Sarcoplasmic reticulum K(+) (TRIC) channel does not carry essential countercurrent during Ca(2+) release

Abstract

The charge translocation associated with sarcoplasmic reticulum (SR) Ca(2+) efflux is compensated for by a simultaneous SR K(+) influx. This influx is essential because, with no countercurrent, the SR membrane potential (Vm) would quickly (<1 ms) reach the Ca(2+) equilibrium potential and SR Ca(2+) release would cease. The SR K(+) trimeric intracellular cation (TRIC) channel has been proposed to carry the essential countercurrent. However, the ryanodine receptor (RyR) itself also carries a substantial K(+) countercurrent during release. To better define the physiological role of the SR K(+) channel, we compared SR Ca(2+) transport in saponin-permeabilized cardiomyocytes before and after limiting SR K(+) channel function. Specifically, we reduced SR K(+) channel conduction 35 and 88% by replacing cytosolic K(+) for Na(+) or Cs(+) (respectively), changes that have little effect on RyR function. Calcium sparks, SR Ca(2+) reloading, and caffeine-evoked Ca(2+) release amplitude (and rate) were unaffected by these ionic changes. Our results show that countercurrent carried by SR K(+) (TRIC) channels is not required to support SR Ca(2+) release (or uptake). Because K(+) enters the SR through RyRs during release, the SR K(+) (TRIC) channel most likely is needed to restore trans-SR K(+) balance after RyRs close, assuring SR Vm stays near 0 mV.

Figure 1

SR K⁺ channel function in our cell-like salt solutions. (A) Single SR K⁺ channel recordings (filtered at 0.5 kHz) at different membrane potentials. Open events shown upward from marked zero current level (dash). Sample sojourns to the subconductance level are marked (arrows). (B) Current amplitude (mean ± SE, n ≥ 6) plotted as a function of membrane potential. (Solid and open circles) Full open (178 pS) and subconductance (110 pS) levels, respectively. Both conductance levels reversed at 0 mV. (Dashed line) Current-voltage relationship of single RyR2 channels in the same solutions (190 pS, reversal potential −2.7 mV; from Gillespie and Fill (4)). (C) Open probability plotted as a function of membrane potential. Open probability (P_o) was calculated as P_o = 1−P_c, where P_c is the probability the channel is fully closed. (Dashed line) P_o single RyR2 channels in the same solutions (49).

Figure 2

(A) Single SR K⁺ channel current (full open state) is plotted as a function of membrane potential (mean ± SE; n > 5). (Solid circles) Data collected when solutions on both sides of the channel contained 120 mM K⁺ (no Mg²⁺, ATP, or added Ca²⁺). (Solid line) Fit has a slope of 186 pS. (Open circles) Current with 120 mM cytosolic Na⁺ and 120 mM luminal K⁺. (Open squares) 120 mM cytosolic Cs⁺ and 120 mM luminal K⁺. (Dashed line) Cs⁺ results from Cukierman et al. (16) show that our results are consistent with previous published data. Additional analyses of these data are shown in Fig. S1B in the Supporting Material. (B) Single RyR2 channel current plotted as a function of membrane potential (mean ± SE; n > 5). (Solid circles) (610 pS) Current with 120 mM K⁺ on both sides of the channel. (Open circles) (587 pS) 120 mM cytosolic Na⁺ and 120 mM luminal K⁺. (Open squares) (544 pS) 120 mM cytosolic Cs⁺ and 120 mM luminal K⁺. (Lines) Fit to the Na⁺ and Cs⁺ data are not shown. The reversal potentials of the three data sets were not significantly different. Cytosolic solution contained ∼5 μM Ca²⁺ (no Mg²⁺ or ATP). (C) Action of reduced SR K⁺ channel conductance on spontaneous Ca²⁺ sparks in permeabilized ventricular myocytes. The cytosolic solution contained 150 nM free Ca²⁺. Sample line-scan images before (left) and after (right) 120 mM cytosolic K⁺ was replaced by 120 mM Cs⁺. (D) Average full width at half-maximum (FWHM; ms), amplitude (F/F_o), full duration at half-width (FDHW; ms) and Ca²⁺ spark frequency (CaSpF; 100 μm⁻¹ s⁻¹) are shown with different cytosolic cations present. These data (mean ± SE) were collected from 15 different cells and represent 5685 (K⁺), 1549 (Na⁺), 1618 (Cs⁺), and 113 (Tris⁺) sparks. Values were statistically compared (t-test) to the K⁺ values (ns = not significant, ^∗p < 0.05, ^∗∗p < 0.001).

