Coupling of L-type Ca2+ channels to KV7/KCNQ channels creates a novel, activity-dependent, homeostatic intrinsic plasticity

Abstract

Experience-dependent modification in the electrical properties of central neurons is a form of intrinsic plasticity that occurs during development and has been observed following behavioral learning. We report a novel form of intrinsic plasticity in hippocampal CA1 pyramidal neurons mediated by the KV7/KCNQ and CaV1/L-type Ca2+ channels. Enhancing Ca2+ influx with a conditioning spike train (30 Hz, 3 s) potentiated the KV7/KCNQ channel function and led to a long-lasting, activity-dependent increase in spike frequency adaptation-a gradual reduction in the firing frequency in response to sustained excitation. These effects were abolished by specific blockers for CaV1/L-type Ca2+ channels, KV7/KCNQ channels, and protein kinase A (PKA). Considering the widespread expression of these two channel types, the influence of Ca2+ influx and subsequent activation of PKA on KV7/KCNQ channels may represent a generalized principle in fine tuning the output of central neurons that promotes stability in firing-an example of homeostatic regulation of intrinsic membrane excitability.

FIG. 1.

Protocol for inducing intrinsic plasticity. A: Alexa594 (10 μM) was included in the patch solution for a subset of neurons to confirm the integrity of the perforated-patch configuration. As illustrated, establishment of stable series resistance in the perforated-patch configuration for 25 min did not introduce Alexa594 into the cell. B: sudden membrane rupturing, however, was accompanied by an instantaneous fluorescent signal in the soma. C: topographical location and morphological features of the recorded neuron. D, top: intrinsic membrane excitability was first assessed with a 1-s depolarizing current test pulse. A brief, suprathreshold conditioning pulse train (CS: 30 Hz, 3-s) was then delivered via the recording electrode. Subsequently, membrane excitability was monitored for as long as the recordings remained in the perforated patch configuration. Bottom: a representative voltage trace from one neuron illustrating that CS triggered the corresponding number of action potentials, followed by a pronounced postburst afterhyperpolarization (AHP). E: 1st temporal derivative of the 1st action potential triggered by the test pulse. Neither the threshold nor the maximum rate of rise of the action potential was altered immediately following CS presentation, suggesting that CS-induced enhancement of spike frequency adaptation is not due to an alteration in the Na_V channel availability that had developed during CS.

FIG. 2.

Repetitive firing triggers a persistent reduction in membrane excitability. A: representative voltage traces in response to 1-s somatic current test pulse acquired before (−5 min) and after (1–44 min) presentation of the conditioning spike train (CS; 30 Hz, 3-s). CS induced a gradual, sustained reduction in intrinsic membrane excitability in the form of an enhanced spike frequency adaptation over tens of minutes. Partial recovery was sometimes observed with extended recording periods. Time = 0 denotes presentation of CS. 4 traces, acquired starting from the time indicated, were shown. For clarity only the early portion of the membrane response to the test pulse was illustrated here and for the rest of the figures. B: the number of spikes evoked by the test pulse decreased progressively following CS presentation (n = 10/12). In the absence of CS, membrane excitability remained stable over time (n = 4). Data are normalized to the baseline excitability prior to CS presentation and presented as means ± SE here and for the rest of the figures. C: summary of changes in membrane excitability and firing pattern induced by CS. Adaptation index was computed as: 1-(ISI_min/ISI_n); adaptation index of 1 signifies maximal adaptation, i.e., cessation of firing. Note the leftward shift of the curve following CS presentation (20–25 min). Numbers denote the interspike interval number. D, left: phase plane analysis illustrating the threshold for the 2nd and the 7th spikes before (−5 min, black) and after (20–25 min, red) CS presentation. Spike threshold was defined by the discontinuity in the relationship between dV/dt and V. Right: spike threshold before (−5 min, black) and after (20–25 min, red) CS presentation plotted against spike number.

FIG. 3.

CS-induced intrinsic plasticity requires the K_V7/KCNQ channels. K⁺ channel blockers were applied for ≥15 min prior to CS presentation and maintained throughout the recordings. A–C and E: bath applications of linopirdine (1 μM, n = 4; 3 μM, n = 3) and XE991 (1 μM, n = 8; 3 μM, n = 12) dose-dependently reduced and prevented CS-induced intrinsic plasticity. D: prolonged application of XE991 (3 μM, n = 2; 10 μM, n = 4; pooled data) in the absence of CS did not alter intrinsic membrane excitability. E: histogram summarizing the effects of K⁺ channel blockers on CS-induced intrinsic plasticity.

FIG. 4.

