Calcium-Activated Big-Conductance (BK) Potassium Channels Traffic through Nuclear Envelopes into Kinocilia in Ray Electrosensory Cells.

Electroreception through ampullae of Lorenzini in the little skate, Leucoraja erinacea, involves functional coupling between voltage-activated calcium channels (CaV1.3, cacna1d) and calcium-activated big-conductance potassium (BK) channels (BK, kcnma1). Whole-mount confocal microscopy was used to characterize the pleiotropic expression of BK and CaV1.3 in intact ampullae. BK and CaV1.3 are co-expressed in electrosensory cell plasma membranes, nuclear envelopes and kinocilia. Nuclear localization sequences (NLS) were predicted in BK and CaV1.3 by bioinformatic sequence analyses. The BK NLS is bipartite, occurs at an alternative splice site for the mammalian STREX exon and contains sequence targets for post-translational phosphorylation. Nuclear localization of skate BK channels was characterized in heterologously transfected HEK293 cells. Double-point mutations in the bipartite NLS (KR to AA or SVLS to AVLA) independently attenuated BK channel nuclear localization. These findings support the concept that BK partitioning between the electrosensory cell plasma membrane, nucleus and kinocilium may be regulated through a newly identified bipartite NLS.

Further studies of ion channels in the electroreceptor of the skate through deep sequencing, cloning and cross species comparisons.

Our comparative studies seek to understand the structure and function of ion channels in cartilaginous fish that can detect very low voltage gradients in seawater. The principal channels of the electroreceptor include a calcium activated K channel whose α subunit is Kcnma1, and a voltage-dependent calcium channel, Cacna1d. It has also been suggested based on physiological and pharmacological evidence that a voltage-gated K channel is present in the basal membranes of the receptor cells which modulates synaptic transmitter release. Large conductance calcium-activated K channels (BK) are comprised of four α subunits, encoded by Kcnma1 and modulatory ß subunits of the Kcnmb class. We recently cloned and published the skate Kcnma1 gene and most of Kcnmb4 using purified mRNA of homogenized electroreceptors. Bellono et al. have recently performed RNA sequencing (RNA-seq) on purified mRNA from skate electroreceptors and found several ion channels including Kcnma1. We searched the Bellono et al. RNA-seq repository for additional channels and subunits. Our most significant findings are the presence of two Shaker type voltage dependent K channel sequences which are grouped together as isoforms in the data repository. The larger of these is a skate ortholog of the voltage dependent fast potassium channel Kv1.1, which is expressed at appreciable levels. The second ortholog is similar to Kv1.5 but has fewer N-terminal amino acids than other species. The sequence for Kv1.5 in the skate is very strongly aligned with the recently reported sequence for potassium channels in the electroreceptors of the cat shark, S. retifer, which also modulate synaptic transmission. The latter channel was designated as Kv1.3 in the initial report, but we suggest that these channels are actually orthologs of each other, and that Kv1.5 is the prevailing designation. We also found a beta subunit sequence (Kcnab2) which may co-assemble with one or both of the voltage gated channels. The new channels and subunits were verified by RT-PCR and the Kv1.1 sequence was confirmed by cloning. We also searched the RNA-seq repository for accessory subunits of Kcnma1, and found a computer-generated assembly that contained a complete sequence of its ß subunit, Kcnmb2. Skate Kcnmb2 has a total of 279 amino acids, with 51 novel amino acids at the N-terminus which may play a specific physiological role. This sequence was confirmed by PCR and cloning. However, skate Kcnmb2 is expressed at low levels in the electroreceptor compared to Kcnma1 and skate Kcnmb1 is absent. The evolutionary origin of the newly described K channels and their subunits was studied by alignments with mammalian sequences, including human, and also those in related fish: the whale shark (R. typus), the ghost shark (C.milii), and (S. retifer). There are also orthologous K channels of the lamprey, which has electroreceptors. Tree building and bootstrap programs were used to confirm phylogenetic inferences. Further research should focus on the subcellular locations of these channels, their gating behavior, and the effects of accessory subunits on gating.

Calcium activated K⁺ channels in the electroreceptor of the skate confirmed by cloning. Details of subunits and splicing.

Elasmobranchs detect small potentials using excitable cells of the ampulla of Lorenzini which have calcium-activated K(+) channels, first described in 1974. A distinctive feature of the outward current in voltage clamped ampullae is its apparent insensitivity to voltage. The sequence of a BK channel α isoform expressed in the ampulla of the skate was characterized. A signal peptide is present at the beginning of the gene. When compared to human isoform 1 (the canonical sequence), the largest difference was absence of a 59 amino acid region from the S8-S9 intra-cellular linker that contains the strex regulatory domain. The ampulla isoform was also compared with the isoform predicted in late skate embryos where strex was also absent. The BK voltage sensors were conserved in both skate isoforms. Differences between the skate and human BK channel included alternative splicing. Alternative splicing occurs at seven previously defined sites that are characteristic for BK channels in general and hair cells in particular. Skate BK sequences were highly similar to the Australian ghost shark and several other vertebrate species. Based on alignment of known BK sequences with the skate genome and transcriptome, there are at least two isoforms of Kcnma1α expressed in the skate. One of the ß subunits (ß4), which is known to decrease voltage sensitivity, was also identified in the skate genome and transcriptome and in the ampulla. These studies advance our knowledge of BK channels and suggest further studies in the ampulla and other excitable tissues.

