Molecular and biophysical mechanisms of Ca2+ sparklets in smooth muscle

Abstract

In this article, we review the biophysical basis and functional implications of a novel Ca(2+) signal (called "Ca(2+) sparklets") produced by Ca(2+) influx via L-type Ca(2+) channels (LTCCs) in smooth muscle. Ca(2+) sparklet activity is bimodal. In low activity mode, Ca(2+) sparklets are produced by random, brief openings of solitary LTCCs. In contrast, small clusters of LTCCs can function in a high activity mode that creates sites of continual Ca(2+) influx called "persistent Ca(2+) sparklets". Low activity and persistent Ca(2+) sparklets contribute to Ca(2+) influx in arterial, colonic, and venous smooth muscle. Targeting of PKCalpha by the scaffolding protein AKAP150 to specific sarcolemmal domains is required for the activation of persistent Ca(2+) sparklets. Calcineurin, which is also associated with AKAP150, opposes the actions of PKCalpha on Ca(2+) sparklets. At hyperpolarized potentials, Ca(2+) sparklet activity is low and hence does not contribute to global [Ca(2+)](i). Membrane depolarization increases low and persistent Ca(2+) sparklet activity, thereby increasing local and global [Ca(2+)](i). Ca(2+) sparklet activity is increased in arterial myocytes during hypertension, thus increasing Ca(2+) influx and activating the transcription factor NFATc3. We discuss a model for subcellular variations in Ca(2+) sparklet activity and their role in the regulation of excitation-contraction coupling and excitation-transcription coupling in smooth muscle.

Figure 1. Ca²⁺ sparklets in arterial and venous myocytes

(A) TIRF images of Ca²⁺ sparklets from representative arterial (left) and portal vein (right) smooth muscle cells (-70 mV; external Ca²⁺ = 20 mM). Traces below each image show the time-course of [Ca²⁺]_i in the sites marked by the green circles. (B) Simultaneous recordings of Ca²⁺ sparklets and unitary LTCC currents. Redrawn from [8].

Figure 2. Ca²⁺ sparklet activity is quantal

All-points histogram generated from 25 representatives blank TIRF traces (A) or Ca²⁺ sparklet traces (B) similar to those in the inserts. (C) Description of Ca²⁺ sparklet nP_s analysis. Solid lines represent fit of the [Ca²⁺]_i traces using the single channel detection and analysis algorithms of Clampex 9.0. Dotted lines represent quantal levels. Figure redrawn from [9].

Figure 3. Proposed model for heterogeneous Ca²⁺ sparklet activity and NFATc3 activation in arterial myocytes

Activation of hetero-trimeric G_q/11 proteins by a membrane receptor like the angiotensin receptor type 1 (AT1R) activates phospholipase C (PLC), which increases diacylglycerol (DAG) levels and activates PKCα AKAP150 targets PKCα and calcineurin to specific regions of the sarcolemma where they can modulate the activity of nearby LTCCs. Ca²⁺ entering the cell recruits calmodulin, thus releasing PKCα from its interaction with AKAP150 allowing it to phosphorylate neighboring LTCCs. LTCCs associated with the AKAP150/PKCα/calcineurin complex can develop persistent Ca²⁺ sparklet activity, forming a signaling unit that modulates Ca²⁺ influx and NFATc3-dependent gene expression in smooth muscle. Initiation or continuation of this signaling unit could be prevented by inhibiting PKCα, calcineurin, or NFATc3 activation or with L-type Ca²⁺ channel blockers. Green arrows indicate activation; red arrows indicate inhibition/down-regulation.

Figure 4. Signal mass of Ca²⁺ sparklets in smooth muscle

Amplitude histograms of ΔQ_Ca from low and high nP_s Ca²⁺ sparklet sites at −70 (A) and −40 mV (B). (C) Scatter plot of ΔQ_Ca amplitudes from low and high nP_s Ca²⁺ sparklet sites at −70 and −40 mV; the red line represents the median value for each column of values. Figure redrawn from [10].

Figure 5. The effects of blocking Ca²⁺ sparklets on global [Ca²⁺]_i at -70 mV

Simulation of the effects of blocking persistent Ca²⁺ sparklets on global [Ca²⁺]_i, measured in a cell loaded with Fluo-5F using epifluorescence (A) or TIRF (B) microscopy. Right side panels show all-points histograms from the [Ca²⁺]_i records undergoing a decrease of 14 nM [Ca²⁺]_i. Solid lines in the histograms represent the best fit of the data using a Gaussian function.