Functional characterization of a small conductance GIRK channel in rat atrial cells

Abstract

Muscarinic K+ (KACh) channels are key determinants of the inhibitory synaptic transmission in the heart. These channels are heterotetramers consisting of two homologous subunits, G-protein-gated inwardly rectifying K+ (GIRK)1 and GIRK4, and have unitary conductance of approximately 35 pS with symmetrical 150 mM KCl solutions. Activation of atrial KACh channels, however, is often accompanied by the appearance of openings with a lower conductance, suggesting a functional heterogeneity of G-protein-sensitive ion channels in the heart. Here we report the characterization of a small conductance GIRK (scGIRK) channel present in rat atria. This channel is directly activated by Gbetagamma subunits and has a unitary conductance of 16 pS. The scGIRK and KACh channels display similar affinities for Gbetagamma binding and are frequently found in the same membrane patches. Furthermore, Gbetagamma-activated scGIRK channels--like their KACh counterparts--exhibit complex gating behavior, fluctuating among four functional modes conferred by the apparent binding of a different number of Gbetagamma subunits to the channel. The electrogenic efficacy of the scGIRK channels, however, is negligible compared to that of KACh channels. Thus, Gbetagamma subunits employ the same signaling strategy to regulate two ion channels that are apparently endowed with very different functions in the atrial membrane.

FIGURE 1

Two populations of G-protein-activated single-channel openings in neonatal rat atrial myocytes. Activation of a single 34-pS K_ACh channel in a cell-attached patch is accompanied by the appearance of 16-pS openings. In this and all following figures, the downward deflections correspond to inward currents. Membrane potential: −90 mV; 1 μM ACh is added to the pipette solution. Excision of the membrane patch in GTP-free solution instantaneously and completely eliminated both 16- and 34-pS events. The membrane potential continued to be clamped at −90 mV during the entire experiment. Perfusion of the experimental chamber with 0.4 nM Gβ₁γ₇ restored both the 16- and 34-pS single-channel openings. The single-channel recording in the presence of Gβ₁γ₇ begins 10 min after Gβ₁γ₇ application.

FIGURE 2

Small conductance atrial GIRK channel. Single-channel activity recorded in cell-attached configuration from an atrial myocyte in the presence of 1 μM ACh. Channel activity ceased after patch excision in GTP-free solution. Application of 12 nM Gβ₁γ₇ restored the channel activity. Membrane potential: −90 mV.

FIGURE 3

Voltage dependence of scGIRK and K_ACh channels. (A) Gβ₁γ₇-activated single-channel currents were recorded from the same membrane patch at the indicated potentials. All-points histograms were generated from the recordings and fitted by a sum of Gaussian functions to determine the mean current amplitudes. (B) I−V curves for the scGIRK (gray) and K_ACh (black) channels were constructed from the single-channel current amplitudes determined at each membrane potential. Each data point represents the mean ± SEM of currents recorded from 5 to 14 channels.

FIGURE 4

Characteristics of scGIRK channels. (A) Cumulative open-time distributions with superimposed fits are shown for two different scGIRK channels activated by 4 nM Gβ₁γ₇ in the absence (left panel) and presence (right panel) of Mg-ATP/PKA_c (the scGIRK channel shown in B). Membrane potential: −90 mV. Two exponential components were readily identified in both histograms. The time constants of the fast and slow components are τ_o1 = 0.12 ms (57.7%) and τ_o2 = 0.94 ms (42.3%) in the presence of Gβ₁γ₇ alone, and τ_o1 = 0.49 ms (55.3%) and τ_o2 = 3.49 ms (44.7%) in the presence of Gβ₁γ₇/Mg-ATP/PKA_c. The relative areas of individual components are given in parentheses. (B) Effect of PKA on scGIRK channel gating. A pair of an scGIRK and a K_ACh channel residing in the same membrane patch was activated in the presence of 4 nM Gβ₁γ₇, and then PKA_c was applied to the cytoplasmic side of the patch in the presence of Mg-ATP. Application of PKA triggered clustering of scGIRK channel openings into long bursts of activity. Expanded current traces recorded from the same patch before and after application of PKA are shown in the right panel to illustrate the kinetic behavior of the channel in greater detail. (C) All-points histograms generated from data recorded from the same scGIRK channel in the presence of Gβγ (left panel) and Gβγ/Mg-ATP/PKA_c (right panel). The histograms were fitted by the sum of two Gaussian functions to determine the unitary conductance of scGIRK channel (14.4 pS and 14.2 pS, respectively).

