1. Individual cells were isolated from adult rats ventricular myocardium by a collagenase digestion procedure. 2. Steady membrane potentials recorded with conventional intracellular glass micro-electrodes from cells in a modified Krebs solution containing 3 . 8 mM-KCl and 0 . 5 mM-CaCl2 were less negative than -40 mV in most cells (-25 . 3 +/- 10 . 9 mV, mean +/- S.D., 211 cells). 3. After addition of the potassium selective ionophore valinomycin (60 nM) to the bathing solution all recorded membrane potentials were more negative than -60 mV (-74 . 8 +/- 7 . 0 mV, sixty-three cells). 4. The internal concentration of potassium in the cells was determined as 120 . 8 +/- 1 . 7 mM (+/- S.E., n = 24) by flame emission spectrometry after centrifugation through silicone oil, using tritiated water and D-[1-14C] mannitol to estimate total and extracellular water in the pellet. 5. In the majority of cells in the standard solution the membrane potential recorded within a few msec of penetration was more negative than -70 mV (-78 . 4 +/- 9 . 7 mV, seventy-three cells). In sixty-six cells penetration initiated an action potential which overshot zero by 31 . 3 +/- 7 . 1 mV. This overshoot was abolished by reducing the external sodium to 0 . 1 of the normal value, and reduced or abolished by addition of tetrodotoxin (30 microM). 6. Modifications of the standard bathing solution which increased the number of cells with steady recorded membrane potentials more negative than -60 mV were: isosmotic substitution of sucrose for NaCl; replacement of NaCl and KCl by sodium isethionate and potassium methyl sulphate; addition of 5 or 10 mM-CaCl2; addition of 10 mM-MnCl2. 7. For cells in solution containing 2 . 5 or 5 . 5 mM-CaCl2, input resistances estimated from the amplitude of hyperpolarizations evoked by 200 msec current pulses were approximately 40 M omega at a resting potential close to -80 mV and became much greater as cells were depolarized. Time constants measured at the resting potential were approximately 8 msec. 8. In certain conditions, repeated spontaneous action potentials were recorded from contracting cells, and in quiescent cells evoked action potentials could be initiated by applying brief depolarizing pulses through the micro-electrode. Action potentials were coincident with contractions. 9. It is concluded that the resting potential of these isolated cells is normally more negative than -70 mV, and that the cells retain the ionic mechanisms necessary for the generation of active currents.

Rapid antigen tests, such as the Abbott BinaxNOW COVID-19 Ag Card (BinaxNOW), offer results more rapidly (approximately 15-30 minutes) and at a lower cost than do highly sensitive nucleic acid amplification tests (NAATs) (1). Rapid antigen tests have received Food and Drug Administration (FDA) Emergency Use Authorization (EUA) for use in symptomatic persons (2), but data are lacking on test performance in asymptomatic persons to inform expanded screening testing to rapidly identify and isolate infected persons (3). To evaluate the performance of the BinaxNOW rapid antigen test, it was used along with real-time reverse transcription-polymerase chain reaction (RT-PCR) testing to analyze 3,419 paired specimens collected from persons aged ≥10 years at two community testing sites in Pima County, Arizona, during November 3-17, 2020. Viral culture was performed on 274 of 303 residual real-time RT-PCR specimens with positive results by either test (29 were not available for culture). Compared with real-time RT-PCR testing, the BinaxNOW antigen test had a sensitivity of 64.2% for specimens from symptomatic persons and 35.8% for specimens from asymptomatic persons, with near 100% specificity in specimens from both groups. Virus was cultured from 96 of 274 (35.0%) specimens, including 85 (57.8%) of 147 with concordant antigen and real-time RT-PCR positive results, 11 (8.9%) of 124 with false-negative antigen test results, and none of three with false-positive antigen test results. Among specimens positive for viral culture, sensitivity was 92.6% for symptomatic and 78.6% for asymptomatic individuals. When the pretest probability for receiving positive test results for SARS-CoV-2 is elevated (e.g., in symptomatic persons or in persons with a known COVID-19 exposure), a negative antigen test result should be confirmed by NAAT (1). Despite a lower sensitivity to detect infection, rapid antigen tests can be an important tool for screening because of their quick turnaround time, lower costs and resource needs, high specificity, and high positive predictive value (PPV) in settings * Specimens were used to perform a limiting-dilution inoculation of Vero CCL-81 cells, and cultures showing evidence of cytopathic effect were tested by real-time RT-PCR for the presence of SARS-CoV-2 RNA. Viral recovery was defined as any culture in which the first passage had an N1 Ct value at least two Ct values lower than the corresponding clinical specimen. † https://www.biorxiv

1. Rapid inward Na current (INa) was studied in isolated cells from rat ventricular myocardium by a double-suction-pipette voltage clamp technique. All experiments were carried out at 20-22 degrees C. 2. INa elicited by single depolarizing voltage steps from a holding potential, VH, of -80 mV had a threshold between -70 and -60 mV and was maximal at -30 to -20 mV. Peak currents in Krebs-Ringer solution containing 145 mM Na were of the order 0.9-1.8 mA cm-2, assuming an average cell surface area of 8000 square micrometers. 3. The reversal potential for INa was predicted by the Nernst equation for external Na in the range 1.45-145 mM with 16 mM-Na solution perfusing the interior of the cell. 4. Instantaneous I-V plots were linear for potentials of -100 to + 10 mV. Maximum Na conductance (-gNa) was calculated to be 25 mS cm-2 in 145 mM-Na solutions and gNa was constant for potentials positive to -10 mV. 5. INa activated with a time constant of 0.7 msec at -55 mV, decreasing to 100 microsec on depolarizations positive to + 10 mV. 6. Two time constants (tau h1, tau h2) were required to describe INa inactivation during a maintained depolarization, with tau h2 three to four times as long as tau h1. tau h1 was about 2 msec at -50 mV, decreasing to 0.9 msec at -10 mV. 7. The time course for recovery of INa from inactivation also exhibited two time constants (tau r1, tau r2), with the longer tau r2 having a maximum value of the order 100 msec in the potential range -60 to -80 mV. 8. INa in isolated rat cardiac cells has a low sensitivity to tetrodotoxin, requiring a concentration of 30 micrometers for complete blockade.

The second messenger NAADP triggers Ca2+ release from endo‐lysosomes. Although two‐pore channels (TPCs) have been proposed to be regulated by NAADP, recent studies have challenged this. By generating the first mouse line with demonstrable absence of both Tpcn1 and Tpcn2 expression (Tpcn1/2 −/− ), we show that the loss of endogenous TPCs abolished NAADP‐dependent Ca2+ responses as assessed by single‐cell Ca2+ imaging or patch‐clamp of single endo‐lysosomes. In contrast, currents stimulated by PI(3,5)P2 were only partially dependent on TPCs. In Tpcn1/2 −/− cells, NAADP sensitivity was restored by re‐expressing wild‐type TPCs, but not by mutant versions with impaired Ca2+‐permeability, nor by TRPML1. Another mouse line formerly reported as TPC‐null likely expresses truncated TPCs, but we now show that these truncated proteins still support NAADP‐induced Ca2+ release. High‐affinity [32P]NAADP binding still occurs in Tpcn1/2 −/− tissue, suggesting that NAADP regulation is conferred by an accessory protein. Altogether, our data establish TPCs as Ca2+‐permeable channels indispensable for NAADP signalling.

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