K_V7.2/KCNQ2-containing channels constitute the K_V7/KCNQ current in adult CA1 pyramidal neurons. A: representative current traces elicited by voltage-steps from a holding potential of −30 to −40 mV in the presence of 0, 0.1, 1, and 10 mM TEA. B: TEA dose-dependently suppressed the K_V7/KCNQ current. TEA dose-response plot was fit with a single Hill-Langmuir equation: I = 1/[1 + (x/x₀)]^k, where I is the K_V7/KCNQ current amplitude; I_max is full inhibition; x is the TEA concentration; x₀ is the IC₅₀ (the concentration at which I/I_max = 0.5); and k is the slope factor. All parameters were allowed to free float. This yielded an IC₅₀ of 1.25 mM and a slope constant of 0.35. Thus data were also fit (not shown) with an extended, two-site Hill-Langmuir equation: I = q(1 + x/x₀)^k + (1 –q)/(1 + x/x₁)^k, where x₀ and x₁ are the IC₅₀s for 2-channel populations with proportional contributions of q and (1 − q), respectively (other definitions same as in the preceding text). In this case, maximum inhibition was assumed to be 100%, and slope constants were constrained at unity. This yielded IC₅₀ values of 10 μM and 7.0 mM with proportional contributions of the high and the low TEA-sensitivity components to be 0.33 and 0.67, respectively. These data suggest the presence of a small population of K_V7.2/KCNQ2 homomeric channels in adult CA1 pyramidal neurons. C and D: scRT-PCR revealed that CA1 pyramidal neurons (n = 22) co-expressed transcripts for K_V7.2/KCNQ2 (21/22) and K_V7.3/KCNQ3 (20/22) subunits. K_V7.5/KCNQ5 transcripts were only detected in a subset of hippocampal CA1 pyramidal neurons (12/22) at the same level of sensitivity.

FIG. 5.

K_V7.2/KCNQ2 and K_V7.3/KCNQ3 proteins in the hippocampus. Light micrographs depicting K_V7.2/KCNQ2 (top left) and K_V7.3/KCNQ3 (bottom left) immunoreactivities in the hippocampus and adjacent structures in rat sagittal sections. Specific labeling was found in the stratum pyramidale of the CA1 and CA3 subfields, granule cell layer of the dendate gyrus, thalamus (Th), and superior colliculus (SC). At higher magnification, K_V7.2/KCNQ2 and K_V7.3/KCNQ3 immunoreactivities were found to associate with the perisomatic region. Diffuse neuropilar labeling was also evident in both st. oriens and st. radiatum of the CA1 subfield.

FIG. 6.

CS-induced intrinsic plasticity requires activity-dependent rise in intracellular Ca²⁺. BAPTA-AM, a membrane-permeable Ca²⁺ chelator, was applied for ≥15 min prior to CS presentation and maintained throughout the recordings. A and B: bath application of BAPTA-AM (20 μM; n = 5) prevented CS-induced intrinsic plasticity. C: histogram summarizing the effect of BAPTA-AM on CS-induced intrinsic plasticity.

FIG. 7.

CS-induced intrinsic plasticity requires Ca²⁺ influx through the Ca_V1/L-type Ca²⁺ channels. Voltage-gated Ca²⁺ channel blockers were applied for ≥15 min prior to CS presentation and maintained throughout the recordings. A–C, left: bath applications of the nondihydropyridine Ca_V1/L-type Ca²⁺ channel blockers SR33805 (5 μM; n = 5), calciseptine (1 μM; n = 4), and the dihydropyridine Ca_V1/L-type Ca²⁺ channel blocker nimodipine (1 μM; n = 4) all prevented CS-induced intrinsic plasticity. B and C, right: in the presence of Ca_v2/N- and P, Q-type Ca²⁺ channel blocker ω-conotoxin MVIIC (1 μM; n = 4), CS still triggered a persistent reduction in membrane excitability. B: histogram summarizing the effects of voltage-gated Ca²⁺ channel blockers on CS-induced intrinsic plasticity.

FIG. 8.

CS-induced intrinsic plasticity requires PKA activity and likely involves homomeric K_V7.2/KCNQ2 channels. TEA was applied for ≥15 min prior to CS presentation and maintained throughout the recordings. Slices were preincubated in H-89 for ≥30 min prior to electrophysiological measurements, and H-89 was maintained throughout the recording. A, B, and D: Blocking homomeric K_V7.2/KCNQ2 channels with low concentration of TEA (0.5 mM; n = 6) or PKA activity with H-89 (5 μM; n = 5) prevented CS-induced intrinsic plasticity. C: histogram summarizing the effects of these treatments on CS-induced intrinsic plasticity.

FIG. 9.

Modulation of the K_V7/KCNQ current by Ca²⁺ influx through the Ca_V1/L-type Ca²⁺ channels. A: representative current traces elicited by 4-s voltage steps from a holding potential of −30 to −40 mV in control (black), 10 μM BayK8644 (green), and 10 μM BayK8644 + 10 μM XE991 (blue). Traces were taken at time points 1–3 as indicated in B. B: averaged time course of normalized K_V7/KCNQ current during sequential applications of 10 μM BayK8644 and 10 μM BayK8644 + 10 μM XE991 (n = 6). Data were collected at 45-s intervals and shown as means ± SE. Bath application of BayK8644 potentiated the K_V7/KCNQ current by over twofold that was readily suppressed by 10 μM XE991. C: representative current traces elicited in control (black), 300 nM (pink), and 10 μM nimodipine (red). Traces were taken at time points 1–3 as indicated in D. D: averaged time course of normalized K_V7/KCNQ current during sequential applications of 300 nM and 10 μM nimodipine (n = 6). E: voltage dependence of XE991-sensitive current in the presence of BayK8644. Smooth line represents best fit with a Boltzmann function, yielding V_½ of −31mV and a slope factor of 7.5 mV. F: maximal percentage inhibition of the K_V7/KCNQ current in the presence of 300 nM and 10 μM nimodipine.