Constitutively active L-type Ca2+ channels

Abstract

Ca(2+) influx through L-type Ca(2+) channels (LTCCs) influences numerous physiological processes ranging from contraction in muscle and memory in neurons to gene expression in many cell types. However, the spatiotemporal organization of functional LTCCs has been nearly impossible to investigate because of methodological limitations. Here, we examined LTCC function with high temporal and spatial resolution using evanescent field fluorescence microscopy. Surprisingly, we found that LTCCs operated in functionally organized clusters, not necessarily as individual proteins. Furthermore, LTCC function in these clusters does not appear to be controlled by simple stochastic gating but instead by a PKC-dependent switch mechanism. This work suggests that resting intracellular free calcium concentration in arterial myocytes is predominantly controlled by this process in combination with rare voltage-dependent openings of individual LTCCs. We propose that Ca(2+) influx via persistent LTCCs may be an important mechanism regulating steady-state local and global Ca(2+) signals.

Fig. 1.

Persistent Ca²⁺ influx in arterial smooth muscle. (A) Surface plot of an image from a cell showing three active Ca²⁺ sparklet sites ([Ca²⁺]_o = 2 mM) ([Ca²⁺]_o, external calcium concentration). The image below shows an expanded 2D view of the region demarked by the white square in the surface plot. Traces show the time course of [Ca²⁺]_i in regions a-d. (B) Amplitude histogram of Ca²⁺ sparklets in 2 mM [Ca²⁺]_o.(C) Images from two representative cells exposed to 20 mM [Ca²⁺]_o. Traces show the time course of [Ca²⁺]_i in regions a and b. Traces c and c′ show consecutive recordings of [Ca²⁺]_i in region c. (D) Amplitude histogram of Ca²⁺ sparklets at 20 mM [Ca²⁺]_o. The solid black line is the best fit (χ² = 6.0) to the data with the following multicomponent Gaussian function: , where a and b are constants, and [Ca²⁺]_i and q (37.9 nM) are intracellular Ca²⁺ and the quantal unit of Ca²⁺ influx, respectively (n = 2,995 events). The dotted line in the histograms shows the amplitude threshold. (E) Bar plot of the mean ± SEM nP_s of Ca²⁺ sparklet sites.

Fig. 2.

LTCCs underlie persistent Ca²⁺ entry. (A) Relationship between Ca²⁺ sparklet amplitude and their associated Ca²⁺ currents. The solid line is a linear fit to the data. (Inset) A simultaneous record of [Ca²⁺]_i and i_Ca at -70 mV. (B) Simultaneous recordings of quantal Ca²⁺ sparklets and unitary Ca²⁺ channel activity at -90 and -70 mV (Left). (Center) Sample Ca²⁺ channel records from a cell-attached patch. Red lines mark the quantal level for Ca²⁺ sparklets and the unitary channel level of the full and subconductance Ca²⁺ channel. Voltage dependencies of Ca²⁺ sparklets (in pA units) and the sub- and full-conductance Ca²⁺ channel (Right). Solid lines are linear fits to the data. (C) Sample [Ca²⁺]_i traces of silent (Left), low (Center), and high (Right) nP_s sites before and after application of Bay-K 8644. (D) [Ca²⁺]_i records from Ca²⁺ sparklet site before and after nifedipine. (E) Mean ± SEM nP_s of Ca²⁺ sparklet sites before and after application of Bay-K 8644 or nifedipine. (F) Amplitude histogram of Ca²⁺ sparklets in control conditions and after Bay-K 8644 treatment. The solid lines show best fits to the data with the Gaussian function in Fig. 1 using a q value of 38.5 nM (χ² = 3.8) and 39.2 nM (χ² = 4.3) for control (n = 818 events) and Bay-K 8644 (n = 1453 events) data, respectively.

Fig. 3.

PKC induces persistent Ca²⁺ sparklet activity. (A) Images of cells with silent (Left), low (Center), and high (Left) nP_s sites. Traces below each image show the time course of [Ca²⁺]_i in the green circle before (Upper) and after PDBu (200 nM; Lower) treatment. (B) Mean ± SEM nP_s of Ca²⁺ sparklet sites in control conditions and in the presence of PDBu or PKC inhibitory peptide (100 μM). (C) Amplitude histogram of Ca²⁺ sparklets in control conditions and in the presence of PDBu. Solid lines are the best fits to the data with the Gaussian function described in Fig. 1, using a q value of 38.7 nM (χ² = 1.6) and 39.4 nM (χ² = 2.9) for control (n = 779 events) and PDBu (n = 2247 events) data, respectively. (D) Sample traces of the fraction of the membrane with [Ca²⁺]_i above threshold (resting [Ca²⁺] + 40 nM) in a control cell (Upper) and a cell dialyzed with activated PKC (Lower). [Ca²⁺]_i was recorded ≈1, 2, or 3 min after gaining access into the cell interior. The images at Left were taken at the mentioned time points. The green line in the images outlines the cell.

Fig. 4.

Low- and high-nP_o LTCCs. (A) Representative cell-attached records of low-activity LTCCs before (Left) and after (Right) application of 200 nM PDBu (-70 mV; 20 mM Ca²⁺). Dotted lines show the full-conductance single-channel current level. The graph to the right of these traces plots the mean ± SEM nP_o of low- and high-activity LTCCs before and after PDBu. (B) Representative cell-attached LTCCs records of high-activity LTCCs channels under control conditions (Left) and after 200 nM bisindolylmaleimide (BIM) (Right). The bar plot shows the mean ± SEM nP_o of high-activity LTCCs before and after BIM.