Voltage dependence of the Ca²⁺-activated K⁺ channel K_Ca3.1 in human erythroleukemia cells

Abstract

We have isolated a K(+)-selective, Ca(2+)-dependent whole cell current and single-channel correlate in the human erythroleukemia (HEL) cell line. The whole cell current was inhibited by the intermediate-conductance KCa3.1 inhibitors clotrimazole, TRAM-34, and charybdotoxin, unaffected by the small-conductance KCa2 family inhibitor apamin and the large-conductance KCa1.1 inhibitors paxilline and iberiotoxin, and augmented by NS309. The single-channel correlate of the whole cell current was blocked by TRAM-34 and clotrimazole, insensitive to paxilline, and augmented by NS309 and had a single-channel conductance in physiological K(+) gradients of ~9 pS. RT-PCR revealed that the KCa3.1 gene, but not the KCa1.1 gene, was expressed in HEL cells. The KCa3.1 current, isolated in HEL cells under whole cell patch-clamp conditions, displayed an activated current component during depolarizing voltage steps from hyperpolarized holding potentials and tail currents upon repolarization, consistent with voltage-dependent modulation. This activated current increased with increasing voltage steps above -40 mV and was sensitive to inhibition by clotrimazole, TRAM-34, and charybdotoxin and insensitive to apamin, paxilline, and iberiotoxin. In single-channel experiments, depolarization resulted in an increase in open channel probability (Po) of KCa3.1, with no increase in channel number. The voltage modulation of Po was an increasing monotonic function of voltage. In the absence of elevated Ca(2+), voltage was ineffective at inducing channel activity in whole cell and single-channel experiments. These data indicate that KCa3.1 in HEL cells displays a unique form of voltage dependence modulating Po.

Inhibition of KCa3.1 by depolarisation and 2-aminoethoxydiphenyl borate (2-APB) during Ca2+ release activated Ca2+ (CRAC) entry in human erythroleukemia (HEL) cells: Implications for the interpretation of 2-APB inhibition of CRAC entry

“…Application of thapsigargin was accompanied by a marked increase in the 0 mV current magnitude (Panel A) consistent with the pronounced increase in ramp current shown in Panel B2. This ramp current has the well defined characteristics of KCa3.1 in HEL cells as previously reported by our laboratory [14]. Changing the holding potential between ramps from −80 to 0 mV was accompanied by a large decline in the current to a new steady-state value near that recorded before thapsigargin application (as seen in Panels A and B3).…”

“…The calculated amplitude histograms were fit to the sum of multiple Gaussian distributions using the multi-peak fitting routine within IGOR Pro as previously reported [14,15]. The calculated areas, A, of the individual Gaussian peaks in the multipeak fit were taken directly from the output of the peak fitting routine and used to calculate NP o were N is the number of channels and P o is the open channel probability as previously reported by our laboratory [14,15]. Assuming all channels are identical and behave independently, NP o can be determined from the areas of individual Gaussian peaks using the following relationship [16] as modified from Selyanko et al [17] and Li et al [18]:…”

“…The role of membrane potential modulation of CRAC-mediated Ca 2+ entry in HEL cells is of particular importance given the depolarised resting membrane potential and the expression of the Ca 2+ -activated K + channel, KCa3.1 [12,14,15,23]. We have investigated the role of the resting membrane potential and subsequent hyperpolarisation driven by activation of the Ca 2+ -activated K + channel, KCa3.1, in the activation and modulation of the elevation in [Ca 2+ ] i mediated by thapsigargin.…”

“…In Fig. 1B, 200 nM charybdotoxin, a potent inhibitor of KCa3.1 in HEL cells [14,23] was added near the peak of the rise in Ca 2+…”

“…Charybdotoxin significantly reduced this increase to 153 ± 15 nM, a 51 ± 3% decrease in the thapsigarginmediated rise in [Ca 2+ ] i (p ≤ 0.05, n = 9). Charybdotoxin's inhibitory influence on the Ca 2+ response most likely arises from the inhibition of the hyperpolarising influence of KCa3.1 activated by the rise in [Ca 2+ ] i [14,23]. Assuming that 200 nM charybdotoxin maximally inhibits KCa3.1, the residual elevation in [Ca 2+ ] i is accounted for by the maintenance of a residual membrane potential sufficient to support net Ca 2+ entry.…”