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Phosphatidylinositol bisphosphate (PIP2) directly regulates functions as diverse as the organization of the cytoskeleton, vesicular transport and ion channel activity. It is not known, however, whether dynamic changes in PIP2 levels have a regulatory role of physiological importance in such functions. Here, we show in both native cardiac cells and heterologous expression systems that receptor-regulated PIP2 hydrolysis results in desensitization of a GTP-binding protein-stimulated potassium current. Two receptor-regulated pathways in the plasma membrane cross-talk at the level of these channels to modulate potassium currents. One pathway signals through the betagamma subunits of G proteins, which bind directly to the channel. Gbetagamma subunits stabilize interactions with PIP2 and lead to persistent channel activation. The second pathway activates phospholipase C (PLC) which hydrolyses PIP2 and limits Gbetagamma-stimulated activity. Our results provide evidence that PIP2 itself is a receptor-regulated second messenger, downregulation of which accounts for a new form of desensitization.

Transient increase in intracellular free Ca2؉ concentration generated by the voltage-gated Ca v 1.2 channels acts as an important intracellular signal. By using fluorescence resonance energy transfer combined with patch clamp in living cells, we present evidence for voltage-gated mobility of the cytoplasmic tails of the Ca v 1.2 channel and for its regulatory role in intracellular signaling. Anchoring of the C-terminal tail to the plasma membrane caused an inhibition of its state-dependent mobility, channel inactivation, and CREB-dependent transcription. Release of the tail restored these functions suggesting a direct role for voltage-gated mobility of the C-terminal tail in Ca 2؉ signaling.The ion conductance of voltage-dependent ion channels is tightly regulated by the gating mechanism encoded in membrane voltage sensors and cytoplasmic gates of the pore-forming ␣ 1 subunit (1). This voltage gating is due to the displacement of membrane charges (2) that has been documented as gating current (3) and further characterized as conformational rearrangements detected by spectroscopy of the attached fluorescent groups (4 -6). However, little is known about a functional role for state-dependent mobility of the channel cytoplasmic tails (7). Such motion of the L-type (Ca v 1.2) Ca 2ϩ channel C-terminal tail that binds calmodulin (CaM) 1 may serve to transfer a regulatory signal (8, 9). Thus, investigation of the mobility of cytoplasmic regions of Ca 2ϩ channels associated with their gating is important for understanding of the molecular correlates of channel regulation and mechanisms of Ca 2ϩ signaling. Spectral properties of the enhanced cyan (ECFP) and yellow fluorescent proteins (EYFP) (10) are well suited for measurements of molecular rearrangements by FRET (11). Here we genetically fused the cytoplasmic N and C termini of the human Ca v 1.2 channel ␣ 1C pore-forming subunits with EYFP and ECFP, respectively. The labeled channels were then functionally expressed in COS1 cells. The rearrangements of the tails due to transition into the distinct functional states of the channel were monitored using FRET (12) in living cells under voltage clamp conditions. A regulatory role for the voltage-gated mobility of the C-terminal tail was then characterized. EXPERIMENTAL PROCEDURESMolecular Biology-(EYFP) N -␣ 1C,77 and (EYFP) N -␣ 1C,IS-IV expression plasmids were prepared in pcDNA3 vector essentially as described earlier (13, 14) using pEYFP vector (Clontech). To prepare the PH-EYFP and PH-ECFP expression plasmids, the 518-bp EcoRI/BamHI fragment of pPLCPH-enhanced green fluorescent protein (15) was ligated into the pECFP-1 and pEYFP-1 plasmids, respectively, at EcoRI/ BamHI sites. To prepare the ␣ 1C,77 -(ECFP) C expression plasmid, the 1029-bp AatII/NotI fragment of 77pcDNA3 (13) Electrophysiology-COS1 cells were grown on poly-D-lysine-coated coverslips (MatTek) in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. Cells were transfected with pcDNA3 vectors coding for ␣ 1C , ␤ 1 (17) (or ␤ 2a (1...

