To determine the role of cellularly generated protons in synaptic signaling, we recorded GABA miniature IPSCs (mIPSCs) from cultured rat cerebellar granule cells (CGCs) while varying the extracellular pH buffering capacity. Consistent with previous reports, we found that increasing pH from 7.4 to 8.0 sped mIPSC rise time and suppressed both amplitude of the current and total charge transferred. Conversely, acidification (from pH 7.4 to 6.8) slowed the rise time and increased current amplitude and total charge transferred. In a manner consistent with alkalinization, increasing the buffering capacity from 3 to 24 mmHEPES at pH 7.4 resulted in faster mIPSC rise time, a 37% reduction in amplitude, and a 48% reduction in charge transferred. Supplementing the normal physiological buffers (24 mmHCO₃⁻/5%CO₂) with 10 mmHEPES similarly diminished mIPSCs in a manner consistent with alkalinization, resulting in faster rise time, a 39% reduction in amplitude, and a 51% reduction in charge transferred. These findings suggest the existence of an acidifying synaptic force that is overcome by commonly used concentrations (10 mm) of HEPES buffer. Here we show that Na⁺/H⁺exchanger (NHE) activity appears to, in part, contribute to this synaptic acidification because inhibition of NHE by amiloride or lithium under physiological or weak buffering conditions alters mIPSCs in a manner consistent with alkalinization. These results suggest that acidification of the synaptic cleft occurs physiologically during GABAergic transmission and that NHE plays a critical role in generating the acidic nano-environment at the synapse.

The sinoatrial (SA) node is the primary site from which the mammalian heart is paced, but the mechanisms underlying the pacemaking still remain clouded. It is generally believed that the hyperpolarization-activated current I_f, encoded by hyperpolarization-activated cyclic nucleotide–gated (HCN) genes, contributes significantly to pacing, which in tandem with inward current generated by efflux of Ca²⁺ via the Na⁺–Ca²⁺ exchanger (NCX), resulting from the released Ca²⁺, mediates the diastolic depolarization. Here, we review the data that implicate I_f as the “pacemaker current” and conclude that there is not only a significant discrepancy between the range of diastolic depolarization potential (–60 to –40 mV) and the activation potential of I_f (negative to –70 mV), but that also the kinetics of I_f and its pharmacology are incompatible with the frequency of a heartbeat in rodents and humans. We propose that I_f serves as a functional insulator, which protects the SA-nodal cells against the large negative electrical sink of atrial tissue connected to it with connexins. We also evaluate the role of I_f and calcium signaling in mediating the diastolic depolarization in rat neonatal cardiomyocytes (rN-CM), and human induced pluripotent stem-cell derived cardiomyocytes (hiPSC-CM), and provide evidence for a possible involvement of mitochondrial Ca²⁺ in initiating the oscillatory events required for the spontaneous pacing.