurons were separated into two components, transient and sustained currents. The extracellular application of HpTX2 weakly but significantly reduced transient currents. However, when HpTX2 was added to internal solution, the significant reduction of amplitudes were observed in sustained currents but not in transient currents. This indicates the non-specificity of HpTX2 effects on Kv4 family. Compared with the effect of cytosolic 4-AP to block transient currents, it is possible that cytosolic HpTX2 is pharmacologically specific to sustained currents in CA1 neurons. These results suggest that distinctive actions of HpTX2 inside and outside of neurons are very efficient to selectively reduce specific K+ outward currents.

Keyword

CA1; Heteropodatoxin2; Sustained K+ current; Transient K+ current; Voltage-dependent K+ channel

MeSH Terms

Animals
Brain
Carisoprodol
Cytosol
Humans
Membranes
Neurons
Rats
Carisoprodol

Figure

• 

  Fig. 1 Extracellular HpTX2 reduces transient K+ outward currents in CA1 neurons. (A) The microscopic IR view of a nucleated-patch and a protocol of voltage-clamping to induce K+ outward currents. The scale bar inserted in the picture indicates 10 µm. Data acquired from experiments in which nucleated-patches were pulled within 2 min after making whole-cells, were used in this study. A pre-pulse (dotted line) to activate only sustained currents was applied for isolating transient currents from total outward currents. In all experiments, peaks of transient and sustained currents were generated at +60 mV holding potential. The time scale bar for voltage-clamping is 100 ms. (B) Example traces of transient and sustained currents recorded at +60 mV clamping in CA1 neurons. Currents were recorded at 10 min after pulling a nucleated-patch with (HpTX2) or without (control) HpTX2 in external solution. Scale bars indicate 0.2 nA and 100 ms. (C) All individual (circles) and averaged (square bars) amplitudes of transient (left panel) and sustained (right panel) currents are plotted for each group (control and HpTX2). Error bars represent standard error of mean (SEM). *: p<0.05, compared with control.

• 

  Fig. 2 Cytosolic HpTX2 reduces sustained but not transient K+ outward currents in CA1 neurons. (A) Example traces of both currents recorded immediately (0), 5, 10 and 15 min after pulling nucleated-patches. HpTX2 (200 nM) was added to internal pipette solution and normal external solution was perfused during recording. Scale bars indicate 0.2 nA and 100 ms. (B) Averaged peak amplitudes of transient (left panel) and sustained currents (right panel) recorded with intracellular 100 (gray circles) or 200 nM (dark circles) HpTX2. (C) Averaged current densities of transient and sustained currents measured at 0 min and 15 min after making nucleated-patches. The changes of current densities were acquired from data shown in (B) (100 nM: n=5, 200 nM: n=11). To calculate current density, the whole cell capacitance of 0 min was checked before recording currents, and that of 15 min was checked again right after completing the recording. Error bars represent SEM. **: p<0.01, compared with 0 min.

• 

  Fig. 3 Blocking effects of cytosolic 4-AP and Cs-gluconate internal solution on K+ outward currents. (A) Normalized individual and averaged values of peak amplitudes of transient and sustained currents recorded with 4-AP (3 mM) in internal pipette solution. (B) Normalized individual and averaged values of peak amplitude of transient and sustained currents recorded with Cs-gluconate internal solution. Error bars represent SEM. *: p<0.05 or **: p<0.01, compared with 0 min.

• 

  Fig. 4 Comparison of blocking effects on transient (gray bar) and sustained (open bar) currents induced by cytosolic drugs in hippocampal CA1 neurons. The reduction rate was calculated by the ratio of decreased amplitudes at 15 min after pulling nucleated-patches, compared with "0 min" peak amplitudes. Error bars represent SEM. *: p<0.05 or **: p<0.01, compared with "0 min" values shown in Fig. 2 and 3.

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