Growth of calcium-blind mutants of Yersinia pestis at 37 °C in permissive Ca2+-deficient environments

Cells of wild-type Yersinia pestis exhibit a low-calcium response (LCR) defined as bacteriostasis with expression of a pCD-encoded type III secretion system (T3SS) during cultivation at 37 °C without added Ca2+ versus vegetative growth with downregulation of the T3SS with Ca2+ (≥2.5 mM). Bacteriostasis is known to reflect cumulative toxicity of Na+, l-glutamic acid and culture pH; control of these variables enables full-scale growth (‘rescue’) in the absence of Ca2+. Several T3SS regulatory proteins modulate the LCR, because their absence promotes a Ca2+-blind phenotype in which growth at 37 °C ceases and the T3SS is constitutive even with added Ca2+. This study analysed the connection between the LCR and Ca2+ by determining the response of selected Ca2+-blind mutants grown in Ca2+-deficient rescue media containing Na+ plus l-glutamate (pH 5.5), where the T3SS is not expressed, l-glutamate alone (pH 6.5), where l-aspartate is fully catabolized, and Na+ alone (pH 9.0), where the electrogenic sodium pump NADH : ubiquinone oxidoreductase becomes activated. All three conditions supported essentially full-scale Ca2+-independent growth at 37 °C of wild-type Y. pestis as well as lcrG and yopN mutants (possessing a complete but dysregulated T3SS), indicating that bacteriostasis reflects a Na+-dependent lesion in bioenergetics. In contrast, mutants lacking the negative regulator YopD or the YopD chaperone (LcrH) failed to grow in any rescue medium and are therefore truly temperature-sensitive. The Ca2+-blind yopD phenotype was fully suppressed in a Ca2+-independent background lacking the injectisome-associated inner-membrane component YscV but not peripheral YscK, suggesting that the core translocon energizes YopD.

Voltage Dependence of the Apparent Affinity for External Na+ of the Backward-Running Sodium Pump

The steady-state voltage and [Na+]o dependence of the electrogenic sodium pump was investigated in voltage-clamped internally dialyzed giant axons of the squid, Loligo pealei, under conditions that promote the backward-running mode (K+-free seawater; ATP- and Na+-free internal solution containing ADP and orthophosphate). The ratio of pump-mediated 42K+ efflux to reverse pump current, Ipump (both defined by sensitivity to dihydrodigitoxigenin, H2DTG), scaled by Faraday's constant, was −1.5 ± 0.4 (n = 5; expected ratio for 2 K+/3 Na+ stoichiometry is −2.0). Steady-state reverse pump current-voltage (Ipump-V) relationships were obtained either from the shifts in holding current after repeated exposures of an axon clamped at various Vm to H2DTG or from the difference between membrane I-V relationships obtained by imposing Vm staircases in the presence or absence of H2DTG. With the second method, we also investigated the influence of [Na+]o (up to 800 mM, for which hypertonic solutions were used) on the steady-state reverse Ipump-V relationship. The reverse Ipump-V relationship is sigmoid, Ipump saturating at large negative Vm, and each doubling of [Na+]o causes a fixed (29 mV) rightward parallel shift along the voltage axis of this Boltzmann partition function (apparent valence z = 0.80). These characteristics mirror those of steady-state 22Na+ efflux during electroneutral Na+/Na+ exchange, and follow without additional postulates from the same simple high field access channel model (Gadsby, D.C., R.F. Rakowski, and P. De Weer, 1993. Science. 260:100–103). This model predicts valence z = nλ, where n (1.33 ± 0.05) is the Hill coefficient of Na binding, and λ (0.61 ± 0.03) is the fraction of the membrane electric field traversed by Na ions reaching their binding site. More elaborate alternative models can accommodate all the steady-state features of the reverse pumping and electroneutral Na+/Na+ exchange modes only with additional assumptions that render them less likely.

Pharmacological Block of the Electrogenic Sodium Pump Disrupts Rhythmic Bursting Induced by Strychnine and Bicuculline in the Neonatal Rat Spinal Cord

Ballerini, Laura, Enrico Bracci, and Andrea Nistri. Pharmacological block of the electrogenic sodium pump disrupts rhythmic bursting induced by strychnine and bicuculline in the neonatal rat spinal cord. J. Neurophysiol. 77: 17–23, 1997. The cellular mechanisms underlying rhythmic bursts induced in the isolated neonatal rat spinal cord by bath application of strychnine and bicuculline (which block glycine- and γ-aminobutyric acid-A-receptor-mediated inhibition, respectively) were probed with pharmacological tools. Such spontaneous bursts were recorded either intracellularly from lumbar motoneurons or extracellularly from ventral roots. As previously described, these network-driven events consisted of large-amplitude depolarizations arising abruptly from baseline with a highly regular period (on average 28 s). Burst episodes (lasting on average 7 s) comprised several oscillations and appeared synchronously on flexor and extensor motoneuron pools of both sides of the spinal cord. Their diffuse location made convenient to use bath-applied substances in the attempt to selectively block distinct membrane processes operating through the network. Application of apamin (0.4 μM) shortened both cycle period and burst duration without changing their regular rhythmicity. Similar results were obtained with carbachol (10 μM). Cs+ (4 mM) reversibly hyperpolarized the motoneuron membrane potential and largely increased burst duration, which was characterized by a long series of repetitive oscillatory waves. Cycle period and rhythmicity remained unaltered. Ouabain (10 μM), strophanthidin (4 μM), or K+-free solutions disrupted rhythmic bursting, which was fragmented into irregularly occurring paroxysmal activity mixed with short depolarizing events, still developing simultaneously on both sides of the spinal cord. Bursting activity eventually ceased after ∼30–40 min of application of ouabain or strophanthidin. Prolonged washout of strophanthidin or K+-free solutions reestablished regular bursting patterns, whereas no recovery from ouabain was observed. At the time of strong depression of bursting, it was still possible to evoke bursts by single electrical pulses applied to the segmental dorsal root. Antidromic spikes of motoneurons could still be evoked by ventral root stimulation. These results demonstrate that, in a spinal bursting network mainly made up by excitatory processes, blockers of slow Ca2+-dependent K+ currents, such as apamin or carbachol, or of the slow inward rectifier, such as Cs+, did not suppress rhythmicity, suggesting that these conductances simply contributed to control cycle period and/or burst duration. Conversely, pharmacological blockers of the electrogenic Na+ pump such as ouabain, strophanthidin, or K+-free solutions severely disrupted all characteristics of rhythmic bursting. It is proposed that the operation of the electrogenic Na+ pump of premotoneurons was a crucial element for rhythmic bursting.