The association of ␣ 1C with the auxiliary ␣ 2 ␦ and ␤ subunits is important for the functional expression of Ca v 1.2 channels. The cytoplasmic ␤ subunit binds to a conserved "␣-interaction domain" in the ␣ 1C subunit cytoplasmic linker between transmembrane repeats I and II (8, 9). The extracellular ␣ 2 subunit is bound via an SS bridge to its post-translationally cleaved transmembrane ␦ peptide (10, 11) that renders association with ␣ 1C . Both ␣ 2 ␦ (12-14) and ␤ subunits (15-19) modulate the channel. In particular, ␤ subunits affect the time course of the Ba 2ϩ current decay up to 3-fold depending on the type of the ␤ subunit.To study conformational rearrangements in the channel in response to depolarization, measurements of differential changes in fluorescence resonance energy transfer (FRET) between the cyan (ECFP) and yellow (EYFP) fluorescent proteins fused to the ␣ 1C and ␤ subunit termini have been an effective approach. The current findings begin to specify the central features of conformational rearrangements associated with the transition of the channel from the resting (Ϫ90 mV) to the inactivated state of Ca v 1. Table 1.¶ To whom correspondence should be addressed: Laboratory of Clinical Investigation, National Institute on Aging, 5600 Nathan Shock Dr., Baltimore, MD 21224. Tel.: 410-558-8343; Fax: 410-558-8318; E-mail: soldatovN@grc.nia.nih.gov. 1 The abbreviations used are: CaM, calmodulin; ADSI, the annular determinant of slow inactivation; CDI, Ca 2ϩ -dependent inactivation; I-V, current-voltage; PH, pleckstrin homology; PIP 2 , phosphatidylinositol bisphosphate; ECFP, enhanced cyan fluorescent protein; EYFP, enhanced yellow fluorescent protein; FRET, fluorescence resonance energy transfer.

Auxiliary beta-subunits bound to the cytoplasmic alpha(1)-interaction domain of the pore-forming alpha(1C)-subunit are important modulators of voltage-gated Ca(2+) channels. The underlying mechanisms are not yet well understood. We investigated correlations between differential modulation of inactivation by beta(1a)- and beta(2)- subunits and structural responses of the channel to transition into distinct functional states. The NH(2)-termini of the alpha(1C)- and beta-subunits were fused with cyan or yellow fluorescent proteins, and functionally coexpressed in COS1 cells. Fluorescence resonance energy transfer (FRET) between them or with membrane-trapped probes was measured in live cells under voltage clamp. It was found that in the resting state, the tagged NH(2)-termini of the alpha(1C)- and beta-subunit fluorophores are separated. Voltage-dependent inactivation generates strong FRET between alpha(1C) and beta(1a) suggesting mutual reorientation of the NH(2)-termini, but their distance vis-à-vis the plasma membrane is not appreciably changed. These voltage-gated rearrangements were substantially reduced when the beta(1a)-subunit was replaced by beta(2). Differential beta-subunit modulation of inactivation and of FRET between alpha(1C) and beta were eliminated by inhibition of the slow inactivation. Thus, differential beta-subunit modulation of inactivation correlates with the voltage-gated motion between the NH(2)-termini of alpha(1C)- and beta-subunits and targets the mechanism of slow voltage-dependent inactivation.

Abstract-Fluorescence microscopy imaging has become one of the most useful techniques to assess the activity of individual cells, subcellular trafficking of signals to and between organelles, and to appreciate how organelle function is regulated. The past 2 decades have seen a tremendous advance in the rational design and development in the nature and selectivity of probes to serve as reporters of the intracellular environment in live cells. These probes range from small organic fluorescent molecules to fluorescent biomolecules and photoproteins ingeniously engineered to follow signaling traffic, sense ionic and nonionic second messengers, and report various kinase activities. These probes, together with recent advances in imaging technology, have enabled significantly enhanced spatial and temporal resolution. This review summarizes some of these developments and their applications to assess intracellular organelle function. Key Words: microscopy Ⅲ calcium Ⅲ redox Ⅲ mitochondria Ⅲ fluorescent proteins R esponses of single cells undergoing physiological activation processes or pathological stresses can be quite heterogeneous in time and space. Measurements of these parameters in bulk (such as in tissue or cellular suspension), using conventional physiological and biochemical methods, can often fail to resolve discrete differences in the kinetics and magnitudes of the responses between cells, as well as complex intracellular and subcellular dynamical processes, critical to understanding how cells actually work. For example, during embryonic development or periods of environmental stress, the decision for a certain cell to undergo apoptosis may be triggered in a moment and distinct from its neighbors, the resulting intracellular processes may proceed along a more or less protracted time frame, and these events may occur heterogeneously among (and inside) other cells. Yet, bulk measures of the progression of apoptosis, such as by DNA-laddering, although undeniably valuable, would show only the overall slow progression with time. All-ornone phenomenon occurring in discrete organelles, such as the mitochondrial permeability transition (MPT), appears as a graded response when examined in bulk suspensions of cells or mitochondria. Similar arguments can be made for resolving questions of Ca 2ϩ signaling and gene expression. Ca and, unless they are synchronized (such as in the beating heart), their occurrence or nature (eg, frequency, amplitude, etc) might not be apparent from ensemble measurements. Significant information about these processes would obviously need to use a single cells studies. Currently, one of the most useful techniques to assess the activities of individual cells, subcellular trafficking of signals to and between organelles, and to appreciate how organelle function is regulated is based on fluorescence microscopy imaging. Each of the various imaging techniques requires that the signaling molecule(s), compartment(s), or organelle(s) be labeled in a specific fashion such that they can be tracked in time...