FIGURE 5

Classification of the scGIRK channel gating. (A) Continuous single-channel recordings were divided into consecutive 400-ms segments and the channel open probability and the frequency of openings were determined for the individual segments. Plots of open probability (left panel) and frequency of gating (right panel) versus time, derived from the initial 20 s of the scGIRK channel recording illustrated in Fig. 2, are shown to illustrate the heterogeneous channel behavior. (B) Modal classification of scGIRK channel gating. Open probability histograms generated from scGIRK channel activity recorded at two different Gβ₁γ₇ concentrations. The histogram shown at left was generated from pooled data recorded from four different scGIRK channels activated in the presence of 0.4 nM Gβ₁γ₇. The histogram shown at right was generated from data recordings from a single scGIRK channel activated in the presence of 12 nM Gβ₁γ₇. The histograms shown in this and the following figures leave out the blank 400-ms data segments and include the active data segments only. The p_o histograms were fitted by a sum of gamma components to determine the mean p_o values and the relative occupancy of different gating components. The mean p_o values and relative areas (given in parentheses) of different components are p_o1 = 0.00045 (100%) in the presence of 0.4 nM Gβ₁γ₇; and p_o1 = 0.00046 (29.3%), p_o2 = 0.0018 (37.7%), p_o3 = 0.0043 (27.2%), and p_o4 = 0.0085 (5.8%) in the presence of 12 nM Gβ₁γ₇. The inset shows the right part of the histogram on a different scale to illustrate the minor contribution of the component representing mode 4 (gray line) to the total fit of the histogram. This component was frequently too small to be accurately distinguished from the component representing mode 3, and therefore, modes 3 and 4 were analyzed together in the p_o histograms.

FIGURE 6

Gating equilibrium among four functional modes of GIRK channels. (A) A model assuming four functional modes of GIRK channels rendered active by the independent binding of an increasing number of Gβγ subunits to four equivalent Gβγ sensors in the channel structure. (B) For each channel, the relative occupancy of different gating modes was estimated from the fraction of the p_o (gray symbols) and f (black symbols) histograms fitted by the corresponding gamma components. The sojourns of the channels to gating modes 3 and 4 are jointly represented. In each experiment, the probability of Gβ₁γ₇-binding to one of the Gβγ sensors, P, was computed from the relative occupancy of mode 1, F₁, according to Eq. 2. The solid lines represent the predicted occupancy of the individual gating modes. The symbols represent the occupancy of mode 1 (circles), mode 2 (diamonds), and modes 3 and 4 (triangles) determined from the individual p_o and f distributions for different scGIRK (n = 14) and K_ACh (n = 14) channels. The graph in panel B, bottom, illustrates the discrepancy between the predicted occupancy of mode 0 and the experimentally determined fractions of blank data segments for the scGIRK (blue) and K_ACh (red) channels.

FIGURE 7

Gating equilibrium among three functional modes of GIRK channels. (A) A model assuming three functional modes of GIRK channels rendered active by the independent binding of two, three, and four Gβγ subunits to four equivalent Gβγ sensors in the channel structure. (B) For each channel, the relative occupancy of different gating modes was estimated from the fraction of the p_o (gray symbols) and f (black symbols) histograms fitted by the corresponding gamma components. In each experiment, the probability of Gβ₁γ₇-binding to one of the Gβγ sensors, P, was computed from the relative occupancy of mode 0, F₀, according to the equation: F₀ = 4P(1 − P)³ + (1 − P)⁴ (red line). The solid lines represent the predicted occupancy of the individual gating modes. The symbols represent the occupancy of mode 1 (diamonds), mode 2 (triangles), and mode 3 (squares) determined from the individual p_o and f distributions for different scGIRK (n = 14) and K_ACh (n = 14) channels.

FIGURE 8

Comparison of the Gβ₁γ₇ affinity and electrogenic efficacy of the scGIRK and K_ACh channels. (A) Gβ₁γ₇ binding to scGIRK and K_ACh channels. The probability of Gβ₁γ₇ binding, P, is plotted against Gβ₁γ₇ concentration for the scGIRK (left panel) and K_ACh (right panel) channels. The parameter P was estimated at three standard time points of each experiment: 10 (black circle), 20 (gray circle) and 30 (empty circle) min after Gβ₁γ₇ perfusion. The points and error bars represent the mean ± SEM of four to six separate experiments. The solid lines through the data represent the least-squares fit with the Hill equation (Eq. 3), with a Hill coefficient, N, of 4 for both channels. The P_max constant is 0.52 for the K_ACh channel and 0.36 for the scGIRK channel, whereas the dissociation constant K_d is 1.65 nM for the K_ACh channel and 1.55 nM for the scGIRK channel. (B) Open probability of different gating modes. The mean open probabilities determined from the analysis of scGIRK p_o histograms (left panel) are p_o1 = 0.00060 ± 0.00006, p_o2 = 0.0022 ± 0.0002, and p_o3&4 = 0.0054 ± 0.0005. The values for the K_ACh channel functional modes (right panel) are p_o1 = 0.0019 ± 0.0005, p_o2 = 0.0086 ± 0.0018, and p_o3&4 = 0.0234 ± 0.0063. Data are mean ± SEM (n = 10–14).

