Na,K pumps behave like channels controlled by extracellular- and cytoplasmic-side gates driven to open and close alternately by coupling to a cycle of Na-mediated phosphorylation and K-mediated dephosphorylation. The marine agent palytoxin disrupts that coupling, allowing both gates to sometimes be open, thereby transforming pumps into channels. So, palytoxin enables ion-pathway characterization and assays of gating mechanisms with single-molecule resolution. Palytoxin-bound Na,K pump-channels exposed to Na solutions and millimolar cytoplasmic ATP spend ∼90% of the time open, each channel conducting millions of Na ions per second. Cysteine scanning of the transmembrane domain with small hydrophilic reagents reveals an unbranched cation-selective pathway crossing the entire membrane. Replacing external Na with K promptly shuts the extracellular-side gate, like during K transport by unmodified pumps. The high open probability of palytoxin-bound pump-channels in Na solutions falls ≥5 fold if ATP is withdrawn. Cytoplasmic AMPPNP or ADP can replace ATP - all acting with low affinity - in supporting this cytoplasmic-side gate opening, echoing acceleration of cytoplasmic K release by these nucleotides in unmodified Na,K pumps. This low-affinity, modulatory, nucleotide binding occurs electrostatically distant from the normally-phosphorylated Asp, as its mutation to Asn, Ala, or Glu leaves nucleotide affinity unaltered. The phosphate analogs BeFx or AlFx close palytoxin-bound pump-channels, apparently in all-or-none manner, quickly when ATP is absent, but slowly in millimolar ATP. This antagonism reflects modulatory nucleotide binding because ADP or AMPPNP mimic it, and it persists after mutating the phosphate-acceptor Asp to Asn. Thus BeFx or AlFx binds most tightly to pump-channel conformations with the cytoplasmic-side gate shut, despite bound palytoxin. Modulatory nucleotide binding, therefore, apparently favors cytoplasmic-side gate opening by weakening the cytoplasmic N-, P-, and A-domain interactions that stabilize gate closure, but modulation fails when BeFx strengthens those interactions. [HL36783]