The large-conductance Ca 2+ - and voltage-activated K + channel (BK Ca, MaxiK), which is encoded by the Kcnma1 gene, is generally expressed at the plasma membrane of excitable and nonexcitable cells. However, in adult cardiomyocytes, a BK Ca -like channel activity has been reported in the mitochondria but not at the plasma membrane. The putative opening of this channel with the BK Ca agonist, NS1619, protects the heart from ischemic insult. However, the molecular origin of mitochondrial BK Ca (mitoBK Ca ) is unknown because its linkage to Kcnma1 has been questioned on biochemical and molecular grounds. Here, we unequivocally demonstrate that the molecular correlate of mitoBK Ca is the Kcnma1 gene, which produces a protein that migrates at ∼140 kDa and arranges in clusters of ∼50 nm in purified mitochondria. Physiological experiments further support the origin of mitoBK Ca as a Kcnma1 product because NS1619-mediated cardioprotection was absent in Kcnma1 knockout mice. Finally, BK Ca transcript analysis and expression in adult cardiomyocytes led to the discovery of a 50-aa C-terminal splice insert as essential for the mitochondrial targeting of mitoBK Ca .

The basic functional unit of the large-conductance, voltage-and Ca 2+ -activated K + (MaxiK, BK, BK Ca ) channel is a tetramer of the pore-forming α-subunit (MaxiKα) encoded by a single gene, Slo, holding multiple alternative exons. Depending on the tissue, MaxiKα can associate with modulatory β-subunits (β1-β4) increasing its functional diversity. As MaxiK senses and regulates membrane voltage and intracellular Ca 2+ , it links cell excitability with cell signalling and metabolism. Thus, MaxiK is a key regulator of vital body functions, like blood flow, uresis, immunity and neurotransmission. Epilepsy with paroxysmal dyskinesia syndrome has been recognized as a MaxiKα-related disorder caused by a gain-of-function C-terminus mutation. This channel region is also emerging as a key recognition module containing sequences for MaxiKα interaction with its surrounding signalling partners, and its targeting to cell-specific microdomains. The growing list of interacting proteins highlights the possibility that associations with the C-terminus of MaxiKα are dynamic and depending on each cellular environment. We speculate that the molecular multiplicity of the C-terminus (and intracellular loops) dictated by alternative exons may modulate or create additional interacting sites in a tissue-specific manner. A challenge is the dissection of MaxiK macromolecular signalling complexes in different tissues and their temporal association/dissociation according to the stimulus.

Neurons and neural networks often extend hundreds to thousands of micrometers in three dimensions. To capture all the calcium transients associated with their activity, we need volume imaging methods with sub-second temporal resolution. Such speed is challenging for conventional two-photon laser scanning microscopy (2PLSM) to achieve, because of its dependence on serial focal scanning in 3D and the limited brightness of indicators. Here we present an optical module that can be easily integrated into standard 2PLSMs to generate an axially elongated Bessel focus. Scanning the Bessel focus in 2D turned frame rate into volume rate and enabled video-rate volumetric imaging. Using Bessel foci designed to maintain lateral resolution that resolves synapses in sparsely labeled brains in vivo, we demonstrated the power of this approach in enabling discoveries for neurobiology by imaging the calcium dynamics of volumes of neurons and synapses in fruit flies, zebrafish larvae, mice, and ferrets in vivo.

The large conductance, voltage-and Ca 2؉ -activated potassium (MaxiK, BK) channel and caveolin-1 play important roles in regulating vascular contractility. Here, we hypothesized that the MaxiK ␣-subunit (Slo1) and caveolin-1 may interact with each other. Slo1 and caveolin-1 physiological association in native vascular tissue is strongly supported by (i) detergent-free purification of caveolin-1-rich domains demonstrating a pool of aortic Slo1 co-migrating with caveolin-1 to light density sucrose fractions, (ii) reverse co-immunoprecipitation, and (iii) double immunolabeling of freshly isolated myocytes revealing caveolin-1 and Slo1 proximity at the plasmalemma. In HEK293T cells, Slo1-caveolin-1 association was unaffected by the smooth muscle MaxiK ␤1-subunit. Sequence analysis revealed two potential caveolin-binding motifs along the Slo1 C terminus, one equivalent, 1007 YNMLCFGIY 1015 , and another mirror image, 537 YT-EYLSSAF 545 , to the consensus sequence, XXXX XX . Deletion of 1007 YNMLCFGIY 1015 caused ϳ80% loss of Slo1-caveolin-1 association while preserving channel normal folding and overall Slo1 and caveolin-1 intracellular distribution patterns. 537 YTEYLSSAF 545 deletion had an insignificant dissociative effect. Interestingly, caveolin-1 coexpression reduced Slo1 surface and functional expression near 70% without affecting channel voltage sensitivity, and deletion of 1007 YNMLCF-GIY 1015 motif obliterated channel surface expression. The results suggest 1007 YNMLCFGIY 1015 possible participation in Slo1 plasmalemmal targeting and demonstrate its role as a main mechanism for caveolin-1 association with Slo1 potentially serving a dual role: (i) maintaining channels in intracellular compartments downsizing their surface expression and/or (ii) serving as anchor of plasma membrane resident channels to caveolin-1-rich membranes. Because the caveolin-1 scaffolding domain is juxtamembrane, it is tempting to suggest that Slo1-caveolin-1 interaction facilitates the tethering of the Slo1 C-terminal end to the membrane.Large conductance, voltage-and Ca 2ϩ -activated potassium (MaxiK, BK) 4 channels play important roles in vascular, neuronal, and urinary functions. In vascular smooth muscle, MaxiK channel appears to be a unique signaling protein because of its ability to mediate the effects of several vasoconstricting as well as vasodilating agents. The ability of MaxiK protein to complete with high fidelity these opposite tasks calls for specific associations and subcellular compartmentalization with corresponding signaling partners (1). Recently, it has been appreciated that many signaling molecules are segregated primarily in specialized microdomains like caveolae (plasma membrane invaginations enriched with cholesterol and caveolin protein), thereby, optimizing signal transduction between agonists and specific effectors (2).Three caveolin proteins have been identified, caveolin-1, -2, and -3. All of them seem to be expressed in smooth muscle (3, 4). However, gene ablation experiments have shown that caveolin-1 p...

Imaging neurons and neural circuits over large volumes at high speed and subcellular resolution is a difficult task. Incorporating a Bessel focus module into a two-photon fluorescence mesoscope, we achieved rapid volumetric imaging of neural activity over mesoscale with synaptic resolution. We applied it to calcium imaging of entire dendritic spans of neurons as well as neural ensembles within multiple cortical regions over two hemispheres of the awake mouse brain.