A new molecular biological epoch in amiloride-sensitive Na+ channel physiology has begun. With the application of these new techniques, undoubtedly a plethora of new information and new questions will be forthcoming. First and foremost, however, is the question of how many discrete amiloride-sensitive Na+ channels exist. This question is important not only for elucidating structure-function relationships, but also for developing strategies for pharmacological or, ultimately, genetic intervention in such diseases as obstructive nephropathy, Liddle's syndrome, or salt-sensitive hypertension where amiloride-sensitive Na+ channel dysfunction has been implicated [17, 62]. Epithelia Na+ channels purified from kidney are multimeric. However, it is not yet clear which subunits are regulatory and which participate directly as a part of the Na+ conducting core and what is the nature of the gate. The combination of electrophysiologic techniques such as patch clamp and the ability to study reconstituted channels in planar lipid bilayers along with molecular biology techniques to potentially manipulate the individual subunits should provide the answers to questions that have puzzled physiologists for decades. It seems clear that the robust versatility of the channel in responding to a wide range of differing and potentially synergistic regulatory inputs must be a function of its multimeric structure and relation to the cytoskeleton. Multiple mechanisms of regulation imply multiple regulatory sites. This hypothesis has been validated by the demonstration that enzymatic carboxyl methylation and phosphorylation have both individual and synergistic effects on the purified channel in planar lipid bilayers. Of the multiple mechanisms proposed for channel regulation, evidence is now available to support the ideas that channels may be activated (or inactivated) by direct modifications including phosphorylation and carboxyl methylation, by activation or association of regulatory proteins such as G proteins, and by recruitment from subapical membrane domains. The observation that channel gating is achieved primarily through regulation of open probability without alterations in conductance may simplify future understanding of the molecular events involved in gating once the regulatory sites have been identified. As more Na+ channels or Na+ channel subunits are cloned from different epithelia, it will become possible to piece together the puzzle of epithelial Na+ channels. It is interesting to observe that renal Na+ channel proteins contain a subunit which falls into the 70 kD range. This size protein is in the range reported for the aldosterone-induced proteins [12, 46, 153].(ABSTRACT TRUNCATED AT 400 WORDS)