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Contribution of BK channels to action potential repolarisation at minimal cytosolic Ca2+ concentration in chromaffin cells

  • Ion Channels, Receptors and Transporters
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Abstract

BK channels modulate cell firing in excitable cells in a voltage-dependent manner regulated by fluctuations in free cytosolic Ca2+ during action potentials. Indeed, Ca2+-independent BK channel activity has ordinarily been considered not relevant for the physiological behaviour of excitable cells. We employed the patch-clamp technique and selective BK channel blockers to record K+ currents from bovine chromaffin cells at minimal intracellular (about 10 nM) and extracellular (free Ca2+) Ca2+ concentrations. Despite their low open probability under these conditions (V50 of +146.8 mV), BK channels were responsible for more than 25% of the total K+ efflux during the first millisecond of a step depolarisation to +20 mV. Moreover, BK channels activated about 30% faster (τ = 0.55 ms) than the rest of available K+ channels. The other main source of fast voltage-dependent K+ efflux at such a low Ca2+ was a transient K+ (IA-type) current activating with V 50 = −14.2 mV. We also studied the activation of BK currents in response to action potential waveforms and their contribution to shaping action potentials both in the presence and the absence of extracellular Ca2+. Our results show that BK channels activate during action potentials and accelerate cell repolarisation even at minimal Ca2+ concentration, and suggest that they could do so also in the presence of extracellular Ca2+, before Ca2+ entering the cell facilitates their activity.

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Abbreviations

AP:

Action potential

APW:

Action potential waveform

BKi :

Inactivating BK current or channel

BKs :

Non-inactivating BK current or channel

Ca 2+i :

Cytosolic Ca2+

Ca 2+o :

Extracellular Ca2+

ChTx:

Charybdotoxin

HAD:

Half-amplitude duration

IA :

Fast inactivating potassium current

IbTx:

Iberiotoxin

k i :

Rate constant of inactivation

N-methyl-d-glucamine:

NMDG

τ:

Time constant

TEA:

Tetraethylammonium

V h :

Holding potential

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Acknowledgments

We thank Dr. Antonio G. García for the generous supply of chromaffin cell cultures, and Drs. Paola Imbrici and Arnaud Ruiz for their valuable comments on the manuscript. Useful discussions with Drs. Juan Antonio Gilabert and Guido Ulate are also greatly acknowledged. This work was supported by grants from the Spanish Ministerio de Ciencia e Innovación (BFU2005-06034; Spanish Ion Channel Initiative (SICI) grant CSD2008-00005) to A.R.A. and (RYC-2009-03979 and SAF2010-20604) to R.S.S.

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Author notes

  1. Ricardo S. Scott & Inmaculada Cuchillo-Ibañez

    Present address: Instituto de Neurociencias UMH-CSIC, Campus de San Juan, Sant Joan d’Alacant, Alicante, Spain

Authors and Affiliations

  1. Departamento de Farmacología y Terapéutica, Universidad Autónoma de Madrid, 28029, Madrid, Spain

    Ricardo S. Scott & Inmaculada Cuchillo-Ibañez

  2. Departamento de Toxicología y Farmacología & Instituto Universitario de Investigación en Neuroquímica, Universidad Complutense de Madrid, 28040, Madrid, Spain

    Diego Bustillo, Luis Alcides Olivos-Oré, Maria Victoria Barahona & Antonio R. Artalejo

  3. Dipartimento di Neuroscienze, Università degli Studi di Torino, 10125, Torino, Italy

    Emilio Carbone

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Correspondence to Antonio R. Artalejo.

Electronic supplementary material

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Supplemental Figure 1

Effect of paxilline on K+ currents from bovine chromaffin cells at minimal Ca 2+i . A Effect of paxilline (1 μM, 10 min) on K+ currents activated by 600 ms-long depolarisations from a V h of −70 mV (left panels) or 0 mV (right panels). Upper panels show the corresponding voltage protocols. B Effect of paxilline on peak current amplitudes at the different test potentials from cells with the membrane voltage held at −70 mV (left panel) or 0 mV (right panel). Data are mean±SEM of five to eight experiments. *p < 0.001 (PDF 189 kb)

