Voltage-dependent inward currents in smooth muscle cells of skeletal muscle arterioles

<div><p>Voltage-dependent inward currents responsible for the depolarizing phase of action potentials were characterized in smooth muscle cells of 4^th order arterioles in mouse skeletal muscle. Currents through L-type Ca²⁺ channels were expected to be dominant; however, action potentials were not eliminated in nominally Ca²⁺-free bathing solution or by addition of L-type Ca²⁺ channel blocker nifedipine (10 μM). Instead, Na⁺ channel blocker tetrodotoxin (TTX, 1 μM) reduced the maximal velocity of the upstroke at low, but not at normal (2 mM), Ca²⁺ in the bath. The magnitude of TTX-sensitive currents recorded with 140 mM Na⁺ was about 20 pA/pF. TTX-sensitive currents decreased five-fold when Ca²⁺ increased from 2 to 10 mM. The currents reduced three-fold in the presence of 10 mM caffeine, but remained unaltered by 1 mM of isobutylmethylxanthine (IBMX). In addition to L-type Ca²⁺ currents (15 pA/pF in 20 mM Ca²⁺), we also found Ca²⁺ currents that are resistant to 10 μM nifedipine (5 pA/pF in 20 mM Ca²⁺). Based on their biophysical properties, these Ca²⁺ currents are likely to be through voltage-gated T-type Ca²⁺ channels. Our results suggest that Na⁺ and at least two types (T- and L-) of Ca²⁺ voltage-gated channels contribute to depolarization of smooth muscle cells in skeletal muscle arterioles. Voltage-gated Na⁺ channels appear to be under a tight control by Ca²⁺ signaling.</p></div

Tail currents of low-voltage Ca²⁺ channels are slow.

<p><b>A)</b> Test voltage protocol (top) and representative tail currents for pre-pulses to -20 mV (tracings a) and +30 mV (tracings b). <b>B)</b> Averaged time constants of tail currents recorded at different voltages (n = 4). <b>C</b>) The average amplitudes of tail currents recorded at different voltages (n = 4). Tail currents from -20 mV were fitted by a single exponential. Tail currents from 30 mV were fitted by the sum of two exponentials. Kinetics of the slow component elicited after pre-pulse to 30 mV was similar to the tail current elicited after pre-pulse to -20 mV.</p

Two types of voltage-gated Ca²⁺ channels.

<p><b>A)</b> Whole-cell Ba²⁺ currents were recorded in the presence of 1μM TTX. Two kinetically different components were observed. Voltage steps up to –10 mV produced fast inactivating current (tracings a, n = 6). Further depolarization activated slow inactivating current of greater amplitude (tracings b, n = 6). <b>B)</b> In the presence of 10 μM L-type Ca²⁺ channel blocker nifedipine (n = 6), the rapidly activated component inactivated notably faster with Ba²⁺ (left panel) than with Ca²⁺ (right panel). <b>C)</b> Averaged peak current-voltage relationships determined in the presence of 1 μM TTX and 10 μM nifedipine as indicated. The nifedipine-resistant current peaked at about 0 mV. Its magnitude was nearly the same with Ba²⁺ and with Ca²⁺ (compare open and filled squares).</p

Inactivation of low-voltage activated Ca²⁺ currents.

<p><b>A)</b> 100 ms long pre-pulse to -20 mV inactivates Ca²⁺ current that peaks at –20 mV (tracings a) but not the one that peaks at +30 mV (tracings b). <b>B)</b> Voltage-dependence of inactivation of low- (filled circles) and high- (open circles) voltage- activated Ca²⁺ currents (n = 3). The smooth lines are the best fits by equation .</p

Electrical properties of smooth muscle cells.

