T-Type Calcium Channel: A Privileged Gate for Calcium Entry and Control of Adrenal Steroidogenesis

Abstract

Intracellular calcium plays a crucial role in modulating a variety of functions such as muscle contraction, hormone secretion, gene expression, or cell growth. Calcium signaling has been however shown to be more complex than initially thought. Indeed, it is confined within cell microdomains, and different calcium channels are associated with different functions, as shown by various channelopathies. Sporadic mutations on voltage-operated L-type calcium channels in adrenal glomerulosa cells have been shown recently to be the second most prevalent genetic abnormalities present in human aldosterone-producing adenoma. The observed modification of the threshold of activation of the mutated channels not only provides an explanation for this gain of function but also reminds us on the importance of maintaining adequate electrophysiological characteristics to make channels able to exert specific cellular functions. Indeed, the contribution to steroid production of the various calcium channels expressed in adrenocortical cells is not equal, and the reason has been investigated for a long time. Given the very negative resting potential of these cells, and the small membrane depolarization induced by their physiological agonists, low threshold T-type calcium channels are particularly well suited for responding under these conditions and conveying calcium into the cell, at the right place for controlling steroidogenesis. In contrast, high threshold L-type channels are normally activated by much stronger cell depolarizations. The fact that dihydropyridine calcium antagonists, specific for L-type channels, are poorly efficient for reducing aldosterone secretion either in vivo or in vitro, strongly supports the view that these two types of channels differently affect steroid biosynthesis. Whether a similar analysis is transposable to fasciculata cells and cortisol secretion is one of the questions addressed in the present review. No similar mutations on L-type or T-type channels have been described yet to affect cortisol secretion or to be linked to the development of Cushing syndrome, but several evidences suggest that the function of T channels is also crucial in fasciculata cells. Putative molecular mechanisms and cellular structural organization making T channels a privileged entry for the "steroidogenic calcium" are also discussed.

Keywords: ACTH; T-type calcium channels; adrenal cortex; aldosterone; calcium signaling; cortisol; electrophysiology; steroidogenesis.

Figure 1

Activation and inactivation of voltage-operated calcium channels and steady-state “window” currents. (A) Examples of slowly deactivating (T-type) Ba²⁺ currents recorded in the whole cell configuration of the patch clamp technique. Left. Voltage protocol for determining the activation curve: tail currents were evoked by repolarizing the cell to −65 mV after a short period (20 ms) of depolarization at various voltages (−45 to +5 mV for this selection of traces) from a holding potential of −90 mV. Right. Voltage protocol for determining the steady-state inactivation curve: tail currents were elicited in the same cell at −65 mV, but after steady-state inactivation of the channels for 10 s at various holding potentials (here from −80 to −30 mV) and 20 ms activation at +20 mV. Current amplitude upon cell repolarization was then determined by fitting tail currents to an exponential function (the time constant was approximately 7 ms). (B) Comparison of low (T-type) versus high (L-type) threshold voltage-operated calcium channels. Left panel shows normalized activation and inactivation curves determined for T-type channels using the same type of protocol as shown in panel A. Tail current amplitudes were plotted as a function of the test voltage, fitted to Boltzmann’s equation, and normalized to the maximal current (I_o). Curves for L-type channels were similarly defined from L-current amplitudes determined with a different voltage protocol, including the inactivation of T currents. Right panel displays the calculated normalized steady-state current (I_stst) expected through T-type and L-type channels within their respective permissive window of voltage. The theoretical steady-state currents were obtained from the activation and inactivation curves according to Ohm’s law and expressed as a percentage of the maximal current. The white rectangle on the voltage axis indicates the range of membrane potentials reached in naive glomerulosa cells and in cells stimulated with physiological concentrations of angiotensin II or extracellular potassium. (C) Effect of the Ile770Met mutation described in the CACNA1D L-type calcium channel (13) on the channel activation, inactivation, and steady-state current. Curves have been determined as in (B) for the wild-type channel (continuous line, Ca_V1.3) and for the mutant channel (dotted line, mut) and show the significant shift of the channel permissive window toward lower voltages. (D) Effect of PKC activation on CACNA1H T channel activation, inactivation, and steady-state current. Curves have been determined as in (B) for the naive channel (continuous line, Ca_V3.2) and for the channel in glomerulosa cells treated with the PKC activator phorbol 12 myristate 13-acetate ester (dotted line, +PMA) and show the significant reduction of the amplitude of the maximal steady-state current with the slight shift of the permissive window toward higher voltages. The graphs of this figure have been constructed based on data available in Ref. (14, 13).

Figure 2

Common structure of the alpha 1 subunit of voltage-operated calcium channels. The main, pore-forming α₁ subunits of the various voltage-operated calcium channels share a common general structure, with four homologous repeats (I–IV), each composed of six hydrophobic, putative membrane-spanning alpha helix domains (S1–S6). The three large loops connecting repeats together, as well as the N- and C-terminal extremities are located in the cytosol. The positions of Gly403 and Ile770 mutated in CACNA1D (13), and of Met1549 in CACNA1H (35), are indicated at the end of the S6 segments in repeats I, II, and III, respectively.

Figure 3

The concept of intracellular calcium pipeline: a model for explaining the selective transport of calcium from the T channels into the mitochondria. Electron microscopy reveals the presence of close apposition in many places of the endoplasmic reticulum (ER) with the plasma membrane or the mitochondria (white arrows), within various cell types, including rat parotid cells (A), rat spinal cord neurons (B), or bovine adrenal glomerulosa cells (C). Scale = 1 μm; m indicates mitochondria, nu nucleus and ld lipid droplets. See Ref. (112) for additional information. (D). A hypothetical model for the cellular transport of calcium into mitochondria. At the pipeline filling site, T-type calcium channels and, to a lesser extent, L-type channels are activated upon cell depolarization by potassium or angiotensin II. Several experimental data suggest that calcium entering the cell through T-type channels could be selectively pumped into the lumen of the ER, while calcium entering through L-type channels would be poured into the cytosol. At the pipeline delivery site, InsP3 receptors are maintained in proximity of the mitochondria within “quasi synaptic” structures. Calcium released upon activation of the InsP3 receptors, due to calcium overloading of the ER and/or to InsP3 production by AT₁ receptor-activated PLC, is rapidly internalized into the very negatively charged matrix, through the mitochondrial inner membrane calcium uniporter. Intramitochondrial calcium elevation then stimulates limiting steps of aldosterone biosynthesis. AT₁, angiotensin II receptor, type 1; G_q/11, heterotrimeric G protein of the q/11 family; PLC, phospholipase C β; PKC, protein kinase C; InsP₃, inositol 1,4,5-trisphosphate.