Supplemental Figure 2

Effect of TEA on K+ currents from chromaffin cells at minimal Ca 2+i . A Effect of TEA (30 mM) on K+ currents activated by 600 ms-long depolarisations from a V h of −70to 0 mV up to +120, in 20 mV steps (see the adjoining protocol). B Effect of different concentrations of TEA (3, 10 and 30 mM) on peak currents evoked by the voltage protocol employed in A. C, left panel. Effect of TEA (30 mM) on the rate of inactivation (v i) of K+ currents elicited V h of −70 mV to the indicated potentials; right panel. Effect of TEA (30 mM) on the rate of inactivation (k i) of K+ currents elicited by 600 ms-long depolarisations from a V h of 0 mV to the indicated potentials. Data are means±SEM of eight experiments. *p < 0.05, **p < 0.01 with respect to control values (PDF 23 kb)

Supplemental Figure 3

K+-tail currents from bovine chromaffin cells at minimal Ca 2+i . A, left panel. Tail currents (arrow) observed upon repolarisation to −70 mV from different test potentials in a chromaffin cell held at a V h of 0 mV (see the adjoining protocol); tail current amplitudes were subsequently used to build the activation curve for BK channels (right panel). The Boltzmann function fitted to the experimental data gave a value of +146.8 mV for V 50, and of 31.71 mV for K. Data were normalised with respect to the maximal current derived from curve fitting. B, left panel. Effect of IbTx (150 nM, 5 min) on K+ currents elicited by graded depolarisations from a V h of −70 mV (see the adjoining protocol); right panel. Tail current amplitudes before (Control) and after treatment with IbTx (IbTx) were used to build the corresponding activation curves. A Boltzmann function fitted to tail currents in the presence of IbTx gave values for V 50 and K of −14.2 and 11.94 mV, respectively. Values were normalised with respect to the maximal current derived from curve fitting to data in the presence of IbTx. Data are means ± SEM of eight and seven cells for panel A and B, respectively (PDF 202 kb)

Supplemental Figure 4

Time course of recovery from inactivation of I A and BK current at minimal Ca 2+i . A, upper panel. BK currents activated by a series of variably spaced pairs of of 600 ms-long voltage depolarisations to +100 mV from a V h of 0 mV (see voltage protocol); lower panel. Peak currents (I 1 and I 2) in response to paired pulses were ratioed (I 2/I 1) and plotted against the interpulse interval. Experimental data were fitted by a double exponential function with two rate constants: k fast = 23 s−1 and k slow = 0.77 s−1; an expanded view of the faster recovery component is shown as an inset. B, upper panel. I A currents were elicited by variably spaced pairs of 600 ms-long voltage depolarisations to +20 mV from a V h of −70 mV; lower panel. Peak current responses (I 1 and I 2) to paired pulses were ratioed (I 2/I 1) and plotted against the interpulse interval. The result of fitting a double exponential function with rate constants k fast = 27 s−1 and a k slow = 1.84 s−1, is shown; an expanded view of the faster recovery component is also shown as an inset. Data are means ± SEM of seven cells for each type of current (PDF 29 kb)

Supplemental Figure 5

Effect of paxilline on K+ currents induced by action potential waveforms in the presence of 2 mM Ca 2+o . A Perforated-patch recordings of K+ currents (black traces) induced by a fast (left panel) or a slow (right panel) action potential waveform (APW; dashed lines) from a chromaffin cell bathed in a solution containing 2 mM Ca2+. Blue traces depict the current remaining after the administration of paxilline (1 μM, 10 min). Red traces result from subtracting the blue traces (in the presence of paxilline) from the black ones (Control), hence corresponding to the BK current. Dotted lines denote the 0 mV level. Notice the existence of two peaks of BK current: the first peak coincides with the peak of the APW whereas the second delayed one occurs in the late repolarising phase of the APW. B Example of Ca2+ currents induced by a fast ramp-based APW from a chromaffin cell bathed in a solution containing 2 mM Ca2+ and 140 mM NMDG. The cell was dialysed with a solution containing 140 mM CsCl (PDF 56 kb)

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Scott, R.S., Bustillo, D., Olivos-Oré, L.A. et al. Contribution of BK channels to action potential repolarisation at minimal cytosolic Ca2+ concentration in chromaffin cells. Pflugers Arch - Eur J Physiol 462, 545–557 (2011). https://doi.org/10.1007/s00424-011-0991-9

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