<p><b>A</b>, Skeletal muscle arterioles were loaded with 10 μM Fluo-4 and visualized under the microscope while gently pressed to a cover slip by a holding pipette prior to patch-clamp experiments (left panel). Fourth-order arterioles were indentified based on their morphology, with a single layer of smooth muscle cells (middle panel) and endothelial cells (right panel) oriented perpendicular to each other). <b>B</b>, Capacitive transients were measured in response to the voltage step from –70 to –60 mV. The electrically coupled cells (trace a) became uncoupled after treatment with the gap-junction inhibitory peptides during gramicidin-perforated patch clamp experiments (trace b-c) and after establishing the conventional whole-cell approach (trace d); <b>C</b>, Distribution of the resting membrane potential values was fitted by a single Gaussian function peaking at –77 ± 2 mV, n = 81 (smooth line). The average resting potential was V_rest = –68 ± 2 mV, n = 81.</p

TTX affected action potentials in the absence of extracellular Ca²⁺.

<p><b>A)</b> Hyperpolarizing current steps produced only passive voltage responses during gramicidin-perforated patch clamp experiments whereas depolarizing current steps induced action potentials starting from the threshold of about –50 mV. <b>B)</b> Effects of 1 μM TTX (n = 6, blue trace) and 10 μM nifedipine (n = 6, red trace) are shown in panel a at normal physiological conditions (2Ca solution, n = 10); in nominally Ca²⁺-free (0Ca, n = 4) solution, application of 1 μM TTX (0Ca + 1TTX, n = 4) had much greater effect as shown in panel b. <b>C)</b> Averaged effects of TTX and nifedipine on the maximal rate of upstroke (as recorded in Fig 2B). Maximal rates of upstrokes recorded in different solutions were normalized to that recorded in 2Ca. TTX and nifedipine significantly reduced the maximal rate of upstroke in 2Ca solution. The effect of TTX was significantly greater in 0Ca than in 2Ca solutions. Asterisks indicate significance of the difference from control values in the 2Ca solution (*, <i>P</i><0.05; **, <i>P</i><0.01).</p

TTX-sensitive Na⁺ currents.

<p><b>A)</b> Steps to different voltages (indicated) produced inward currents with at least two kinetically distinct components (2Ca solution, n = 6). The fast component was through voltage-gated Na⁺ channels as it was blocked by application of 1 μM TTX (n = 6). It was partially blocked by extracellular Ca²⁺ (10Ca solution, n = 6). The thick lines highlight the maximal currents recorded at 10 mV. <b>B)</b> Averaged peak current–voltage relationships of inward currents recorded in 2Ca (n = 6) and 10Ca (n = 4) bath solutions.</p

Image1_Single cell phototransfection of mRNAs encoding SARS-CoV2 spike and nucleocapsid into human astrocytes results in RNA dependent translation interference.TIF

Multi-RNA co-transfection is starting to be employed to stimulate immune responses to SARS-CoV-2 viral infection. While there are good reasons to utilize such an approach, there is little background on whether there are synergistic RNA-dependent cellular effects. To address this issue, we use transcriptome-induced phenotype remodeling (TIPeR) via phototransfection to assess whether mRNAs encoding the Spike and Nucleocapsid proteins of SARS-CoV-2 virus into single human astrocytes (an endogenous human cell host for the virus) and mouse 3T3 cells (often used in high-throughput therapeutic screens) synergistically impact host cell biologies. An RNA concentration-dependent expression was observed where an increase of RNA by less than 2-fold results in reduced expression of each individual RNAs. Further, a dominant inhibitory effect of Nucleocapsid RNA upon Spike RNA translation was detected that is distinct from codon-mediated epistasis. Knowledge of the cellular consequences of multi-RNA transfection will aid in selecting RNA concentrations that will maximize antigen presentation on host cell surface with the goal of eliciting a robust immune response. Further, application of this single cell stoichiometrically tunable RNA functional genomics approach to the study of SARS-CoV-2 biology promises to provide details of the cellular sequalae that arise upon infection in anticipation of providing novel targets for inhibition of viral replication and propagation for therapeutic intervention.</p