Coordinate regulation of gonadotropin-releasing hormone neuronal firing patterns by cytosolic calcium and store depletion

Abstract

Elevation of cytosolic free Ca2+ concentration ([Ca2+]i) in excitable cells often acts as a negative feedback signal on firing of action potentials and the associated voltage-gated Ca2+ influx. Increased [Ca2+]i stimulates Ca2+-sensitive K+ channels (IK-Ca), and this, in turn, hyperpolarizes the cell and inhibits Ca2+ influx. However, in some cells expressing IK-Ca the elevation in [Ca2+]i by depletion of intracellular stores facilitates voltage-gated Ca2+ influx. This phenomenon was studied in hypothalamic GT1 neuronal cells during store depletion caused by activation of gonadotropin-releasing hormone (GnRH) receptors and inhibition of endoplasmic reticulum (Ca2+)ATPase with thapsigargin. GnRH induced a rapid spike increase in [Ca2+]i accompanied by transient hyperpolarization, followed by a sustained [Ca2+]i plateau during which the depolarized cells fired with higher frequency. The transient hyperpolarization was caused by the initial spike in [Ca2+]i and was mediated by apamin-sensitive IK-Ca channels, which also were operative during the subsequent depolarization phase. Agonist-induced depolarization and increased firing were independent of [Ca2+]i and were not mediated by inhibition of K+ current, but by facilitation of a voltage-insensitive, Ca2+-conducting inward current. Store depletion by thapsigargin also activated this inward depolarizing current and increased the firing frequency. Thus, the pattern of firing in GT1 neurons is regulated coordinately by apamin-sensitive SK current and store depletion-activated Ca2+ current. This dual control of pacemaker activity facilitates voltage-gated Ca2+ influx at elevated [Ca2+]i levels, but also protects cells from Ca2+ overload. This process may also provide a general mechanism for the integration of voltage-gated Ca2+ influx into receptor-controlled Ca2+ mobilization.

Figure 1

Store depletion and membrane excitability in GT1 cells. (A) Simultaneous measurements of membrane potential (V_m) and [Ca²⁺]_i in GnRH-stimulated cells. (B) Simultaneous measurements of V_m and [Ca²⁺]_i in TG-treated cells. In A and B, perforated-patch recording techniques were used. (C) AP firing in GnRH-stimulated cells with [Ca²⁺]_i clamped at ≈20 nM, using conventional whole-cell recording techniques. Arrows indicate the moment of application of GnRH and TG. In this and the following figures, dotted lines represent baseline potential (upper tracing) and basal [Ca²⁺]_i (bottom tracing).

Figure 2

Characterization of the current underlying GnRH-induced hyperpolarization in GT1 neurons. (A Left) Representative current traces of the initial GnRH-induced transient current at the indicated holding potentials (V_h). (Right) Current–voltage relation of the GnRH-induced current. Dotted line represents the reversal potential. (B) Simultaneous measurement of V_m (solid lines) and [Ca²⁺]_i (dotted lines) in response to GnRH in the absence (Left) or presence (Right) of 100 nM apamin. (C) Characterization of currents activated by store depletion in GT1 neurons. Simultaneous measurement of current (solid lines) and [Ca²⁺]_i (dotted lines) in response to GnRH in the absence (Left) or presence (Right) of 100 nM apamin.

Figure 3

Role of SK channels in unstimulated and store-depleted cells. (A) Lack of effect of apamin on AP firing in a spontaneously active cell. (B and C) Activation of SK channels during sustained store depletion by GnRH or TG. Simultaneous measurements of V_m and [Ca²⁺]_i changes in response to GnRH (B Left) or TG (C Left) before and during apamin addition. (Right) Asterisk denotes significant difference (P < 0.05; n = 3) from control. Double asterisks denote significant difference (P < 0.05; n = 3) between responses to TG or GnRH and apamin.

Figure 4

Effects of external Cs⁺ and GnRH on inwardly rectifying K⁺ current and membrane excitability in GT1 cells. (A) Current–voltage relation of the inwardly rectifying K⁺ current before (○) and during (●) the application of 5 mM CsCl. Under perforated-patch voltage-clamp recording conditions, K_ir were activated by 1-sec hyperpolarizing voltage steps from –70 to –140 mV (holding potential = −40 mV). The mean current between 0.9 and 1 sec was measured and plotted against the V_m. (B) Simultaneous measurements of V_m and [Ca²⁺]_i during application of 5 mM CsCl. (C) I–V characteristics of the K_ir before (○) and during 100 nM GnRH application (●). (D) Simultaneous measurements of V_m and [Ca²⁺]_i in response to 100 nM GnRH (arrow) in the presence of 5 mM CsCl (bar).

Figure 5

GnRH-induced activation of a Ca²⁺-conducting inward current in GT1 cells. (A) Effects of extracellular Ca²⁺ depletion (shaded area) on GnRH-induced current and [Ca²⁺]_i in a cell voltage-clamped at –60 mV. (B) Application of 100 nM GnRH in the presence or absence of Ca²⁺-deficient medium in cells voltage-clamped at –85 mV. In A and B, the bath contained 1 μM nifedipine, 50 μM Ni²⁺, and 100 nM apamin. Simultaneous measurements of current and [Ca²⁺]_i were performed by using perforated-patch, voltage-clamp recording techniques.

Figure 6

Thapsigargin-induced Ca²⁺-conducting inward current in GT1 cells. Simultaneous measurements of current and [Ca²⁺]_i were performed by using perforated-patch, voltage-clamp recording techniques. (A) Effects of extracellular Ca²⁺ depletion (shaded area) on thapsigargin-induced current and [Ca²⁺]_i in a cell voltage-clamped at –60 mV. (B) Thapsigargin-induced inward current in cells voltage-clamped at –85 mV. In A and B, the bath contained 1 μM nifedipine, 50 μM Ni²⁺, and 100 nM apamin.