FIGURE 9

Synchronized slow modal transitions in GIRK channel gating. Single-channel recordings from a pair of scGIRK and K_ACh channel activated by 4 nM Gβ₁γ₇. After an initial period (∼7 min) of steady gating, the activity of both channels increased simultaneously.

FIGURE 10

Biphasic activation of GIRK channels. Time courses of Gβγ activation for the scGIRK (A) and the K_ACh (B) channels shown in Fig. 9. To identify the different phases of scGIRK and K_ACh channel activation, the F-t plots were generated as described in Materials and Methods. The horizontal lines indicate the mean F values obtained within apparently homogeneous sections of channel activity. After an initial period (∼7 min) of steady gating, the activity of both channels increased simultaneously. (C) Two representative 15-s segments of single-channel recordings are shown to illustrate the changes in the channel behavior associated with different phases of this experiment.

FIGURE 11

The slow modal transitions in scGIRK and K_ACh channel gating are associated with changes in the equilibrium among the four functional modes of these channels. (A) Open probability histograms generated from the initial (left panel) and late (right panel) phases of scGIRK activity shown in Fig. 9. The mean p_o values and the relative areas of different gamma components (shown in parentheses) are p_o1 = 0.00037 (65.5%), p_o2 = 0.0015 (28.7%), and p_o3 = 0.0032 (5.8%) for the initial phase; and p_o1 = 0.00072 (58.7%), p_o2 = 0.0026 (32.8%), and p_o3 = 0.0045 (8.5%) for the late phase of channel activation. The P values computed from these histograms are P^initial = 0.225, and P^late = 0.270. In this experiment, the changes in the kinetic behavior of the scGIRK were associated with both a slight shift in modal equilibrium and a significant increase in the open probability of different functional modes. (B) Open probability histograms generated from the initial (left panel) and late (right panel) phases of K_ACh channel activity shown in Fig. 9. The mean p_o values and the relative areas of different components are p_o1 = 0.0033 (50.0%), p_o2 = 0.012 (37.5%), and p_o3 = 0.031 (12.5%) for the initial phase; and p_o1 = 0.0033 (20.3%), p_o2 = 0.015 (37.4%), and p_o3 = 0.053 (42.3%) for the late phase of channel activation. The P values computed from these histograms are P^initial = 0.328 and P^late = 0.555 for the K_ACh channel. Thus, the changes in the gating behavior for this K_ACh channel were mostly associated with a shift in the modal equilibrium toward gating modes 3 and 4.

FIGURE 12

Opposing regulation of scGIRK and K_ACh channels. Single-channel activity recorded in the presence of 12 nM Gβ₁γ₇ from a membrane patch harboring a scGIRK and a K_ACh channel. In this experiment, an initial phase of efficient K_ACh channel gating and infrequent scGIRK openings was replaced with a second phase, characterized by increased scGIRK activity and infrequent K_ACh channel gating.

FIGURE 13

Modal distributions of scGIRK and K_ACh channels associated with the opposing regulation of these channels. Open probability histograms generated from the homogeneous phases of scGIRK (top panels) and K_ACh (bottom panels) channel gating shown in Fig. 12. For the scGIRK channel the mean p_o values and relative areas (given in parentheses) of different components are p_o1 = 0.00060 (87.2%) and p_o2 = 0.0030 (12.8%) for the initial phase; and p_o1 = 0.00061 (34.3%), p_o2 = 0.0026 (40.3%), and p_o3 = 0.0072 (25.4%) for the late phase of channel activation. For the K_ACh channel the mean p_o values and the relative areas of different components are p_o1 = 0.0073 (26.3%), p_o2 = 0.0268 (40.0%), and p_o3 = 0.0780 (33.7%) for the initial phase; and p_o1 = 0.00068 (79.4%), p_o2 = 0.0032 (18.7%), and p_o3 = 0.0080 (1.9%) for the late phase of channel activation. The P values, determined from these distributions, are P^initial = 0.084 and P^late = 0.440 for the scGIRK channel; and P^initial = 0.503, and P^late = 0.130 for the K_ACh channel. The K_ACh channel gating during the late phase is impaired not only by its confinement within the low-efficient modes 1 and 2, but also by the significant reduction in the open probability of individual kinetic components contributing to channel activity.