Basic concepts of optical mapping techniques in cardiac electrophysiology.

Optical mapping is a tool used in cardiac electrophysiology to study the heart's normal rhythm and arrhythmias. The optical mapping technique provides a unique opportunity to obtain membrane potential recordings with a higher temporal and spatial resolution than electrical mapping. Additionally, it allows simultaneous recording of membrane potential and calcium transients in the whole heart. This article presents the basic concepts of optical mapping techniques as an introduction for students and investigators in experimental laboratories unfamiliar with it.

Mechanisms of calcium transient and action potential alternans in cardiac cells and tissues.

Alternation of cardiac action potential duration (APD) from beat to beat and concurrent alternation of the amplitude of the calcium transient are regarded as important arrhythmia mechanisms. These phenomena are causally interrelated and can be reliably evoked by an increase in beat frequency or by ischemia. The first part of this historical review deals with the physiology of APD alternans. Sections recounting the evolution of knowledge about calcium-activated ion currents and calcium transient alternans are interspersed among sections describing the growth of the so-called "restitution hypothesis," which involves time-dependent recovery of potassium channels (including their passage through pre-open states) as a function of diastolic interval. Major developments are generally in chronological order, but it is necessary to move back and forth between the two theories to respect the overall time line, which runs from about l965 to the present. The concluding two sections deal with the pathophysiology of calcium transient and APD alternans during ischemia, which may be the basis for out-of-hospital cardiac arrest during the initial stages of acute myocardial infarction.

Calcium and cardiac arrhythmias: DADs, EADs, and alternans.

Rapid progress has been made in understanding the molecular mechanisms by which calcium ions mediate certain cardiac arrhythmias. Principal advances include imaging of cytosolic calcium in isolated cells and in intact tissues, use of fluorescent indicators and monophasic action potentials to record membrane potentials in isolated tissue, and sequencing of the genes that encode critical ion channel proteins. In this review, five types of arrhythmias are discussed where calcium ion currents, or currents controlled by calcium, appear to be responsible for arrythmogenesis. These include: (1) the delayed afterpotential that occurs in conditions of intracellular calcium overload such as digitalis toxicity; (2) the early afterdepolarization that occurs when action potential duration is prolonged; (3) the slowly conducted calcium-dependent action potential (the slow response) in the SA and AV nodes; (4) the phenomenon of calcium transient alternans during ischemia, which is related to action potential duration alternans and t-wave alternans; (5) catecholamine-induced cardiac arrhythmias in families with mutations of the sarcoplasmic reticulum calcium-release channel. For each type of arrhythmia, the clinical implications of emerging knowledge are discussed. An especially important issue is whether ventricular fibrillation during acute coronary artery occlusion is due to calcium transient alternans. Ventricular fibrillation due to acute ischemia is an important subset of the 400,000 sudden cardiac deaths that occur annually in the U.S. Certain drugs, including beta blockers, fish oils, verapamil, and diltiazem, seem to specifically prevent ventricular fibrillation in this setting, and in most cases an effect of the drug on cytosolic calicum appears to be involved.

Spatial heterogeneity of action potential alternans during global ischemia in the rabbit heart.

Cardiac ischemia causes beat-to-beat fluctuation in action potential duration (APD) alternans, which leads to T wave alternans and arrhythmias. Occurrence of APD alternans that is out of phase at two sites is especially important, but most APD alternans studies have involved rapid pacing of normal myocardium rather than ischemia. To determine the spatial features of APD alternans during ischemia, blood-perfused rabbit hearts were stained with 4-[beta-[2(di-n-butylamino)-6-napthyl]vinyl]pyridinium (di-4-ANEPPS) and imaged with a high-resolution camera. Hearts were perfused with oxygenated Tyrode solution at 37 degrees C for staining and then switched to a 50:50% blood/Tyrode mixture. Hearts were paced from the right ventricle at 3/s, and made ischemic by stopping flow for 6 min. Images of 10,000 pixels were obtained at 300 frames/s. Motion artifact was controlled by immobilization and by manual selection of undistorted single-pixel records. Upstroke propagation and conduction isochrones were displayed by computerized image processing. APD alternans was demonstrated in six of seven hearts, and was out of phase in different regions of the image in three hearts. The largest spatial variation in the onset of depolarization to 50% repolarization (APD50) was 155%. This caused beat-to-beat reversal of repolarization. An alternans map could be constructed for well-immobilized portions of the image. There were discrete regions of APD alternans separated by a boundary, as occurs with intracellular Ca2+ concentration alternans. Pixels as close together as 1.1 mm showed an APD alternans that was out of phase. The out-of-phase APD alternans was not due to conduction alternans, as shown by upstroke intervals and conduction isochrones. This contrasts with rapid pacing, where a causal relationship appears to exist. These new observations suggest distinct mechanisms for the genesis of arrhythmias during ischemia.