It is generally accepted that to generate calcium currents in response to depolarization, Cav1.2 calcium channels require association of the pore-forming ␣1C subunit with accessory Cav␤ and ␣2␦ subunits. A single calmodulin (CaM) molecule is tethered to the C-terminal ␣1C-LA/IQ region and mediates Ca 2؉ -dependent inactivation of the channel. Cav␤ subunits are stably associated with the ␣1C-interaction domain site of the cytoplasmic linker between internal repeats I and II and also interact dynamically, in a Ca 2؉ -dependent manner, with the ␣1C-IQ region. Here, we describe a surprising discovery that coexpression of exogenous CaM (CaMex) with ␣1C/␣2␦ in COS1 cells in the absence of Cav␤ subunits stimulates the plasma membrane targeting of ␣1C, facilitates calcium channel gating, and supports Ca 2؉ -dependent inactivation.

Constitutive Ca2+/calmodulin (CaM)-activation of adenylyl cyclases (ACs) types 1 and 8 in sinoatrial nodal cells (SANC) generates cAMP within lipid-raft-rich microdomains to initiate cAMP–protein kinase A (PKA) signaling, that regulates basal state rhythmic action potential firing of these cells. Mounting evidence in other cell types points to a balance between Ca2+-activated counteracting enzymes, ACs and phosphodiesterases (PDEs) within these cells. We hypothesized that the expression and activity of Ca2+/CaM-activated PDE Type 1A is higher in SANC than in other cardiac cell types. We found that PDE1A protein expression was 5-fold higher in sinoatrial nodal tissue than in left ventricle, and its mRNA expression was 12-fold greater in the corresponding isolated cells. PDE1 activity (nimodipine-sensitive) accounted for 39% of the total PDE activity in SANC lysates, compared to only 4% in left ventricular cardiomyocytes (LVC). Additionally, total PDE activity in SANC lysates was lowest (10%) in lipid-raft-rich and highest (76%) in lipid-raft-poor fractions (equilibrium sedimentation on a sucrose density gradient). In intact cells PDE1A immunolabeling was not localized to the cell surface membrane (structured illumination microscopy imaging), but located approximately within about 150 nm inside of immunolabeling of hyperpolarization-activated cyclic nucleotide-gated potassium channels (HCN4), which reside within lipid-raft-rich microenvironments. In permeabilized SANC, in which surface membrane ion channels are not functional, nimodipine increased spontaneous SR Ca2+ cycling. PDE1A mRNA silencing in HL-1 cells increased the spontaneous beating rate, reduced the cAMP, and increased cGMP levels in response to IBMX, a broad spectrum PDE inhibitor (detected via fluorescence resonance energy transfer microscopy). We conclude that signaling via cAMP generated by Ca2+/CaM-activated AC in SANC lipid raft domains is limited by cAMP degradation by Ca2+/CaM-activated PDE1A in non-lipid raft domains. This suggests that local gradients of [Ca2+]–CaM or different AC and PDE1A affinity regulate both cAMP production and its degradation, and this balance determines the intensity of Ca2+-AC-cAMP-PKA signaling that drives SANC pacemaker function.