Spermine blocks connexin40 (Cx40) gap junctions, and two cytoplasmic amino-terminal domain glutamate residues are essential for this inhibitory activity. To further examine the molecular basis for block, we mutated a portion of a basic amino acid (HKH) motif on the Cx40 amino-terminal domain. Replacement of the Cx40 H15 + K16 residues with the Q15 + A16 sequence native to spermine-insensitive connexin43 (Cx43) gap junctions increased the equilibrium dissociation constant (K(d)) and reduced the maximum inhibition by spermine. The corresponding electrical distance (delta) approximation was decreased by about 50%. The transjunctional voltage (V(j))-dependent gating of homotypic Cx40 H15Q + K16A mutant gap junctions was also significantly reduced. The minimum normalized steady-state junctional conductance (G(min)) increased from 0.17 to 0.72, with an increase in the half-inactivation voltage from 48 to 60 mV. However, the unitary junctional conductance (gamma(j); 160 pS) was only slightly altered, and the relative cation/anion conductance and permeability ratios were unchanged from wild-type Cx40 gap junction channels. The relative K(+)/Cl(-) permeability (P(K)/P(Cl)) ratio increased from six to ten when [KCl] was reduced to 25% of normal. These data suggest that the HKH motif at positions 15-17 is important to the conformational structure of the putative voltage sensor and spermine receptor of Cx40, without causing significant alteration of the electrostatic surface charge potentials that contribute to the ion selectivity of this gap junction channel.