Figure 3

Caffeine-evoked SR Ca²⁺ release and load in permeabilized acutely dissociated ventricular myocytes. A. Mean peak caffeine-evoked release measured using cytosolic Fluo-4 fluorescence (n = 43, 4, 20, 9, and 10 cells for the K⁺, Na⁺, Cs⁺, and Tris⁺ bars, respectively). Each cytosolic cation was present for <4 min before caffeine (10 mM) was applied. Values were statistically compared (^∗ indicates p < 0.05; ns = not significant) to the K⁺ value (solid bar). (B) Maximum rate of caffeine-evoked release in the same experimental conditions as panel A. (C) Confocal x-y images of intra-SR Fluo-5N fluorescence in representative myocytes segments. Resting SR free Ca²⁺ concentration is proportional to the intensity of the striations. Bright striations are visible at normal resting SR Ca²⁺ load (control). Top pair of images show Fluo-5N fluorescence 1 min (1′) after caffeine application. Bottom pair of images show Fluo-5N fluorescence 1 min after cytosolic K⁺ (120 mM) was replaced by Cs⁺ (120 mM). (D) Time course of Fluo-5N fluorescence (mean ± SE; n = 5–8 cells) before (solid circles) and after (open circles) cytosolic K⁺ (120 mM) was replaced by Cs⁺ (120 mM). Data was normalized to the value at 0 min. (Star) Fluo-5N fluorescence after application of 10 mM caffeine (cytosolic K⁺ present). (Points marked by bracket) Not statistically different (t-test).

Figure 4

SR Ca²⁺ leak and uptake in permeabilized acutely dissociated ventricular myocytes. (A) Rate of SR Ca²⁺ leak measured using intra-SR Fluo-5N fluorescence. Thapsigargin was applied at time zero. (Open squares) Data collected with cytosolic K⁺ and ruthenium red (RuRed) present. (Solid circles) (n = 8) are with cytosolic K⁺ present (no RuRed). (Open circles) (n = 6) Cytosolic Cs⁺ present (no RuRed). (Lines) Single exponential fits. (B) Spark amplitude recovery after a 10-mM caffeine-evoked Ca²⁺ release (arrow) with cytosolic K⁺ (solid circles) or Cs⁺ (open circles) present. Cytosolic K⁺ was replaced for Cs⁺ immediately after the caffeine application. (Lines) Fit to the post-caffeine data and their slopes were not significantly different (f-test, p = 0.74; 0.025 ΔF/F₀ per min for K⁺, 0.028 ΔF/F₀ per min for Cs⁺). (C) SR Ca²⁺ uptake by permeabilized myocyte population. Single RyR2 function was blocked RuRed. Uptake was initiated by addition of cytosolic ATP at time zero and monitored as changing cytosolic Fluo-4 fluorescence (left panel). Uptake time constants (right panel), determined by single exponential fitting, with cytosolic K⁺ (solid), Cs⁺ (hatched), or Tris⁺ (shaded) present were 11.5 ± 0.9, 13.5 ± 2.2, and 29.5 ± 1.7 min (respectively). The Tris⁺ time constant was significantly larger than the K⁺ time constant (t-test; ^∗∗p > 0.01).